CN112649406A - Method for detecting glutathione by fluorescence enhancement method - Google Patents
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Abstract
The invention relates to a method for detecting glutathione by using a fluorescence enhancement method, which comprises the following steps: the method comprises the steps of putting a fluorescent material and glutathione into water, and uniformly mixing; adding a surfactant, cations and anions into the mixed solution to form a system for detecting glutathione by fluorescence; thirdly, the prepared fluorescence detection system is uniformly mixed and then placed in an environment with the temperature of 20-40 ℃ to be detected; and fourthly, exciting and irradiating a fluorescence detection system by adopting 980nm, and recording the fluorescence intensity F value at 550nm to obtain the fluorescent material. The method utilizes the characteristic that the up-conversion nano material can emit visible light under the excitation of near infrared light, and can enhance the fluorescence of the up-conversion nano material in a system containing a surfactant, cations and anions, so that the glutathione in the high-sensitivity sensing detection system is high in selectivity and good in stability.
Description
Technical Field
The invention belongs to the technical field of detection of small molecular biological thiol compounds, and particularly relates to a method for detecting glutathione by using a fluorescence enhancement method.
Background
Glutathione is the most important non-protein thiol compound in living cells of mammals and eukaryotic cells, is an important endogenous antioxidant and antidote, and plays an important role in eliminating free radicals in a biological system. Glutathione is the first line of defense against toxic substances and can be widely involved in human immune regulation, metabolism, energy transportation and other physiological processes. It plays a key role in maintaining cellular redox homeostasis, cell signal transduction, detoxification and the like. In general, an imbalance in glutathione concentration in humans can lead to a number of diseases such as leukopenia, psoriasis, liver damage, cancer, Human Immunodeficiency Virus (HIV), alzheimer's disease, diabetes, parkinson's disease, cardiovascular disease and inflammation. In addition to these effects, glutathione is also used as an addictive ingredient and added to foods and natural cosmetics.
To date, many methods for quantitatively detecting glutathione have been developed, such as colorimetry, electrochemistry, enzymology, high performance liquid chromatography, mass spectrometry, electrochemiluminescence, surface-enhanced raman scattering, photoelectrochemical methods, enzyme-linked immunosorbent assay, capillary electrophoresis, voltammetry, and fluorescence spectroscopy. The fluorescence spectroscopy has the advantages of high sensitivity, good flexibility, simple operation, nondestructive detection and the like, and in recent years, the fluorescence spectroscopy becomes a promising glutathione detection method.
To date, researchers have designed a number of organic fluorophores to detect glutathione based on different sensing mechanisms (cyclization, michael addition, disulfide and sulfonamide cleavage, nucleophilic substitution of thiols, and metal complex mismatch coordination, etc.). Although these organic fluorescent materials are practical, most of them still have the limitations of high cost, time consumption, complicated sample purification steps, etc.; in the aspect of biological application, the defects of photobleaching, poor stability, poor biocompatibility and the like also exist. Therefore, development of novel fluorescent nanomaterials having ideal biological and environmental safety, high fluorescence intensity, and low cost is highly desired; the establishment of a new glutathione detection method with high sensitivity, high selectivity, low cost and simple operation has important significance.
Through searching, no patent publication related to the present patent application has been found.
Disclosure of Invention
Aiming at the defects in the prior art, the invention provides a method for detecting glutathione by using a fluorescence enhancement method.
The technical scheme adopted by the invention for solving the technical problems is as follows:
a method for detecting glutathione by using a fluorescence enhancement method comprises the following steps:
the fluorescent material and the glutathione are put in water and uniformly mixed, and the final concentration of the fluorescent material is 0.001-0.1 mg/mL-1The final concentration of glutathione was 6.0X 10-10-1.0×10-3mg·mL-1；
Adding a surfactant, cations and anions into the mixed solution to form a system for detecting glutathione by fluorescence; wherein the surfactant has a final mass concentration of 0.01-0.1% and the cation has a concentration of 10 μ g/mL-1-150μg·mL-1Anion concentration of 2.3X 10-10mol·L-1-2.5×10-1mol·L-1；
Thirdly, the prepared fluorescence detection system is uniformly mixed and then placed in an environment with the temperature of 20-40 ℃ to be detected;
and fourthly, exciting and irradiating a fluorescence detection system by adopting 980nm, and recording the fluorescence intensity F value at 550nm to obtain the fluorescent material.
The fluorescent material is an up-conversion fluorescent nano-material prepared by taking yttrium acetate tetrahydrate as a main luminescent matrix, ytterbium acetate (III) tetrahydrate as a sensitizer, erbium acetate (III) hydrate as an activator, oleic acid as a ligand and a solvent, and 1-octadecene as a high-boiling-point solvent through a solvothermal method.
Moreover, the specific preparation steps of the up-conversion fluorescent nano material are as follows:
placing a luminescent material main body, a sensitizing agent and an activator of an up-conversion material into a three-neck flask, then adding oleic acid and 1-octadecene into the three-neck flask, placing a magnetic stirrer, and then placing the three-neck flask into a temperature control heating sleeve for magnetic stirring; wherein the mass ratio of the luminescent material main body to the sensitizer to the activator of the up-conversion material is 38-40:9-11:1-2, the volume ratio of oleic acid to 1-octadecene is 5-7:16-18, and the ratio of the luminescent material main body to the oleic acid, mg: mL, is 250-270: 5-7;
under the protection of argon, gradually heating the mixture in the three-neck flask to 100 ℃ under magnetic stirring, turning off the argon, vacuumizing, stopping vacuumizing when no bubbles appear on the whole liquid level of the solution, and introducing the argon; continuing heating until the temperature of the solution rises to 160 ℃, maintaining the reaction for 30min, and cooling to room temperature; adding sodium hydroxide and ammonium fluoride solid into methanol, and performing ultrasonic treatment until the sodium hydroxide and ammonium fluoride solid are completely dissolved; slowly dropwise adding a methanol solution of sodium hydroxide and ammonium fluoride into the three-neck flask, and continuously stirring for 30 min; wherein the ratio mg: mg of the ammonium fluoride solid to the sodium hydroxide solid to the methanol is 14-15:10-11:1-2, and the ratio mg: mg of the luminescent material main body to the sodium hydroxide solid is 25-27: 10-11;
heating the solution to 100 ℃ to distill methanol, stopping introducing argon gas and vacuumizing until no methanol is distilled out of the system, turning off a vacuum pump, and continuously introducing argon gas; continuously heating until the temperature of the solution reaches 300 ℃, reacting for 1h at constant temperature, and naturally cooling to room temperature; centrifuging the solution in the three-neck flask for 10min at the rotating speed of 10000r/min, washing the solution for three times by using absolute ethyl alcohol, and carrying out vacuum drying at the temperature of 60 ℃ for 24h to obtain the upconversion fluorescent nano material.
And in the step II, the surfactant is one or more than two of polyvinyl alcohol, gelatin, Tween 80 and Triton X-100.
And in the step II, the cation is Na+、Al3+、Ag+、K+、Cu2+One or more than two of them.
And, the anion in the step II is Cl-、I-、Br-One or more than two of them.
And in the fourth step, a 980nm external source laser is used as an excitation light source to irradiate the fluorescence system to be measured, and the fluorescence intensity value at the position of 550nm is recorded.
The method for detecting glutathione by using the fluorescence enhancement method is applied to the detection of glutathione.
The invention has the advantages and positive effects that:
1. the method utilizes the characteristic that the up-conversion nano material can emit visible light under the excitation of near infrared light, and can enhance the fluorescence of the up-conversion nano material in a system containing a surfactant, cations and anions, so that the glutathione in the high-sensitivity sensing detection system is high in selectivity and good in stability.
2. The method changes the traditional method for determining glutathione by a fluorescence method, firstly puts forward non-modified up-conversion nanoparticles to be directly used as fluorescence detection signal substances, and compared with other fluorescent probes needing complex modification, the method has the advantages of rapidness, simplicity, time saving and the like.
3. According to the method, the surfactant, the positive ions and the negative ions are added into the fluorescence detection system containing the up-conversion fluorescent nano material and the glutathione, so that the fluorescence intensity of the up-conversion nano material is enhanced, the glutathione can be better sensed and detected, the linear range of the detection of the glutathione is 0.75nM-0.75 MuM, the linear range of the detection is wider, and the detection limit is lower.
4. The invention selects 15 common amino acids as glutathione interferents to carry out the determination of the selectivity experiment. Fluorescence tests were performed on L-phenylalanine, L-glutamic acid, tryptophan, L-cystine, tyrosine, L-aspartic acid, L-histidine, L-proline, alanine, L-cysteine, L-arginine, L-lysine, L-serine, L-valine and glycine, respectively. The result shows that the sensing detection method has better selectivity for detecting glutathione.
5. The invention changes the traditional method for measuring glutathione by a fluorescence method, selects an unmodified up-conversion material as a fluorescent material, polyvinyl alcohol as a surfactant and Ag+Is a cation, I-As anion, polyvinyl alcohol, Ag+And I-The method for rapidly and sensitively detecting the glutathione based on the fluorescence enhancement of the up-conversion nano material is established for the sensitization of the up-conversion nano material.
Drawings
FIG. 1 is a graph showing the influence of different concentrations of glutathione on the fluorescence spectrum of an upconversion fluorescent nanomaterial when glutathione is sensed and detected in a solution under optimal conditions in the present invention;
FIG. 2 shows the fluorescence intensity F and the negative logarithm (-log C) of the glutathione concentration of the upconversion nanomaterial of the present inventionGSH) A linear relationship graph of (a);
FIG. 3 is a fluorescence map measured in the selective assay of Glutathione (GSH) in the present invention; wherein, the interfering substances are respectively: l-phenylalanine (Phe), L-glutamic acid (Glu), tryptophan (Trp), L-cystine (Cys), tyrosine (Tyr), L-aspartic acid (Asp), L-histidine (His), L-proline (Pro), alanine (Ala), L-cysteine (Cys), L-arginine (Arg), L-lysine (Lys), L-serine (Ser), L-valine (Val) and glycine (Gly).
Detailed Description
The following detailed description of the embodiments of the present invention is provided for the purpose of illustration and not limitation, and should not be construed as limiting the scope of the invention.
The raw materials used in the invention are conventional commercial products unless otherwise specified; the methods used in the present invention are conventional in the art unless otherwise specified.
A method for detecting glutathione by using a fluorescence enhancement method comprises the following steps:
the fluorescent material and the glutathione are put in water and uniformly mixed, and the final concentration of the fluorescent material is 0.001-0.1 mg/mL-1The final concentration of glutathione was 6.0X 10-10-1.0×10-3mg·mL-1；
Adding a surfactant, cations and anions into the mixed solution to form a system for detecting glutathione by fluorescence; wherein the surfactant has a final mass concentration of 0.01-0.1% and the cation has a concentration of 10 μ g/mL-1-150μg·mL-1Anion concentration of 2.3X 10-10mol·L-1-2.5×10-1mol·L-1；
Thirdly, the prepared fluorescence detection system is uniformly mixed and then placed in an environment with the temperature of 20-40 ℃ to be detected;
and fourthly, exciting and irradiating a fluorescence detection system by adopting 980nm, and recording the fluorescence intensity F value at 550nm to obtain the fluorescent material.
Preferably, the fluorescent material in the first step is yttrium acetate tetrahydrate (Y (CH)3COO)3·4H2O) as main luminescent matrix, ytterbium acetate (III) tetrahydrate (Yb (CH)3COO)3·4H2O) as sensitizer, erbium (III) acetate hydrate (Er (CH)3COO)3·xH2O) is used as an activator, Oleic Acid (OA) is used as a ligand and a solvent, and 1-Octadecene (ODE) is used as a high boiling point solvent, and the up-conversion fluorescent nano material is prepared by a solvothermal method.
Preferably, the specific preparation steps of the up-conversion fluorescent nano material are as follows:
placing a luminescent material main body, a sensitizing agent and an activator of an up-conversion material into a three-neck flask, then adding oleic acid and 1-octadecene into the three-neck flask, placing a magnetic stirrer, and then placing the three-neck flask into a temperature control heating sleeve for magnetic stirring; wherein the mass ratio of the luminescent material main body to the sensitizer to the activator of the up-conversion material is 38-40:9-11:1-2, the volume ratio of oleic acid to 1-octadecene is 5-7:16-18, and the ratio of the luminescent material main body to the oleic acid, mg: mL, is 250-270: 5-7;
under the protection of argon, gradually heating the mixture in the three-neck flask to 100 ℃ under magnetic stirring, turning off the argon, vacuumizing, stopping vacuumizing when no bubbles appear on the whole liquid level of the solution, and introducing the argon; continuing heating until the temperature of the solution rises to 160 ℃, maintaining the reaction for 30min, and cooling to room temperature; adding sodium hydroxide and ammonium fluoride solid into methanol, and performing ultrasonic treatment until the sodium hydroxide and ammonium fluoride solid are completely dissolved; slowly dropwise adding a methanol solution of sodium hydroxide and ammonium fluoride into the three-neck flask, and continuously stirring for 30 min; wherein the ratio mg: mg of the ammonium fluoride solid to the sodium hydroxide solid to the methanol is 14-15:10-11:1-2, and the ratio mg: mg of the luminescent material main body to the sodium hydroxide solid is 25-27: 10-11;
heating the solution to 100 ℃ to distill methanol, stopping introducing argon gas and vacuumizing until no methanol is distilled out of the system, turning off a vacuum pump, and continuously introducing argon gas; continuously heating until the temperature of the solution reaches 300 ℃, reacting for 1h at constant temperature, and naturally cooling to room temperature; centrifuging the solution in the three-neck flask for 10min at the rotating speed of 10000r/min, washing the solution for three times by using absolute ethyl alcohol, and carrying out vacuum drying at the temperature of 60 ℃ for 24h to obtain the upconversion fluorescent nano material.
Preferably, the surfactant in the step II is one or more of polyvinyl alcohol, gelatin, Tween 80 and Triton X-100.
Preferably, in the step II, the cation is Na+、Al3+、Ag+、K+、Cu2+One or more than two of them.
Preferably, the anion in the step II is Cl-、I-、Br-One or more than two of them.
Preferably, in the fourth step, a 980nm external source laser is used as an excitation light source to irradiate the fluorescence system to be measured, and the fluorescence intensity value at the position of 550nm is recorded.
The method for detecting glutathione by using the fluorescence enhancement method is applied to the detection of glutathione.
Specifically, the preparation and detection are as follows:
firstly, detecting glutathione in human serum by a standard recovery method:
the experimental procedure was as follows:
(1) putting the up-conversion nano fluorescent material and glutathione into water, and uniformly mixing, wherein the final concentration of the fluorescent material is 0.01 mg/mL-1The final concentration of glutathione was 7.5X 10, respectively-10mol·L-1，1.2×10-9mol·L-1，7.5×10- 9mol·L-1，1.2×10-8mol·L-1，7.5×10-8mol·L-1，1.2×10-7mol·L-1，7.5×10-7mol·L-1。
(2) Mixing polyvinyl alcohol and Ag+And I-Adding the glutathione into the mixed solution to form a system for detecting the glutathione by fluorescence. Wherein the surfactant has a final mass concentration of 0.04% and Ag+Has a concentration of 100. mu.g.mL-1，I-The concentration is 2.4X 10- 4mol·L-1。
(3) And (3) uniformly mixing the prepared fluorescence detection system, and placing the mixture in an environment with the temperature of 20-40 ℃ for detection.
(4) A 980nm exogenous laser is used as an excitation light source to irradiate a fluorescence detection system, so that a fluorescence spectrum curve graph of the up-conversion nano material under different glutathione concentrations is obtained, as shown in figure 1, the graph shows that the fluorescence intensity of the up-conversion nano material at 550nm is gradually enhanced along with the increase of the glutathione concentration;
recording the fluorescence intensity F value at 550nm, and drawing a standard curve, as shown in FIG. 2, the fluorescence intensity F of the up-conversion nano material and the negative logarithm of the GSH concentration are 7.5 × 10 in the concentration of the GSH water solution-10mol·L-1、1.2×10-9mol·L-1、7.5×10- 9mol·L-1、1.2×10-8mol·L-1、7.5×10-8mol·L-1、1.2×10-7mol·L-1、7.5×10-7mol·L-1The time is linearly related, and the linear regression equation is as follows: f-484.7725 (-log C)GSH) +6586.304, linear correlation coefficient R2Linear range 0.75nM to 0.75 μ M0.9944. According to the 3 σ rule (LOD 3 σ/s), the GSH minimum detection Limit (LOD) was calculated to be 3.5 × 10-10mol·L-1。
(5) And taking out 1mL of human serum sample for dilution by 100 times, centrifuging the diluent, and taking the supernatant for a standard addition recovery experiment. Respectively adding 1 nmol. L-1，10nmol·L-1，100nmol·L-1The glutathione of (4) was manipulated according to the experimental procedures of (1).
The experimental results are shown in the following table:
TABLE 1 spiking recovery experiment
Secondly, calculating a fluorescence change value delta F:
(1) respectively adding the up-conversion nano fluorescent material into aqueous solutions of 16 target substances, and uniformly mixing, wherein the final concentration of the fluorescent material is 0.01 mg/mL-1L-phenylalanine, L-glutamic acid, tryptophan, L-cystine, tyrosine, L-aspartic acid, L-histidine, L-proline, alanine, L-cysteine, L-arginine, L-lysine, L-serine, L-valine, glycine and glutathione all at a final concentration of 7.5X 10-5mol·L-1。
(2) Mixing polyvinyl alcohol and Ag+And I-Adding into the 16 mixed solutions to form a system for detecting the target object by fluorescence. Wherein the surfactant has a final mass concentration of 0.04% and Ag+Has a concentration of 100. mu.g.mL-1，I-The concentration is 2.4X 10- 4mol·L-1。
(3) And (3) uniformly mixing the prepared fluorescence detection system, and placing the mixture in an environment with the temperature of 20-40 ℃ for detection.
(4) A980 nm exogenous laser is used as an excitation light source to irradiate a fluorescence detection system, the fluorescence intensity F value at 550nm is recorded, and each group of fluorescence change values delta F are obtained through calculation, as shown in figure 3, the sensing detection method has good selectivity on GSH detection.
Although the embodiments of the present invention have been disclosed for illustrative purposes, those skilled in the art will appreciate that: various substitutions, changes and modifications are possible without departing from the spirit and scope of the invention and the appended claims, and therefore the scope of the invention is not limited to the disclosure of the embodiments and the accompanying drawings.
Claims (8)
1. A method for detecting glutathione by using a fluorescence enhancement method is characterized by comprising the following steps: the method comprises the following steps:
the fluorescent material and the glutathione are put in water and uniformly mixed, and the final concentration of the fluorescent material is 0.001-0.1 mg/mL-1The final concentration of glutathione was 6.0X 10-10-1.0×10-3mg·mL-1；
Secondly, adding a surfactant, cations and anions into the mixed solutionIn the liquid, a system for detecting glutathione by fluorescence is formed; wherein the surfactant has a final mass concentration of 0.01-0.1% and the cation has a concentration of 10 μ g/mL-1-150μg·mL-1Anion concentration of 2.3X 10-10mol·L-1-2.5×10-1mol·L-1；
Thirdly, the prepared fluorescence detection system is uniformly mixed and then placed in an environment with the temperature of 20-40 ℃ to be detected;
and fourthly, exciting and irradiating a fluorescence detection system by adopting 980nm, and recording the fluorescence intensity F value at 550nm to obtain the fluorescent material.
2. The method for detecting glutathione using a fluorescence enhancement method according to claim 1, wherein: the fluorescent material is an up-conversion fluorescent nano-material prepared by a solvothermal method, wherein yttrium acetate tetrahydrate is used as a main luminescent matrix, ytterbium acetate (III) tetrahydrate is used as a sensitizer, erbium acetate (III) hydrate is used as an activator, oleic acid is used as a ligand and a solvent, and 1-octadecene is used as a high-boiling-point solvent.
3. The method for detecting glutathione using a fluorescence enhancement method according to claim 2, wherein: the specific preparation steps of the up-conversion fluorescent nano material are as follows:
placing a luminescent material main body, a sensitizing agent and an activator of an up-conversion material into a three-neck flask, then adding oleic acid and 1-octadecene into the three-neck flask, placing a magnetic stirrer, and then placing the three-neck flask into a temperature control heating sleeve for magnetic stirring; wherein the mass ratio of the luminescent material main body to the sensitizer to the activator of the up-conversion material is 38-40:9-11:1-2, the volume ratio of oleic acid to 1-octadecene is 5-7:16-18, and the ratio of the luminescent material main body to the oleic acid, mg: mL, is 250-270: 5-7;
under the protection of argon, gradually heating the mixture in the three-neck flask to 100 ℃ under magnetic stirring, turning off the argon, vacuumizing, stopping vacuumizing when no bubbles appear on the whole liquid level of the solution, and introducing the argon; continuing heating until the temperature of the solution rises to 160 ℃, maintaining the reaction for 30min, and cooling to room temperature; adding sodium hydroxide and ammonium fluoride solid into methanol, and performing ultrasonic treatment until the sodium hydroxide and ammonium fluoride solid are completely dissolved; slowly dropwise adding a methanol solution of sodium hydroxide and ammonium fluoride into the three-neck flask, and continuously stirring for 30 min; wherein the ratio mg: mg of the ammonium fluoride solid to the sodium hydroxide solid to the methanol is 14-15:10-11:1-2, and the ratio mg: mg of the luminescent material main body to the sodium hydroxide solid is 25-27: 10-11;
heating the solution to 100 ℃ to distill methanol, stopping introducing argon gas and vacuumizing until no methanol is distilled out of the system, turning off a vacuum pump, and continuously introducing argon gas; continuously heating until the temperature of the solution reaches 300 ℃, reacting for 1h at constant temperature, and naturally cooling to room temperature; centrifuging the solution in the three-neck flask for 10min at the rotating speed of 10000r/min, washing the solution for three times by using absolute ethyl alcohol, and carrying out vacuum drying at the temperature of 60 ℃ for 24h to obtain the upconversion fluorescent nano material.
4. The method for detecting glutathione using a fluorescence enhancement method according to claim 1, wherein: the surfactant in the step II is one or more than two of polyvinyl alcohol, gelatin, Tween 80 and Triton X-100.
5. The method for detecting glutathione using a fluorescence enhancement method according to claim 1, wherein: in the step II, the cation is Na+、Al3+、Ag+、K+、Cu2+One or more than two of them.
6. The method for detecting glutathione using a fluorescence enhancement method according to claim 1, wherein: in the step II, the anion is Cl-、I-、Br-One or more than two of them.
7. The method for detecting glutathione using a fluorescence enhancement method according to claim 1, wherein: and step four, irradiating the fluorescent system to be detected by using a 980nm external source laser as an excitation light source, and recording the fluorescence intensity value at the position of 550 nm.
8. Use of the method for detecting glutathione by fluorescence enhancement according to any one of claims 1 to 7 for glutathione detection.
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| CN113791056A (en) * | 2021-09-12 | 2021-12-14 | 广西师范大学 | Method for detecting ferric ions and glutathione based on HOF-PyTTA fluorescent material |
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| CN107677652A (en) * | 2017-08-24 | 2018-02-09 | 安徽师范大学 | Upper conversion nano rod/dopamine system of additive Mn and preparation method thereof and the detection method of glutathione or cysteine |
| CN108982440A (en) * | 2018-06-10 | 2018-12-11 | 福建医科大学 | The upper building for converting faint photodetector and its detection for glutathione |
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| CN113791056A (en) * | 2021-09-12 | 2021-12-14 | 广西师范大学 | Method for detecting ferric ions and glutathione based on HOF-PyTTA fluorescent material |
| CN113791056B (en) * | 2021-09-12 | 2024-05-14 | 河北健海医学检验实验室有限公司 | Method for detecting ferric ions and glutathione based on HOF-PyTTA fluorescent material |
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