A method for detecting glutathione

Abstract

This invention belongs to the field of biotechnology, specifically a method for detecting glutathione, comprising the following steps: mixing dopamine hydrochloride solution with NaOH solution to obtain a polydopamine nanoparticle solution; replacing DNA with a target that can be adsorbed onto polydopamine and desorbed by glutathione; the resulting polydopamine nanoparticle solution; and Zn-containing... 2+ The solution and buffer solution were mixed and incubated at room temperature to obtain PDANs-Zn. 2+ - Targeted DNA polymer; the sample is added to the resulting PDANs-Zn 2+ In a target-substituted DNA polymer, after incubation at room temperature and centrifugation, the supernatant was collected to obtain the target-substituted DNA strand. The obtained target-substituted DNA underwent an RCA reaction in the reaction system. A fluorescent dye was added to the obtained amplification product, and after incubation, fluorescence detection was performed. Using polydopamine as a "bridge," the detection of GSH was transformed into the detection of DNA, thus improving sensitivity.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and more specifically to a method for detecting glutathione. Background Technology

[0002] Glutathione (GSH), or γ-glutamylcysteine, is commonly represented by the free -SH group. It is a tripeptide compound with important physiological functions, composed of the amino acids glutamic acid, cysteine, and glycine, and exists in both reduced (GSH) and oxidized (GSSG) forms. Glutathione is the most abundant thiol compound in mammalian cells, participating in maintaining cellular redox homeostasis, signal transduction, and gene regulation. Catalyzed by glutathione S-transferase (GST) and glutathione peroxidase (GPX), glutathione participates in the detoxification of various electrophilic compounds and peroxides, converting harmful toxins in the body into harmless substances for excretion. It not only protects hemoglobin and the sulfhydryl groups in corresponding enzymes and restores their activity, but also has anti-aging and toxic compound neutralizing effects. A deficiency of GSH exposes cells to oxidative damage, and abnormal changes in glutathione levels are often associated with diseases such as cancer, cystic fibrosis, Parkinson's disease, and immunodeficiency. Reports indicate that GSH concentrations in tumor tissues are twice as high as in normal tissues, and even up to ten times higher in some drug-resistant tumor tissues. Therefore, GSH levels are crucial for disease diagnosis, medical treatment, and other biomedical applications.

[0003] Currently, there are various methods for detecting glutathione, such as spectrophotometry, high-performance liquid chromatography (HPLC), capillary electrophoresis, electrochemical analysis, colorimetry, mass spectrometry, and fluorescence methods. Spectrophotometry is a method for qualitative or quantitative analysis of a substance based on its absorbance at a specific wavelength or within a certain wavelength range. It is one of the earliest and most widely used methods. However, spectrophotometry has limitations because the commonly used reagents, alloxan or 2-nitrobenzoic acid, lack specificity. HPLC has a wide linear range, high selectivity, good stability, and high accuracy, but low sensitivity. Capillary electrophoresis is simple to operate and highly reliable, but has low sensitivity. Electrochemical analysis has poor selectivity and is easily interfered with by other substances. Colorimetry is simple to operate, but has low sensitivity. Mass spectrometry is complex to operate and has poor sensitivity and selectivity.

[0004] In summary, existing methods for detecting glutathione have low sensitivity and poor selectivity. Therefore, there is a need for a simple, sensitive, and highly specific method for detecting glutathione. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention provides a method for detecting glutathione.

[0006] A method for detecting glutathione includes the following steps:

[0007] S1. Synthesis of polydopamine: Mix dopamine hydrochloride solution with NaOH solution to obtain polydopamine nanoparticle solution;

[0008] S2, DNA adsorption: DNA is replaced by a target that can be adsorbed onto polydopamine and desorbed by glutathione; the polydopamine nanoparticle solution obtained in S1; Zn-containing... 2+ The solution and buffer solution were mixed and incubated at room temperature to obtain PDANs-Zn. 2+ -Target-replaced DNA polymers;

[0009] S3, DNA desorption: The sample is added to the PDAs-Zn obtained in S2. 2+ -The target-substituted DNA polymer was incubated at room temperature and then centrifuged. The supernatant was collected to obtain the target-substituted DNA strand.

[0010] S4, Signal Amplification: The target substitute DNA strand obtained in S3 undergoes an RCA reaction in the reaction system to obtain the amplification product;

[0011] S5. Fluorescence detection: Add fluorescent dye to the amplification product obtained in S4, incubate, and then perform fluorescence detection.

[0012] Preferably, the sequence of the target substitute DNA is 5'-AAAAAAAAACCCAGGTTCTCT-3'.

[0013] Preferably, the concentration of the dopamine hydrochloride solution in S1 is 1-3 mg / mL, the concentration of the sodium hydroxide solution is 350-450 mM, and the volume ratio of the dopamine hydrochloride solution to the sodium hydroxide solution is 50-100:1.

[0014] Preferably, the mixing conditions in S1 are stirring at 45-55°C for 4-6 hours.

[0015] Preferably, S2 contains target-substitute DNA, polydopamine nanoparticle solution, and Zn-containing... 2+ The final volume of the solution and buffer solution is 50 μL. The target-substituted DNA, the polydopamine nanoparticle solution, and the Zn-containing... 2+ The mass concentration ratio of the solutions is 1:2:1.5.

[0016] Preferably, incubate in S2 for 20-50 minutes.

[0017] Preferably, incubate in S3 for 30-60 minutes.

[0018] Preferably, the reaction system in S4 is: 7.5 μL of 320 nM phosphorylated padlock probe, 7.5 μL of target substitute DNA strand, 3 μL of 10×T4 DNA ligase buffer solution, 10.5 μL of sterile water, and 1.5 μL of 350 U / μL T4 DNA ligase to prepare a total volume of 30 μL.

[0019] After mixing them thoroughly, react at 16℃ for 1 hour. Take 10 μL of the reaction system, add 1.5 μL of 10 mM dNTPs, 2 μL of 10×phi29 DNA polymerase reaction buffer, 6 μL of sterile water and 0.5 μL of 10 U / μL phi29 DNA polymerase, and react at 30℃ for 1 hour to carry out the RCA reaction.

[0020] Preferably, the fluorescent dye in S5 is ThT.

[0021] The preferred incubation conditions in S5 are: incubation at 85-105℃ for 3-8 minutes, followed by incubation at 2-6℃ for 3-8 minutes, and finally incubation at room temperature for 0.5-1.5 hours.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] This invention transforms the detection of glutathione into the detection of DNA through the bridging effect of polydopamine. Utilizing the ease with which DNA molecules undergo signal amplification, and combining this with G-trimonic rolling circle amplification technology, it not only improves the sensitivity of enzyme and peptide detection but also avoids cumbersome operational procedures and unstable reaction processes. By using polydopamine as a "bridge," the detection of GSH is transformed into the detection of DNA, thus improving sensitivity.

[0024] In an alkaline, oxygen-rich environment, dopamine hydrochloride (DA) can generate polydopamine nanoparticles (PDANs) through oxidative self-polymerization. At neutral pH, both PDAs and DNA chains are negatively charged, making it difficult for DNA chains to bind to PDAs alone. It has been reported that metal ions can induce ssDNA chains to adsorb onto PDAs. Since the catechol groups of PDAs are strong metal ligands, metal ions can also bind to the bases and phosphate backbone of the DNA chain with varying affinities. Therefore, under the induction of metal ions, DNA chains can adsorb onto polydopamine particles, forming "polydopamine bridges" (PDANs-Zn). 2+ Glutathione is a thiol containing a thiol group and is a strong metal ion chelator. It can act as a ligand to compete for these metal ions, thereby desorbing the ssDNA chain from PDANs. The desorbed target replacement DNA chain is used as a primer for signal amplification under the action of enzymes, padlock probes, and dNTPs. Fluorescent dye is added to the amplification product, which will emit a fluorescent signal under specific conditions.

[0025] Over the past decade, research on PDAs has been very active, with their applications in drug delivery, photothermal therapy, tissue engineering, cell adhesion, and biosensing. In biosensing platforms, PDANs (polydopamine adsorption groups) are widely used as substrates for DNA assembly through π-π stacking interactions. However, this adsorption mode is susceptible to competition from other biomolecules, affecting the performance of biosensors. Since the catechol groups of PDANs are ideal metal ligands, metal coordination is a simple and effective method for PDAN functionalization. This involves the catechol groups on the PDAN surface coordinating with metal ions, followed by the interaction of the phosphate backbone of the DNA strand with the metal ions, inducing DNA bridging onto the PDANs. Therefore, PDANs can serve as nanocarriers for metal-mediated DNA adsorption, forming a "polydopamine bridge" (PDANs-metal ion-DNA). Thiol groups (-SH) can undergo ion exchange and chelation reactions with metal ions, forming strong coordination bonds, while glutathione, a thiol containing a thiol group, is a strong metal ion chelating agent. Attached Figure Description

[0026] Figure 1 This is a schematic diagram illustrating the principle of glutathione detection.

[0027] Figure 2 This is a transmission electron microscope image of polydopamine.

[0028] Figure 3 In the image, A is the Raman spectrum of polydopamine, and B is the ultraviolet spectrum of polydopamine.

[0029] Figure 4 Characterization diagram of polydopamine bridge;

[0030] Figure 5 The image is a polyacrylamide gel electrophoresis image, where M represents a 20bp DNA marker, 1 represents a phosphorylated padlock probe, 2 represents a target substitute DNA strand, 3 represents a phosphorylated padlock probe + target substitute DNA strand, 4 represents a phosphorylated padlock probe + target substitute DNA strand + T4 DNA ligase, and 5 represents a rolling circle amplification product.

[0031] Figure 6 The fluorescence spectra of the amplified products under different RCA reaction conditions are shown.

[0032] Figure 7 The graph shows the changes in fluorescence signal at different padlock probe concentrations.

[0033] Figure 8 The graph shows the changes in fluorescence signal at different ThT concentrations.

[0034] Figure 9The graph shows the changes in fluorescence signal after different ThT incubation times at room temperature;

[0035] Figure 10 Fluorescence spectra of GSH at different concentrations;

[0036] Figure 11 The graph shows the relative fluorescence intensity produced by different concentrations of GSH and the linear relationship between relative fluorescence intensity and GSH concentration.

[0037] Figure 12 Fluorescence intensity maps of different target analytes;

[0038] Figure 13 The oligonucleotide sequence used in this invention;

[0039] Figure 14 The intraday precision results of GSH detection based on PDA and rolling circle amplification strategies;

[0040] Figure 15 The results show the daytime precision of GSH detection based on PDA and rolling circle amplification strategies. Detailed Implementation

[0041] The specific embodiments of the present invention are described in detail below, but it should be understood that the scope of protection of the present invention is not limited to the specific embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention. Unless otherwise specified, the experimental methods described in the embodiments of the present invention are conventional methods.

[0042] The FAM-ssDNA, phosphorylated padlock probe, and target substitute DNA sequences used in this invention were all synthesized and purified by Sangon Biotech Co., Ltd. (Shanghai, China). T4 DNA ligase was purchased from TaKaRa Biotechnology Co., Ltd. (Dalian, China). Dopamine hydrochloride (≥98%) was purchased from Beijing Innovent Biologics Co., Ltd. (Beijing, China); glutathione (GSH), ThT, and HEPES buffer solutions were purchased from Shanghai Maclean Biotechnology Co., Ltd. (Shanghai, China); Phi29 DNA polymerase and dNTPs were purchased from Nanjing Novizan Biotechnology Co., Ltd. (Nanjing, China); all other chemicals used in the experiment were analytical grade and provided by the standard reagent supplier, and all buffers used were prepared from ultrapure water (≥18.25 MΩcm).

[0043] The 20bp DNA marker and 6× loading buffer used in natural polyacrylamide gel electrophoresis were purchased from TaKaRa Biotechnology Co., Ltd. (Dalian, China). The propylene / double 40% solution (19:1), PAGE gel accelerator, ammonium persulfate (APS) and tris(hydroxymethyl)aminomethane (Tris) used were purchased from Sangon Biotech Co., Ltd. (Shanghai, China).

[0044] Example 1

[0045] A method for detecting glutathione includes the following steps:

[0046] S1. Dissolve 150 mg of dopamine hydrochloride in 74 mL of ultrapure water. Mix the above solution with 1 mL of 396 mM NaOH and stir vigorously at 50 °C for 5 h. The obtained polydopamine nanoparticle solution is centrifuged and washed 3 times at 12000 rpm and then redispersed with ultrapure water.

[0047] S2. Add 10 μL of target replacement DNA at a concentration of 600 nM, 6 μL of PDANs at a concentration of 2 mg / mL, and 10 μL of Zn at a concentration of 900 μM. 2+ And 24 μL of a 20 mM buffer solution, the buffer solution being HEPES, pH = 7.6, prepared to a final volume of 50 μL, and incubated at room temperature for 30 min to obtain PDANs-Zn. 2+ - Targeted DNA polymer, the above samples were centrifuged and washed at 12000 rpm;

[0048] The sequence of the target-substituted DNA is 5'-AAAAAAAAACCCAGGTTCTCT-3';

[0049] S3. Add 10 μL of 120 mM glutathione (final concentration 20 mM) to the above pre-adsorbed PDANs-Zn 2+ -The target-substituted DNA polymer was incubated at room temperature for 40 min, then centrifuged and the supernatant was collected to obtain the target-substituted strand;

[0050] S4. Reaction System: Take 7.5 μL of phosphorylated padlock probe (final concentration 80 nM), 7.5 μL of target replacement DNA strand obtained from S3, 3 μL of 10×T4 DNA ligase buffer solution, 10.5 μL of sterile water, and 1.5 μL of T4 DNA ligase at a concentration of 350 U / μL to prepare a total volume of 30 μL. Mix them thoroughly and react at 16℃ for 1 h. Divide the 30 μL reaction system into three equal portions. Add 1.5 μL of 10 mM dNTPs, 2 μL of 10×phi29 DNA polymerase reaction buffer solution, 6 μL of sterile water, and 0.5 μL of phi29 DNA polymerase at a concentration of 10 U / μL to each portion and react at 30℃ for 1 h to perform RCA reaction to obtain the amplification product. The obtained amplification product can be used directly or stored at -20℃ for later use.

[0051] S5. Add 28.5 μL of sterile water and 1.5 μL of ThT (final concentration 20 μM) to the amplification product obtained in S4 to make a final volume of 50 μL. Incubate at 95 °C for 5 min, then at 4 °C for 5 min, and finally at room temperature for 1 h before fluorescence detection (Ex = 425 nm, Em = 450 nm).

[0052] Example 2

[0053] A method for detecting glutathione includes the following steps:

[0054] S1. Dissolve 300 mg of dopamine hydrochloride in 100 mL of ultrapure water. Mix the above solution with 1 mL of 450 mM NaOH and stir vigorously at 55 °C for 6 h. The obtained polydopamine nanoparticle solution is centrifuged and washed 3 times at 12000 rpm and then redispersed with ultrapure water.

[0055] S2. Add 10 μL of target replacement DNA at a concentration of 600 nM, 6 μL of PDANs at a concentration of 2 mg / mL, and 10 μL of Zn at a concentration of 900 μM. 2+ And 24 μL of a 20 mM buffer solution, the buffer solution being HEPES, pH = 7.6, prepared to a final volume of 50 μL, and incubated at room temperature for 50 min to obtain PDANs-Zn. 2+ - Targeted DNA polymer, the above samples were centrifuged and washed at 12000 rpm;

[0056] The sequence of the target-substituted DNA is 5'-AAAAAAAAACCCAGGTTCTCT-3';

[0057] S3. Add 10 μL of 120 mM glutathione (final concentration 20 mM) to the above pre-adsorbed PDANs-Zn 2+-The target-substituted DNA polymer was incubated at room temperature for 60 min, then centrifuged and the supernatant was collected to obtain the target-substituted DNA strand;

[0058] S4. Reaction System: Take 7.5 μL of phosphorylated padlock probe (final concentration 80 nM), 7.5 μL of target replacement DNA strand obtained from S3, 3 μL of 10×T4 DNA ligase buffer solution, 10.5 μL of sterile water, and 1.5 μL of T4 DNA ligase at a concentration of 350 U / μL to prepare a total volume of 30 μL. Mix them thoroughly and react at 16℃ for 1 h. Divide the 30 μL reaction system into three equal portions. Add 1.5 μL of 10 mM dNTPs, 2 μL of 10×phi29 DNA polymerase reaction buffer solution, 6 μL of sterile water, and 0.5 μL of phi29 DNA polymerase at a concentration of 10 U / μL to each portion and react at 30℃ for 1 h to perform RCA reaction to obtain the amplification product. The obtained amplification product can be used directly or stored at -20℃ for later use.

[0059] S5. Add 28.5 μL of sterile water and 1.5 μL of ThT (final concentration 20 μM) to the amplification product obtained in S4 to make a final volume of 50 μL. Incubate at 105 °C for 8 min, then at 6 °C for 8 min, and finally at room temperature for 1.5 h before fluorescence detection (Ex = 425 nm, Em = 450 nm).

[0060] Example 3

[0061] A method for detecting glutathione includes the following steps:

[0062] S1. Dissolve 50 mg of dopamine hydrochloride in 50 mL of ultrapure water. Mix the above solution with 1 mL of 350 mM NaOH and stir vigorously at 45 °C for 4 h. The obtained polydopamine nanoparticle solution is centrifuged and washed 3 times at 12000 rpm and then redispersed with ultrapure water.

[0063] S2. Add 10 μL of target replacement DNA at a concentration of 600 nM, 6 μL of PDANs at a concentration of 2 mg / mL, and 10 μL of Zn at a concentration of 900 μM. 2+ And 24 μL of a 20 mM buffer solution, the buffer solution being HEPES, pH = 7.6, prepared to a final volume of 50 μL, and incubated at room temperature for 20 min to obtain PDANs-Zn. 2+ - Targeted DNA polymer, the above samples were centrifuged and washed at 12000 rpm;

[0064] The sequence of the target-substituted DNA is 5'-AAAAAAAAACCCAGGTTCTCT-3';

[0065] S3. Add 10 μL of 120 mM glutathione (final concentration 20 mM) to the above pre-adsorbed PDANs-Zn 2+ -The target-substituted DNA polymer was incubated at room temperature for 30 min, then centrifuged and the supernatant was collected to obtain the target-substituted DNA strand;

[0066] S4. Reaction System: Take 7.5 μL of phosphorylated padlock probe (final concentration 80 nM), 7.5 μL of target replacement DNA strand obtained from S3, 3 μL of 10×T4 DNA ligase buffer solution, 10.5 μL of sterile water, and 1.5 μL of T4 DNA ligase at a concentration of 350 U / μL to prepare a total volume of 30 μL. Mix them thoroughly and react at 16℃ for 1 h. Divide the 30 μL reaction system into three equal portions. Add 1.5 μL of 10 mM dNTPs, 2 μL of 10×phi29 DNA polymerase reaction buffer solution, 6 μL of sterile water, and 0.5 μL of phi29 DNA polymerase at a concentration of 10 U / μL to each portion and react at 30℃ for 1 h to perform RCA reaction to obtain the amplification product. The obtained amplification product can be used directly or stored at -20℃ for later use.

[0067] S5. Add 28.5 μL of sterile water and 1.5 μL of ThT (final concentration 20 μM) to the amplification product obtained in S4 to make a final volume of 50 μL. Incubate at 85 °C for 3 min, then at 2 °C for 3 min, and finally at room temperature for 0.5 h before fluorescence detection (Ex = 425 nm, Em = 450 nm).

[0068] Verification test

[0069] Here are the results:

[0070] Figure 1 This is a schematic diagram illustrating the principle of glutathione detection. In an alkaline, aerobic environment, dopamine can generate polydopamine nanoparticles through an oxidative self-polymerization reaction. Upon the addition of metal ions, the catechol groups on the surface of PDANs first coordinate with the metal ions, then the adenosine of the DNA interacts with the metal ions, inducing DNA bridging onto the PDANs, thereby forming a "polydopamine bridge" (PDANs-Zn). 2+ -Target Substitute DNA). The thiol group can chelate with metal ions. Upon addition of glutathione, the metal ions desorb from the polydopamine bridge, releasing the target substitute DNA strand. This desorbed target substitute DNA strand is used as a primer for rolling circle amplification by phosphorylated padlock probes, T4 DNA ligase, Phi29 DNA polymerase, and dNTPs. ThT fluorescent dye is added to the amplification product; ThT binds to a large number of G-trisammonia molecules generated by the rolling circle amplification, thus emitting a fluorescent signal.

[0071] The morphology of the nanoparticles was characterized using transmission electron microscopy. The results are as follows: Figure 2 As shown, transmission electron microscopy images reveal that the synthesized polydopamine nanoparticles exhibit good dispersion and uniformity, with an average diameter of approximately 218 nm.

[0072] The absorption spectra of the prepared polydopamine nanoparticles were measured using a UV-Vis spectrophotometer and a Raman spectrometer. The results are as follows: Figure 3 As shown. Figure 3 A is the Raman spectrum, which shows that the characteristic Raman spectrum of DA disappears, consistent with previous reports. Figure 3 B represents the UV-Vis absorption spectrum, indicating that after polymerization, PDANs exhibit strong UV-Vis absorption in the 300–600 nm range.

[0073] The construction and characterization of "polydopamine bridges" were studied by analyzing the fluorescence intensity of FAM-labeled ssDNA oligonucleotide chains. Figure 4 As shown, the fluorescence intensity of 200 nM FAM-ssDNA is approximately 120. When PDANs are added, the fluorescence intensity decreases slightly, indicating that a small portion of the FAM-ssDNA adsorbs onto the PDANs. At lower metal concentrations, the fluorescence intensity decreases when PDANs and Zn are added simultaneously. 2+ Subsequently, a significant decrease in fluorescence intensity was observed, indicating that under the induction of metal ions, most of the FAM-ssDNA strands adsorbed onto PDAs, causing a substantial quenching of fluorescence and thus forming PDAs-Zn. 2+ -FAM-ssDNA bridge. Based on this, when GSH is added to the system, the thiol groups in GSH will chelate PDAs-Zn. 2+ Zn in FAM-ssDNA 2+ This causes FAM-ssDNA to desorb from PDANs, resulting in the restoration of quenched fluorescence.

[0074] To characterize the feasibility of the polydopamine bridging and rolling circle amplification strategy for detecting GSH, natural polyacrylamide gel electrophoresis and fluorescence experiments were performed. The results are as follows: Figure 5 and Figure 6 As shown. Figure 5 As shown, lane M is the DNA Marker standard; bands 1 and 2 represent the linear phosphorylated padlock probe and the target-substitute DNA strand, respectively; band 3 is a mixed band of the phosphorylated padlock probe and the target-substitute DNA strand; after ligation with T4 DNA ligase, only one new band with a large molecular weight appeared (band 4), indicating that the target-substitute DNA strand can effectively hybridize with the phosphorylated padlock probe; a distinct high-molecular-weight RCA product band was observed in band 5, proving that the RCA reaction was successfully performed. Figure 6As shown, no significant fluorescence enhancement was observed within a certain time period without GSH. A significant fluorescence enhancement was observed after adding GSH to a final concentration of 20 mM. Furthermore, without phosphorylated padlock probes, T4 DNA ligase, and phi29 DNA polymerase, the RCA reaction could not be induced throughout the experiment, thus no strong fluorescence signal was generated. Therefore, this experiment requires the addition of GSH, and only when all conditions are met can the RCA reaction proceed. This demonstrates the feasibility of the proposed GSH detection method based on polydopamine bridging and rolling circle amplification.

[0075] To obtain optimal analytical performance, the concentrations of the phosphorylated padlock probe and ThT, as well as the reaction time of ThT, were optimized. The padlock probe concentration plays a crucial role in the RCA process. Figure 7 As shown, the fluorescence response gradually improved with increasing phosphorylated padlock probe concentration, stabilizing at a final concentration of 80 nm. Therefore, we selected 80 nm as the optimal concentration. The concentration of ThT and the reaction time also affect the final fluorescence signal transmission. We selected a final ThT concentration range between 10 μM and 320 μM. Figure 8 As shown, with increasing ThT concentration, the fluorescence intensity gradually increases, reaches a maximum, and then gradually decreases. Therefore, we chose 20 μM as the optimal concentration for the experiment. Furthermore, the entire reaction is also affected by the reaction time. Therefore, to optimize the ThT reaction time, we selected a range of 50 to 90 minutes. Figure 9 As shown, the fluorescence intensity initially increases and then decreases with increasing reaction time, reaching its maximum at 60 min. Therefore, we selected 60 min as the optimal reaction time for ThT.

[0076] Under optimal conditions, different concentrations of GSH were quantitatively detected, such as... Figure 10 As shown, within the range of 10 μM to 40 mM, the fluorescence intensity of the system increases with increasing GSH concentration; Figure 11 When the linear range is 10 μM to 20 mM, the fluorescence intensity shows a good linear relationship with the GSH concentration, and the linear equation is F = 999.85 + 334.02lgC, where C is the GSH concentration. Based on the three-times standard deviation principle (LOD = 3σ / K, where σ is the standard deviation of the blank parallel determination and K is the slope of the calibration curve), the detection limit of this method is calculated to be 5.8 × 10⁻⁶. -3The prior art, authored by Jiayan Zhu, Tifeng Xia, et al., with DOI https: / / doi.org / 10.1016 / j.jssc.2018.11.032 and titled "A Turn-on MOF-Based Luminescent Sensor for Highly Selective Detection of Glutathione," discloses a GSH detection limit of 107.2 μM. The prior art, authored by Yuxia Zou, Mingshun Li, et al., with DOI 10.1021 / acssensors.9b02118 and titled "Bioimaging of Glutathione with a Two-Photon Fluorescent Probe and Its Potential Application for Surgery Guide in Laryngeal Cancer," discloses a GSH detection limit of 90 μM. It can be seen that the detection limit of the method of the present invention is superior to the detection limits of previously reported fluorescence-based GSH detection methods.

[0077] To investigate the specificity of this combined polydopamine bridging and rolling circle amplification strategy for GSH detection, we used different substances for detection, including GInE, phe, AA, and Glu. Figure 12 As shown, only the addition of GSH produces a significant fluorescence intensity in the entire system, while the fluorescence intensity produced by the addition of other substances is relatively low. These results demonstrate that the proposed novel sensing system based on polydopamine bridging and rolling circle amplification strategy for GSH detection exhibits good selectivity.

[0078] To verify the accuracy and repeatability of the combined polydopamine bridging and rolling circle amplification strategy for GSH detection, the repeatability was studied by detecting three samples at concentrations of 100 μM, 1 mM, and 20 mM. The intraday relative standard deviations (RSDs) for the above concentration determinations were 2.73%, 4.78%, and 0.43%, respectively. Figure 14 For the same concentration, the daytime RSDs were 3.67%, 2.05%, and 2.61%, respectively. Figure 15 These results demonstrate that the proposed method for detecting GSH based on a combination of polydopamine bridging and rolling circle amplification strategies exhibits acceptable repeatability and accuracy.

[0079] It should be noted that when numerical ranges are mentioned in the claims of this invention, it should be understood that the two endpoints of each numerical range and any value between the two endpoints can be selected. To avoid redundancy, the present invention describes preferred embodiments.

[0080] Although the preferred embodiments of the present invention have been described, those skilled in the art may make additional changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

[0081] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.

Claims

1. A method for detecting glutathione for non-disease diagnostic and therapeutic purposes, characterized in that, Includes the following steps: S1. Synthesis of polydopamine: Mix dopamine hydrochloride solution with NaOH solution to obtain polydopamine nanoparticle solution; S2, DNA adsorption: DNA is replaced by a target that can be adsorbed onto polydopamine and desorbed by glutathione; the polydopamine nanoparticle solution obtained in S1; Zn-containing... 2+ The solution and buffer solution were mixed and incubated at room temperature to obtain PDANs-Zn. 2+ -Target-replaced DNA polymers; S3, DNA desorption: The sample is added to the PDAs-Zn obtained in S2. 2+ -The target-substituted DNA polymer was incubated at room temperature and then centrifuged. The supernatant was collected to obtain the target-substituted DNA strand. S4. Signal Amplification: The target substitute DNA strand obtained in S3 is used as a primer in the reaction system for RCA reaction to obtain the amplification product; the reaction system is: 7.5 μL of 320 nM phosphorylated padlock probe, 7.5 μL of target substitute DNA, 3 μL of 10×T4 DNA ligase buffer, 10.5 μL of sterile water, and 1.5 μL of 350 U / μL T4 DNA ligase to make a total volume of 30 μL; After mixing thoroughly, react at 16 °C for 1 h. Take 10 μL of the reaction mixture, add 1.5 μL of 10 mM dNTPs, 2 μL of 10×phi29 DNA polymerase reaction buffer, 6 μL of sterile water, and 0.5 μL of 10 U / μL phi29 DNA polymerase, and react at 30 °C for 1 h to perform the RCA reaction. S5. Fluorescence detection: A fluorescent dye is added to the amplification product obtained in S4, and fluorescence detection is performed after incubation; the fluorescent dye is ThT. The sequence of the target-substituted DNA is 5'-AAAAAAAAACCCAGGTTCTCT-3'; The nucleotide sequence of the phosphorylated padlock probe is 5'-GTTTTTTTTTGCTGCCCGCCCTACCCAACTCATCTCCCGCCCTACCCAAAATCAGAGAACCTGG-3'.

2. The method for detecting glutathione according to claim 1, characterized in that, In S1, the concentration of dopamine hydrochloride solution is 1-3 mg / mL, the concentration of sodium hydroxide solution is 350-450 mM, and the volume ratio of dopamine hydrochloride solution to sodium hydroxide solution is 50-100:

1.

3. The method for detecting glutathione according to claim 1, characterized in that, The mixing conditions in S1 are stirring at 45-55℃ for 4-6 hours.

4. The method for detecting glutathione according to claim 1, characterized in that, S2 target-substituted DNA, polydopamine nanoparticle solution, Zn-containing 2+ The final volume of the solution and buffer solution is 50 μL. The target-substituted DNA, the polydopamine nanoparticle solution, and the Zn-containing... 2+ The mass concentration ratio of the solutions is 1:2:1.

5.

5. The method for detecting glutathione according to claim 1, characterized in that, Incubate in S2 for 20-50 minutes.

6. The method for detecting glutathione according to claim 1, characterized in that, Incubate in S3 for 30-60 minutes.

7. The method for detecting glutathione according to claim 1, characterized in that, The incubation conditions for S5 are as follows: incubate at 85-105℃ for 3-8 min, then incubate at 2-6℃ for 3-8 min, and finally incubate at room temperature for 0.5-1.5 h.

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