Specific ratio type detection probe for glutathione as well as preparation method and application of specific ratio type detection probe

Abstract

The invention provides a specific ratio type detection probe of glutathione as well as a preparation method and application thereof, and belongs to the technical field of biological analysis and detection, and the detection probe comprises tetraphenylporphyrin tetrasulfonic acid and fluorescein isothiocyanate. The detection probe is simple to prepare and quick to operate, can be quickly and specifically combined with glutathione, and overcomes the interference of other biological mercaptans. The obvious fluorescence quenching effect caused by the glutathione can be distinguished by naked eyes, and the fluorescence color is changed from orange red to cyan along with the increase of the concentration of the glutathione. In addition, glutathione only quenches the fluorescence of tetraphenylporphyrin tetrasulfonic acid, and does not affect the fluorescence of fluorescein isothiocyanate. By utilizing the differential response mode, the probe realizes ratio type detection of glutathione. The fluorescence detection probe constructed by the invention can be used for detecting glutathione in a blood sample so as to assist in diagnosing malignant tumors, a new way is provided for early detection of malignant tumors, and the fluorescence detection probe has a wide application prospect.

Description

Technical Field

[0001] The present invention belongs to the technical field of bioanalysis and detection, and particularly relates to a specific ratio detection probe for glutathione, a preparation method thereof, and an application thereof. Background Art

[0002] Most cases of malignant tumors are diagnosed at an advanced stage. Therefore, early identification is crucial for improving the prognosis. An ideal blood biomarker should consider specificity, sensitivity, accessibility, and reproducibility. However, the sensitivity or specificity of protein biomarkers widely used in clinical practice is significantly insufficient. For example, the positive rate of carcinoembryonic antigen in gastric cancer is only about 20%, and it is generally elevated in smokers, pancreatitis, and other diseases. In recent years, the emerging nucleic acid-based liquid biopsy is also limited by technical complexity, easy degradation of circulating nucleic acids, and poor accessibility and reproducibility caused by exosome heterogeneity. Therefore, it is necessary to explore new biomarkers with excellent diagnostic efficacy and practical application prospects and their analysis techniques.

[0003] Glutathione (GSH), as a core regulatory molecule of redox homeostasis, its metabolic imbalance is closely related to the occurrence and development of cancer. Studies have shown that the concentration of GSH in tumors is higher than that 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, the blood GSH level of cancer patients is usually reduced. This may be because disease-related oxidative molecules excessively consume GSH in the blood circulation, or tumor tissues excessively uptake GSH from the blood. This is the theoretical basis for GSH to be used as a disease biomarker. However, most studies are from cell or animal models and lack verification with clinical samples. Therefore, based on the above theoretical basis and the problems to be clarified, it is urgent to develop a glutathione probe and construct a clinical cohort to detect GSH in serum specimens of tumor patients and normal people.

[0004] High performance liquid chromatography, mass spectrometry, and surface-enhanced Raman scattering are widely used for GSH detection. The results of these methods are highly reliable, but there are some insurmountable defects, such as large and bulky instruments, complex and time-consuming workflows, high analysis costs, and the need for professional operators. In contrast, optical probe technology has a rapid response and is easy to operate, but the development of GSH optical probes is generally limited by the cross-interference of biological thiols such as cysteine (Cys), homocysteine, or homocysteine (Hcy) in biological samples - these molecules have similar thiol reactivity to GSH, which results in poor specificity of traditional optical probe detection techniques. For example, patents CN107602502B, CN104560027B, and CN109503435B authorize a series of biological thiol detection methods using 2,4-dinitrobenzenesulfonyl as the recognition group, and patents CN116507912A and CN102124337B provide a series of biological thiol detection methods using 5,5'-dithiobis-(2-nitrobenzoic acid) as the recognition group. These probes respond to all three biological thiols (GSH, Cys, and Hcy) and cannot achieve specific detection of GSH. That is to say, most of the so-called GSH probes developed currently can only achieve the total detection of biological thiols (including GSH, Cys, and Hcy) and do not have much specificity for GSH itself. It is worth noting that patent CN106645058B provides a GSH detection method based on TPPS-Hg 2+ Although the probe component of this patent uses the same material as this patent, namely TPPS, the detection principles for GSH are completely different. Specifically, the probe in patent CN106645058B is TPPS-Hg 2+ , where the fluorescence of TPPS is first quenched by Hg 2+ , and then Hg 2+ acts as a GSH recognition group. After GSH is added, it binds to Hg 2+ , resulting in a decrease in free Hg 2+ , so the fluorescence of TPPS gradually recovers. In this patent, the probe is TPPS-FITC. TPPS directly acts as a recognition group to bind to GSH, causing its fluorescence quenching, thereby achieving specific recognition of GSH, while FITC serves as a stable reference signal and is not affected. In addition, many single-signal probes, such as the six patents mentioned above, are relatively sensitive to environmental factors (such as photobleaching, pH fluctuations) because they only use one signal intensity to quantify the concentration of the target substance, and their reliability is relatively low. The ratio fluorescence detection method achieved in this patent improves the accuracy by introducing an internal reference signal FITC and quantifying the target substance through the ratio of the FITC signal to the TPPS signal. Summary of the Invention

[0005] To solve the above technical problems, the present invention provides a specific ratio detection probe for glutathione, its preparation method and application. It can perform fluorescence detection and colorimetric detection simultaneously. As the concentration of GSH increases, the color of the fluorescence detection system changes from orange-red to cyan, and 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 object, this solution first provides a specific ratio detection probe for glutathione, and the probe includes tetraphenylporphyrin tetrasulfonic acid (TPPS) and fluorescein isothiocyanate (FITC).

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

[0008] Dissolve tetraphenylporphyrin tetrasulfonic acid in ultrapure water, dissolve fluorescein isothiocyanate in absolute ethanol, and prepare a tetraphenylporphyrin tetrasulfonic acid solution and a fluorescein isothiocyanate solution with a concentration of 0.1 mg / mL respectively. Mix the two dye solutions in HEPES buffer. Among them, the matrix with a total volume of 1 mL of HEPES contains 11 μL of tetraphenylporphyrin tetrasulfonic acid solution and 9 μL of fluorescein isothiocyanate solution, and a specific ratio detection probe for glutathione with a green-red ratio of 1:2.5 is prepared.

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

[0010] Based on a general inventive concept, this solution also provides an application of a specific ratio detection probe for glutathione in detecting GSH in serum for non-disease diagnosis.

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

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

[0013] The mechanism by which the ratio probe prepared by this solution can detect GSH is as follows:

[0014] Using quantum chemical calculations based on density functional theory, from the perspective of molecular orbital energy level transitions and spatial configuration evolution, the molecular basis for the reactivity of TPPS to GSH rather than to biological thiols such as cysteine (Cys) and homocysteine (Hcy) is revealed, and its resistance to biological thiols such as Cys and Hcy is demonstrated, thus realizing interference-free and highly specific GSH determination.

[0015] In this probe system, there is no intermolecular interaction between TPPS and FITC that affects spectral properties. In addition, the combination of the narrow Stokes shift of FITC and the wide Stokes shift of TPPS effectively prevents cross-interference between the green and red fluorescence channels. Therefore, the combination of TPPS-FITC is a suitable choice for developing a ratio fluorescence probe.

[0016] With TPPS as the specific recognition group for GSH and FITC as the stable reference signal, the ratio visualization detection of GSH is achieved. The sensing system shows a color change from colorless to green in the colorimetric assay and a continuous color change from orange-red to cyan in the fluorescence assay.

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

[0018] (1) This scheme first reports the high responsiveness and high specificity of TPPS to GSH, and develops a GSH detection probe based on this property. As the concentration of GSH 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 the specific detection of GSH, and the probe signal will not be interfered by other biological thiols such as Cys and Hcy, overcoming the problem that the currently developed GSH probes can only focus on the total detection of biological thiols (including GSH, Cys, and Hcy) and have little specificity for GSH itself.

[0020] (3) The ratio fluorescence measurement mode adopted by this probe additionally introduces an internal reference signal FITC, and quantifies the target by the ratio of the signal of the recognition group TPPS to the FITC signal, avoiding the defects that single-emission probes are easily affected by the environment (for example, photobleaching, pH sensitivity), and achieving improved accuracy.

[0021] (4) The probe of this scheme exhibits excellent sensing performance, with characteristics of 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 of this scheme has good reliability because the inherent specificity of TPPS to GSH effectively eliminates cross-interference, and the ratio design effectively improves the resistance of the probe to environmental factors. Therefore, the above anti-interference ability ensures reliable GSH quantification in complex biological samples.

[0022] (5) The probe of this scheme can flexibly change the amount of TPPS or FITC added, thereby preparing probes with different green / red ratios, resulting in different color transitions when GSH is introduced. Description of the Drawings

[0023] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the accompanying drawings required for the description of the embodiments or the prior art. Obviously, the accompanying drawings in the following description are some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other accompanying drawings can also be obtained based on these drawings.

[0024] Figure 1 Schematic diagram of the preparation 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 Spectrograms 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 spectra and absorption spectra of TPPS, FITC, and the ratiometric probe;

[0026] Figure 3 Fluorescence spectrograms of the ratiometric probe after reacting with GSH at different time points in Example 2, (a) Changes in the fluorescence spectrum of the ratiometric probe after reacting with GSH over time, (b) Changes in the ratio of fluorescence intensities at 520 nm and 644 nm of the ratiometric probe after reacting with GSH over time;

[0027] Figure 4 Fluorescence spectra and absorption spectra of the ratiometric probe after reacting with different concentrations of GSH in Example 3, (a) Fluorescence spectra of the ratiometric probe after reacting with different concentrations of GSH, (b) Fitting curve of the ratio of fluorescence intensities at 520 nm and 644 nm of the ratiometric probe after reacting with different concentrations of GSH, (c) Absorption spectra of the ratiometric probe after reacting with different concentrations of GSH, (d) Fitting curve of the ratio of absorbances at 412 nm and 434 nm of the ratiometric probe after reacting with different concentrations of GSH;

[0028] Figure 5 Influence of cysteine (Cys) and homocysteine (Hcy) on the ratiometric probe in Example 4, (a) Fluorescence spectra of the ratiometric probe after reacting with different concentrations of Cys, the inset shows the ratio of fluorescence intensities at 520 nm and 644 nm of the ratiometric probe after reacting with different concentrations of GSH, (b) Fluorescence spectra of the ratiometric probe after reacting with different concentrations of Hcy, the inset shows the ratio of fluorescence intensities at 520 nm and 644 nm of the ratiometric probe after reacting with different concentrations of Hcy, (c) Bar chart of the adsorption energies between TPPS and GSH, Cys, and Hcy, (d) Geometric structures of TPPS and the TPPS-biol-thiol complex for natural population analysis, and the NPA charges of three representative nitrogen atoms;

[0029] Figure 6 It is the ratio of the fluorescence intensities at 520 nm and 644 nm after the ratiometric probe in Example 4 reacts with different interfering substances;

[0030] Figure 7 It is the box plot of the GSH level in serum obtained after the ratiometric probe in Example 5 reacts with the sera of tumor patients and healthy people;

[0031] Figure 8 It is the receiver operating characteristic (ROC) curve constructed for the GSH level in serum detected by the ratiometric probe in Example 5. Detailed implementation manners

[0032] To make the technical problems, technical solutions and advantages to be solved by the present invention clearer, the following will be described in detail with reference to the accompanying drawings and specific embodiments.

[0033] The following examples are used to illustrate the present invention, but are not used to limit the scope of the present invention. Without departing from the spirit and essence of the present invention, any modification or replacement made to the methods, steps or conditions of the present invention belongs to the scope of the present invention.

[0034] If not specifically specified, the technical means used in the examples are conventional means well-known to those skilled in the art; if not specifically specified, the reagents used in the examples are all commercially available.

[0035] Example 1

[0036] Preparation and characterization of a ratiometric probe based on TPPS and FITC

[0037] Preparation: Dissolve TPPS in ultrapure water and dissolve FITC in absolute ethanol to prepare stock solutions with a concentration of 0.1 mg / mL. Both solutions are stored in the dark at 4 °C for subsequent use. Mix the two dyes in HEPES buffer (10 mM, pH 7.4). Specifically, add 11 μL of TPPS and 9 μL of FITC to 980 μL of HEPES buffer to obtain a ratiometric probe with a green / red ratio of 1:2.5.

[0038] Figure 1 It is the preparation process of the ratiometric probe based on TPPS and FITC and a schematic diagram of the principle of using this probe to detect GSH.

[0039] Characterization: Measure the fluorescence spectrum and absorption spectrum by an Agilent Cary Eclipse fluorescence spectrophotometer and a Shimadzu UV-1800 spectrophotometer respectively. Set the excitation wavelength of the instrument to 412 nm, the excitation slit to 10 nm, and the emission slit to 10 nm.

[0040] The results are as Figure 2 shown, Figure 2 where a represents the optical properties of TPPS. The visible absorption spectrum shows a prominent Soret band centered at 412 nm, as well as 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-red region (644 nm) and near-infrared region (702 nm) are the characteristic fluorescence of TPPS. The TPPS solution is colorless under daylight, but exhibits strong red fluorescence under irradiation with 410 nm ultraviolet light.

[0041] Subsequently, FITC with green fluorescence was mixed with TPPS with red fluorescence to form a ratiometric probe. The spectral changes before and after mixing are as Figure 2 shown in b. Before mixing, FITC and TPPS showed absorption peaks at 488 nm and 412 nm respectively, and emission peaks at 520 nm and 644 nm respectively. After mixing, no new absorption peaks and emission peaks were observed in TPPS + FITC, nor was there any weakening or enhancement of the original peaks. It can be seen that there is no intermolecular interaction affecting the spectrum between TPPS and FITC. In addition, the combination of the narrow Stokes shift of FITC and the wide Stokes shift of TPPS 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] Investigate the reaction time of the ratiometric probe for detecting GSH

[0044] GSH with a final concentration of 200 μM was added to the probe system respectively, keeping the total volume of the reaction system at 1 mL, and reacting at 25 °C for 60 minutes. The fluorescence spectrum was recorded at regular intervals during this period. As Figure 3 shown, with the addition of GSH, the fluorescence of TPPS was quickly quenched, while the fluorescence of FITC remained basically unchanged, demonstrating the ratiometric response of the probe to GSH. Using the ratio of the fluorescence intensities of FITC and TPPS (I 520 / I 644 ) as the response signal of the probe to GSH, it can be seen that the reaction reached equilibrium at the 4th second and the signal intensity tended to be stable.

[0045] Example 3

[0046] Investigate the sensitivity of the ratiometric probe for detecting GSH

[0047] For fluorescence detection, GSH was added to the probe system to final concentrations of 0, 20, 40, 80, 100, 120, 140, 160, 180, and 200 μM, while maintaining the total volume of the reaction system at 1 mL. After reacting at 25 °C for 1 minute, the fluorescence spectrum was recorded. For colorimetric detection, GSH was added to the sensing system to 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. After reacting at 25 °C for 1 minute, the absorption spectrum was 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 shows the absorption spectra of the probe at different GSH concentrations and the standard curve for colorimetric detection.

[0048] It can be seen that with the increase in GSH concentration, for fluorescence detection, the red fluorescence of TPPS at 644 nm rapidly decreased, while the green fluorescence of FITC at 520 nm remained stable. Under irradiation with a 410 nm ultraviolet lamp, this process was accompanied by a gradual change in the fluorescence color of the probe from orange - red to cyan; using the ratio of the fluorescence intensities of FITC and TPPS (I 520 / I 644 ) as the response signal of the probe to GSH, in the range of 0 - 200 μM, the GSH concentration showed a good positive correlation with I 520 / I 644 (R 2 = 0.998). For colorimetric detection, with the increase in GSH concentration, the absorption peak of TPPS at 412 nm gradually decreased, accompanied by the formation of a new absorption peak at 434 nm. This process was accompanied by a gradual change in the color of the probe itself from colorless to green; using the absorbance ratio (A 412 / A 434 ) as the response signal of the probe to GSH, in the range of 0 - 100 μM, the GSH concentration showed a good positive correlation with A 412 / A 434 (R 2 = 0.999), and the detection limit was calculated to be 0.75 μM, indicating that the probe had good sensitivity.

[0049] Example 4

[0050] Investigate the specificity of the ratiometric probe for detecting 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. The total volume of the reaction system was maintained at 1 mL. After reacting at 25 °C for 30 minutes, the fluorescence spectrum was recorded. Figure 5 a and Figure 5 b show that after reacting with different concentrations of Cys and Hcy for 30 minutes, the fluorescence of the probe remained unchanged, indicating that the probe effectively overcame the cross-interference caused by biothiols that traditional GSH probes could not avoid.

[0052] Quantum chemical calculations based on density functional theory (DFT) were used to further explain the experimental results, namely why the probe is specific for GSH. All calculations were performed using Gaussian 16 (Revision A.02, Gaussian, Inc). The M06-2X density functional method was used for geometric structure optimization and frequency calculation, and the 6-31G(d) basis set was used for all atoms. As Figure 5 c shows, TPPS-GSH has the largest negative adsorption energy, indicating that TPPS has a stronger binding affinity for GSH than for Cys and Hcy. Natural population analysis was performed using three nitrogen atoms as the representative binding sites between TPPS and biothiols. As Figure 5 d shows, the change in NPA charge 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 for other interfering substances: Various interfering substances were added to the probe system to a final concentration of 100 μM. The total volume of the reaction system was maintained at 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 (Glucose), fructose (Fructose), maltose (Maltose), lactose (Lactose), uric acid (Uric Acid), citric acid (Citric Acid), creatinine (Creatinine), bicarbonate (HCO3 2 -), potassium ion (K + ), ferrous ion (Fe 2+ ). As Figure 6 shown, the interfering substances had little effect on the fluorescence of the probe, and the probe signal value (I 520 / I644 ) has a fluctuation of less than 5%, indicating that the probe has good specificity.

[0054] Experimental Example 5

[0055] Examine the detection of GSH in blood by the ratio probe prepared in Implementation 1

[0056] Collect human whole blood samples in coagulation-promoting tubes and let them stand at room temperature for 1 hour. Then centrifuge the samples at 1000 rpm for 10 minutes at 4°C and collect the upper serum. After serum separation, immediately take 100 μL of serum and add 100 μL of 10% (w / v) trichloroacetic acid solution. Vortex the mixture and incubate it on ice for 10 minutes, then centrifuge at 16000 rpm for 10 minutes at 4°C to remove the precipitated proteins and maintain an acidic environment to prevent GSH degradation. If not detected immediately, store the centrifuged supernatant at -80°C for subsequent detection. In fluorescence and colorimetric assays, mix the supernatant 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 is within the detection range of the probe. The total volume of the liquid after mixing is 1 mL. After reacting at 25°C for 1 minute, record the fluorescence spectrum or absorption spectrum.

[0057] As Figure 7 shown, the GSH concentration in the blood of tumor patients is reduced by 36.1% compared with that of healthy people. To evaluate the efficacy of GSH in diagnosing tumors, a receiver operating characteristic (ROC) curve was constructed. As Figure 8 shown, GSH shows excellent discrimination ability between tumor and healthy states, and the area under the curve (AUC) is 0.980.

[0058] In summary, the detection probe prepared by this scheme can simply and quickly distinguish tumor patients from healthy people by detecting GSH.

[0059] The above is only the preferred implementation mode of this invention patent, and the protection scope of this invention patent is not limited to the above embodiments. For those skilled in the art, the improvements and transformations obtained without departing from the technical concept of this invention patent should also be regarded as the protection scope of this invention patent.

Claims

1. A specific ratio detection probe for glutathione, characterized in that, The probe includes tetraphenylporphyrin tetrasulfonic acid and fluorescein isothiocyanate.

2. A method for preparing a specific ratio detection probe for glutathione as described in claim 1, characterized in that, It includes the following steps: Dissolve tetraphenylporphyrin tetrasulfonic acid in ultrapure water and dissolve fluorescein isothiocyanate in absolute ethanol to prepare a tetraphenylporphyrin tetrasulfonic acid solution and a fluorescein isothiocyanate solution with a concentration of 0.1 mg / mL respectively. Mix the two dye solutions in HEPES buffer. Among them, the matrix with a total volume of 1 mL of HEPES contains 11 μL of tetraphenylporphyrin tetrasulfonic acid solution and 9 μL of fluorescein isothiocyanate solution to obtain a specific ratio detection probe for glutathione with a green-to-red ratio of 1:2.

5.

3. The preparation method according to claim 2, characterized in that, The concentration of the HEPES buffer is 10 mM and the pH is 7.

4.

4. Application of a specific ratio detection probe for glutathione as described in claim 1 or a specific detection ratio probe for glutathione prepared by the preparation method as described in any one of claims 2 - 3 in detecting GSH in serum for non-disease diagnosis.

5. The application according to claim 4, characterized in that, The detection method includes kit detection.

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

Images