A specific ratio detection probe for glutathione and a preparation method and application thereof

Abstract

The scheme provides a specific ratio detection probe of glutathione and a preparation method and application thereof, and belongs to the technical field of biological analysis and detection.The detection probe comprises tetraphenylporphyrin tetrasulfonic acid and fluorescein isothiocyanate.The detection probe is simple to prepare and fast to operate, can be rapidly and specifically combined with glutathione, and overcomes the interference of other biological thiols.The obvious fluorescence quenching effect caused by glutathione can be distinguished by naked eyes, and with the increase of the concentration of glutathione, the fluorescence color changes from orange red to cyan.In addition, glutathione only quenches the fluorescence of tetraphenylporphyrin tetrasulfonic acid, and does not affect the fluorescence of fluorescein isothiocyanate.By using the differential response mode, the probe realizes the ratio detection of glutathione.The fluorescence detection probe constructed by the application can be used for the detection of glutathione in blood samples, and further assists in the diagnosis of malignant tumors, provides a new way for the early detection of malignant tumors, and has a wide application prospect.

Description

Technical Field

[0001] This invention belongs to the field of bioanalytical detection technology, specifically relating to a specific ratiometric detection probe for glutathione, its preparation method, and its application. Background Technology

[0002] Most malignant tumors are diagnosed at an intermediate or advanced stage, making early identification crucial for improving prognosis. Ideal blood biomarkers need to balance specificity, sensitivity, accessibility, and reproducibility. However, the sensitivity or specificity of currently widely used protein biomarkers in clinical practice is significantly insufficient. For example, the positive rate of carcinoembryonic antigen (CEA) in gastric cancer is only about 20%, and it is generally elevated in smokers, pancreatitis, and other diseases. The emerging nucleic acid-based liquid biopsy is also limited by technical complexity, the easy degradation of circulating nucleic acids, and poor accessibility and reproducibility due to exosome heterogeneity. Therefore, it is necessary to explore novel biomarkers and their analytical techniques with superior diagnostic efficacy and practical application prospects.

[0003] Glutathione (GSH), a core regulator of redox homeostasis, is closely associated with cancer development and progression when its metabolism is imbalanced. Studies have shown that GSH concentrations are higher in tumors than in normal tissues, protecting tumor cells from oxidative damage by neutralizing reactive oxygen species, thereby driving tumor proliferation, invasion, and metastasis. Paradoxically, although GSH is overexpressed in tumors, blood GSH levels are typically lower in cancer patients. This may be due to excessive consumption of circulating GSH by disease-associated oxidative molecules, or excessive uptake of blood GSH by tumor tissue. This is the theoretical basis for GSH as a disease biomarker. However, most studies are based on cell or animal models, lacking validation in clinical samples. Therefore, based on the above theoretical foundation and the issues to be clarified, there is an urgent need to develop a glutathione probe and construct a clinical cohort to detect GSH in serum samples from cancer patients and healthy individuals.

[0004] High-performance liquid chromatography (HPLC), mass spectrometry (MS), and surface-enhanced Raman scattering (SERS) are widely used for GSH detection. These methods offer high reliability, but suffer from several inherent drawbacks, such as bulky and cumbersome equipment, complex and time-consuming workflows, high analytical costs, and the need for specialized personnel. In contrast, optical probe techniques offer rapid response and ease of operation. However, the development of GSH optical probes is generally limited by cross-interference from biothiols such as cysteine ​​(Cys), homocysteine, or homocysteine ​​(Hcy) in biological samples—these molecules exhibit similar thiol reactivity to GSH, leading to poor specificity in traditional optical probe detection techniques. For example, patents CN107602502B, CN104560027B, and CN109503435B authorize a series of biothiol detection methods using 2,4-dinitrobenzenesulfonyl as a recognition group, while patents CN116507912A and CN102124337B provide a series of biothiol detection methods using 5,5'-dithiobis-(2-nitrobenzoic acid) as a recognition group. These probes respond to all three biothiols (GSH, Cys, and Hcy), but cannot achieve specific detection of GSH. In other words, most of the currently developed so-called GSH probes can only achieve total detection of biothiols (including GSH, Cys, and Hcy), and do not have much specificity for GSH itself. It is worth noting that patent CN106645058B provides a method based on TPPS-Hg... 2+ Although the GSH detection method in this patent uses the same material, TPPS, for its probe component, the two methods operate on completely different GSH detection principles. Specifically, the probe in patent CN106645058B is TPPS-Hg. 2+ Among them, the fluorescence of TPPS was first detected by Hg. 2+ Quenching, followed by Hg 2+ As a GSH recognition group, it reacts with Hg after GSH is added. 2+ Combining, leading to free Hg 2+ As the fluorescence of TPPS decreases, it gradually recovers. In this patent, the probe is TPPS-FITC. TPPS directly binds to GSH as a recognition group, causing its fluorescence quenching, thus achieving specific recognition of GSH. FITC, as a stable reference signal, remains unaffected. Furthermore, many single-signal probes, such as the six patents mentioned above, are sensitive to environmental factors (such as photobleaching and pH fluctuations) and have relatively low reliability because they use only one signal intensity to quantify the concentration of the target analyte. The ratiometric fluorescence detection method implemented in this patent, however, introduces an internal reference signal FITC and quantifies the target analyte by the ratio of the FITC to TPPS signals, thus improving accuracy. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention provides a specific ratiometric detection probe for glutathione, its preparation method, and its application. It can simultaneously perform fluorescence detection and colorimetric detection. As the GSH concentration increases, the color of the fluorescence detection system changes from orange-red to cyan, while the color of the colorimetric detection system changes from colorless to green. This wide range of color changes is very suitable for visual detection.

[0006] To achieve the above objectives, this solution first provides a specific ratiometric detection probe for glutathione, the probe comprising tetraphenylporphyrin tetrasulfonic acid (TPPS) and fluorescein isothiocyanate (FITC).

[0007] Based on a general inventive concept, this solution also provides a method for preparing a specific ratiometric detection probe for glutathione, comprising the following steps:

[0008] Tetraphenylporphyrin tetrasulfonic acid was dissolved in ultrapure water, and fluorescein isothiocyanate was dissolved in anhydrous ethanol to prepare tetraphenylporphyrin tetrasulfonic acid solution and fluorescein isothiocyanate solution with a concentration of 0.1 mg / mL, respectively. The two dye solutions were mixed in HEPES buffer, wherein 1 mL of HEPES matrix contained 11 μL of tetraphenylporphyrin tetrasulfonic acid solution and 9 μL of fluorescein isothiocyanate solution, thus preparing a specific ratiometric detection probe for glutathione with a green-to-red ratio of 1:2.5.

[0009] Preferably, the HEPES buffer solution has a concentration of 10 mM and a pH of 7.4.

[0010] Based on a general inventive concept, this solution also provides a specific ratiometric detection probe for glutathione for the non-disease diagnostic detection of GSH in serum.

[0011] Preferably, the detection method includes reagent kit detection.

[0012] Preferably, the detection limit of the GSH is 0.75 μM.

[0013] The mechanism by which the ratiometric probe prepared in this scheme can detect GSH is as follows:

[0014] By utilizing quantum chemical calculations based on density functional theory, the molecular basis of TPPS's reactivity to GSH but not to biothiols such as cysteine ​​(Cys) and homocysteine ​​(Hcy) was revealed from the perspective of molecular orbital energy level transitions and spatial configuration evolution. This demonstrated its resistance to biothiols such as Cys and Hcy, thus enabling interference-free and highly specific GSH determination.

[0015] In this probe system, there are no intermolecular interactions between TPPS and FITC that would affect the spectral properties. Furthermore, the narrow Stokes shift of FITC combined with the wide Stokes shift of TPPS effectively prevents cross-interference between the green and red fluorescence channels. Therefore, the TPPS-FITC combination is a suitable choice for developing ratiometric fluorescent probes.

[0016] TPPS, as a specific recognition group for GSH, and FITC, as a stable reference signal, enable ratiometric and visual detection of GSH. The sensing system exhibits a color change from colorless to green in colorimetric measurements and a continuous color change from orange-red to cyan in fluorescence measurements.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] (1) This method is the first to report the high responsiveness and high specificity of TPPS to GSH, and a GSH detection probe was developed based on this characteristic. As the GSH concentration increases, the color of the probe undergoes a series of continuous changes, and this wide range of color changes is very suitable for visual detection.

[0019] (2) This probe achieves specific detection of GSH. The probe signal is not interfered with by other biothiols such as Cys and Hcy, which overcomes the problem that the currently developed GSH probes can only focus on the total detection of biothiols (including GSH, Cys and Hcy) and do not have much specificity for GSH itself.

[0020] (3) The ratiometric fluorescence measurement mode adopted by this probe introduces an additional internal reference signal FITC. The target substance is quantified by the ratio of the signal of the identification group TPPS to the signal of FITC, which avoids the defects of single emission probes that are easily affected by the environment (e.g., photobleaching, pH sensitivity) and achieves improved accuracy.

[0021] (4) The probe in this scheme exhibits excellent sensing performance, with a wide detection range (0-200 μM), a low detection limit (0.75 μM), and a fast kinetic response (reaching reaction equilibrium within 4 seconds). The probe in this scheme has good reliability because the inherent specificity of TPPS to GSH effectively eliminates cross-interference, and the ratiometric design effectively enhances the probe's resistance to environmental factors. Therefore, the above-mentioned anti-interference capabilities ensure reliable GSH quantification in complex biological samples.

[0022] (5) The probes in this scheme can be flexibly modified by changing the amount of added TPPS or FITC, thereby preparing probes with different green / red ratios, resulting in different color changes when GSH is introduced. Attached Figure Description

[0023] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0024] Figure 1 This is a schematic diagram illustrating the fabrication process of the ratiometric probe based on TPPS and FITC in Example 1, and the principle of detecting GSH using this probe.

[0025] Figure 2 The following are the spectra of the ratiometric probe and its components in Example 1: (a) fluorescence excitation spectrum, fluorescence emission spectrum, and absorption spectrum of TPPS; (b) fluorescence emission spectrum and absorption spectrum of TPPS, FITC, and ratiometric probe.

[0026] Figure 3 The fluorescence spectra of the ratiometric probe reacting with GSH at different time points in Example 2 are shown in (a) the change of fluorescence spectrum with time after the ratiometric probe reacts with GSH, and (b) the change of the ratio of fluorescence intensity at 520 nm and 644 nm with time after the ratiometric probe reacts with GSH.

[0027] Figure 4 The following are the fluorescence and absorption spectra of the ratiometric probe reacting with different concentrations of GSH in Example 3: (a) fluorescence spectrum of the ratiometric probe reacting with different concentrations of GSH; (b) fitting curve of the ratio of fluorescence intensity at 520 nm and 644 nm after the ratiometric probe reacting with different concentrations of GSH; (c) absorption spectrum of the ratiometric probe reacting with different concentrations of GSH; and (d) fitting curve of the ratio of absorbance at 412 nm and 434 nm after the ratiometric probe reacting with different concentrations of GSH.

[0028] Figure 5 The effects of cysteine ​​(Cys) and homocysteine ​​(Hcy) on the ratiometric probe in Example 4 are shown in the following figures: (a) Fluorescence spectra of the ratiometric probe after reacting with different concentrations of Cys, with an inset showing the ratio of fluorescence intensity at 520 nm to 644 nm after reacting with different concentrations of GSH; (b) Fluorescence spectra of the ratiometric probe after reacting with different concentrations of Hcy, with an inset showing the ratio of fluorescence intensity at 520 nm to 644 nm after reacting with different concentrations of Hcy; (c) Bar chart of adsorption energies of TPPS with GSH, Cys, and Hcy; and (d) Geometric structures of TPPS and TPPS-biothiol complexes used for natural population analysis, and NPA charges of three representative nitrogen atoms.

[0029] Figure 6 The ratio of fluorescence intensity at 520 nm to 644 nm after the ratiometric probe reacts with different interfering substances in Example 4;

[0030] Figure 7 Box plot of serum GSH levels obtained after the ratiometric probe reacted with the serum of tumor patients and healthy individuals in Example 5;

[0031] Figure 8 The receiver operating characteristic (ROC) curve was constructed for the serum GSH level detected by the ratiometric probe in Example 5. Detailed Implementation

[0032] To make the technical problems, technical solutions and advantages of the present invention clearer, a detailed description will be given below in conjunction with the accompanying drawings and specific embodiments.

[0033] The following embodiments are used to illustrate the present invention, but are not intended to limit the scope of the invention. Any modifications or substitutions made to the methods, steps, or conditions of the present invention without departing from the spirit and essence of the invention are within the scope of the invention.

[0034] Unless otherwise specified, the technical means used in the embodiments are conventional means well known to those skilled in the art; unless otherwise specified, the reagents used in the embodiments are all commercially available.

[0035] Example 1

[0036] Fabrication and Characterization of Ratiometric Probes Based on TPPS and FITC

[0037] Preparation: TPPS was dissolved in ultrapure water, and FITC was dissolved in anhydrous ethanol to prepare a stock solution with a concentration of 0.1 mg / mL. Both solutions were stored at 4°C protected from light until subsequent use. The two dyes were mixed in HEPES buffer (10 mM, pH 7.4). Specifically, 11 μL of TPPS and 9 μL of FITC were added to 980 μL of HEPES buffer to prepare a ratio probe with a green / red ratio of 1:2.5.

[0038] Figure 1 This is a schematic diagram illustrating the fabrication process of a ratiometric probe based on TPPS and FITC, and the principle of detecting GSH using this probe.

[0039] Characterization: Fluorescence and absorption spectra were measured using an Agilent Cary Eclipse fluorescence spectrophotometer and a Shimadzu UV-1800 spectrophotometer, respectively. The excitation wavelength of the instruments was set to 412 nm, the excitation slit to 10 nm, and the emission slit to 10 nm.

[0040] The results are as follows Figure 2 As shown, Figure 2 a represents the optical properties of TPPS. The visible absorption spectrum shows a prominent Soret band centered at 412 nm, and several Q bands at 515 nm, 552 nm, 581 nm, and 635 nm. The optimal excitation wavelength is 412 nm. The emission peaks in the far-infrared region (644 nm) and near-infrared region (702 nm) are characteristic fluorescence features of TPPS. TPPS solution is colorless under sunlight, but exhibits strong red fluorescence under 410 nm ultraviolet light.

[0041] FITC, which exhibits green fluorescence, was then mixed with TPPS, which exhibits red fluorescence, to form a ratiometric probe. The spectral changes before and after mixing are shown below. Figure 2 As shown in b, before mixing, FITC and TPPS exhibit absorption peaks at 488 nm and 412 nm, respectively, and emission peaks at 520 nm and 644 nm, respectively. After mixing, apart from the individual absorption and emission peaks of each dye, no new absorption or emission peaks were observed in TPPS+FITC, nor was there any weakening or enhancement of the existing peaks. This indicates that there are no intermolecular interactions between TPPS and FITC that affect the spectrum. Furthermore, the combination of FITC's narrow Stokes shift and TPPS's wide Stokes shift effectively prevents cross-interference between the green and red fluorescence channels. Therefore, the combination of TPPS and FITC is a suitable choice for developing ratiometric fluorescent probes.

[0042] Example 2

[0043] Examining the reaction time of ratiometric probe detection of GSH

[0044] Add GSH to the probe system to a final concentration of 200 μM, maintaining a total reaction volume of 1 mL. Incubate at 25 °C for 60 minutes, recording the fluorescence spectrum periodically during the reaction. Figure 3 As shown, with the addition of GSH, the fluorescence of TPPS is quickly quenched, while the fluorescence of FITC remains essentially unchanged, demonstrating the ratiomatic response of the probe to GSH. The ratio of FITC to TPPS fluorescence intensity (IF) is used to... 520 / I 644 As the probe's response signal to GSH, it can be seen that the reaction has reached equilibrium at 4 seconds and the signal intensity tends to stabilize.

[0045] Example 3

[0046] Examining the sensitivity of ratiometric probe detection of GSH

[0047] For fluorescence detection, GSH was added to the probe system at final concentrations of 0, 20, 40, 80, 100, 120, 140, 160, 180, and 200 μM, maintaining a total reaction volume of 1 mL. The reaction was carried out at 25°C for 1 minute, and the fluorescence spectra were recorded. For colorimetric detection, GSH was added to the sensing system at final concentrations of 0, 5, 10, 15, 20, 22.5, 25, 27.5, 30, 32.5, 35, 37.5, 40, 42.5, 45, 50, 75, and 100 μM, respectively. The reaction was carried out at 25°C for 1 minute, and the absorption spectra were recorded. All spectral measurements were repeated three times. Figure 4 a, Figure 4 b shows the fluorescence spectra of the probe at different GSH concentrations and the standard curve for fluorescence detection. Figure 4 c. Figure 4 d represents the standard curve of the probe's absorption spectrum and colorimetric detection at different GSH concentrations.

[0048] It can be seen that with the increase of GSH concentration, for fluorescence detection, the red fluorescence of TPPS at 644nm rapidly weakens, while the green fluorescence of FITC at 520nm remains stable. Under 410nm UV light irradiation, this process is accompanied by the probe fluorescence color gradually changing from orange-red to cyan; using the ratio of FITC to TPPS fluorescence intensity (I... 520 / I 644 As a probe for the response signal to GSH, within the range of 0-200 μM, the GSH concentration is related to I... 520 / I 644 A good positive correlation (R) is shown 2 =0.998). For colorimetric detection, as the GSH concentration increases, the absorption peak of TPPS at 412 nm gradually weakens, accompanied by the formation of a new absorption peak at 434 nm. This process is accompanied by the probe itself gradually changing from colorless to green; using the absorbance ratio (A... 412 / A 434 As a probe for the response signal to GSH, within the range of 0-100 μM, the GSH concentration is related to A 412 / A 434 A good positive correlation (R) is shown 2 =0.999), and the detection limit was calculated to be 0.75 μM, indicating that the probe has good sensitivity.

[0049] Example 4

[0050] Investigating the specificity of ratiometric probe detection of GSH

[0051] Specificity of the probe for biothiols other than GSH: Cysteine ​​(Cys) and homocysteine ​​(Hcy) were added to the probe system to final concentrations of 0, 20, 40, 80, 100, 120, 140, 160, 180 and 200 μM, respectively, while maintaining a total reaction volume of 1 mL. After reacting at 25 °C for 30 minutes, the fluorescence spectra were recorded. Figure 5 a and Figure 5 b shows that the fluorescence of the probe remained unchanged after reacting with different concentrations of Cys and Hcy for 30 minutes, indicating that the probe effectively overcomes the cross-interference caused by biothiols, which is unavoidable by traditional GSH probes.

[0052] Quantum chemical calculations based on density functional theory (DFT) were used to further explain the experimental results, specifically why the probe exhibits specificity for GSH. All calculations were performed using Gaussian 16 (Revision A.02, Gaussian, Inc.), with geometry optimization and frequency calculations performed using the M06-2X density functional method. All atoms used the 6-31G(d) basis set. Figure 5 As shown in c, TPPS-GSH exhibits the largest negative adsorption energy, indicating that TPPS has a stronger affinity for GSH than for Cys and Hcy. Natural population analysis was performed using three nitrogen atoms as representative binding sites for TPPS and biothiols, as shown... Figure 5 As shown in d, the NPA charge change of the three atoms in TPPS-GSH is greater than that in TPPS-Cys and TPPS-Hcy, indicating that the interaction between TPPS and GSH is the strongest.

[0053] Specificity of the probe to other interfering substances: Various interfering substances were added to the probe system to a final concentration of 100 μM, maintaining a total reaction volume of 1 mL. After reacting at 25 °C for 1 minute, the fluorescence spectrum was recorded. The interfering substances included: threonine (Thr), arginine (Arg), alanine (Ala), methionine (Met), lysine (Lys), leucine (Leu), tyrosine (Tyr), histidine (His), aspartic acid (Asp), proline (Pro), glucose, fructose, maltose, lactose, uric acid, citric acid, creatinine, bicarbonate (HCO3-), and sulfate (SO4-). 2 -) Potassium ions (K) + ), ferrous ions (Fe) 2+ ).like Figure 6 As shown, the interfering agent has little effect on the probe fluorescence, and the probe signal value (I) 520 / I644 The fluctuation of less than 5% indicates that the probe has good specificity.

[0054] Experimental Example 5

[0055] The ratiometric probe prepared in step 1 was investigated for the detection of GSH in blood.

[0056] Human whole blood samples were collected in coagulation tubes and allowed to stand at room temperature for 1 hour. The samples were then centrifuged at 1000 rpm at 4°C for 10 minutes, and the supernatant serum was collected. Immediately after serum separation, 100 μL of serum was taken and 100 μL of 10% (w / v) trichloroacetic acid solution was added. The mixture was vortexed and incubated on ice for 10 minutes, then centrifuged at 16000 rpm at 4°C for 10 minutes to remove precipitated proteins and maintain an acidic environment to prevent GSH degradation. If not immediately tested, the supernatant obtained from centrifugation was stored at -80°C for subsequent detection. In fluorescence and colorimetric assays, this supernatant was mixed with the fluorescent probe prepared in Example 1 at a volume ratio of 1:10 to 1:25 to ensure that the GSH concentration in the detection system was within the detection range of the probe. The total volume of the mixture was 1 mL. After reacting at 25°C for 1 minute, the fluorescence or absorption spectra were recorded.

[0057] like Figure 7 As shown, the concentration of GSH in the blood of cancer patients was reduced by 36.1% compared to healthy individuals. To evaluate the diagnostic efficacy of GSH for cancer, receiver operating characteristic (ROC) curves were constructed. Figure 8 As shown, GSH exhibits excellent discriminative power between tumor and healthy states, with an area under the curve (AUC) of 0.980.

[0058] In summary, the detection probe prepared by this method can easily and quickly distinguish between cancer patients and healthy individuals by detecting GSH.

[0059] The above description is merely a preferred embodiment of the present invention, and the scope of protection of the present invention is not limited to the above embodiments. For those skilled in the art, any improvements and modifications obtained without departing from the technical concept of the present invention should also be considered within the scope of protection of the present invention.

Claims

1. A specific ratiometric detection probe for glutathione, characterized by, The probe is tetraphenylporphyrin tetrasulfonic acid and fluorescein isothiocyanate.

2. A method for preparing the specific ratiometric detection probe for glutathione according to claim 1, characterized by, The method comprises the following steps: The tetraphenylporphyrin tetrasulfonic acid is dissolved in ultrapure water, and the fluorescein isothiocyanate is dissolved in anhydrous ethanol to prepare a 0.1 mg / mL tetraphenylporphyrin tetrasulfonic acid solution and a 0.1 mg / mL fluorescein isothiocyanate solution respectively; the two dye solutions are mixed in a HEPES buffer, wherein a 1 mL HEPES base contains 11 μL of the tetraphenylporphyrin tetrasulfonic acid solution and 9 μL of the fluorescein isothiocyanate solution, and a specific ratiometric detection probe for glutathione with a green-to-red ratio of 1:2.5 is prepared.

3. The production method according to claim 2, characterized by, The HEPES buffer has a concentration of 10 mM and a pH of 7.

4.

4. Use of the specific ratiometric detection probe for glutathione according to claim 1 or the specific ratiometric detection probe for glutathione prepared by the preparation method of any one of claims 2-3 in non-disease diagnosis for detecting GSH in serum.

5. Use according to claim 4, characterised in that, The detection mode comprises kit detection.

6. Use according to claim 4, characterised in that, The detection limit of the GSH is 0.75 μM.

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