Near-infrared fluorescence photoacoustic double-response detection kit and application thereof

Abstract

The invention belongs to the technical field of detection kits, and relates to a near-infrared fluorescence photoacoustic double-response detection kit and application thereof, the near-infrared fluorescence photoacoustic double-response detection kit comprises a reagent stock solution a and a probe reagent stock solution b; the volume ratio of the reagent stock solution a to the probe reagent stock solution b is 50: 1; the probe reagent stock solution b is prepared from a near-infrared fluorescence / photoacoustic response probe and an organic solvent, and the structural formula of the near-infrared fluorescence / photoacoustic response probe is as follows: # imgabs0 #. The near-infrared fluorescence / photoacoustic double-response detection kit is constructed by utilizing the near-infrared fluorescence / photoacoustic response probe; the method has the characteristics of good selectivity, high sensitivity and strong anti-interference capability, and accurate quantitative detection of polysulfide is realized through near-infrared fluorescence / photoacoustic response analysis.

Description

Technical Field

[0001] The invention belongs to the technical field of detection kits and relates to a near-infrared fluorescence-photoacoustic dual-response detection kit and applications thereof. Background Art

[0002] Polysulfides (such as hydrogen polysulfide) play a crucial role in regulating redox homeostasis in organisms, interacting with numerous targets (such as glyceraldehyde-3-phosphate dehydrogenase and transcription factor complexes that upregulate antioxidant genes). Hydrogen polysulfide can also modulate the activity of ion channels, tumor suppressors, and protein kinases, and its expression levels are associated with inflammation, Parkinson's disease, and cancer. Although some studies have examined polysulfides in vivo, their decomposition products and metabolic mechanisms remain unknown, making their detection crucial.

[0003] There are also many existing polysulfide detection methods. For example, Patent Document No. CN110988254A discloses a method for rapid detection of polysulfides using an oxidation-reduction method. This method involves adding a reducing agent and a catalyst to an alkaline solution to convert polysulfides into ions that can be directly used for analysis and detection at a certain temperature. Although this method can detect polysulfides, it is complex, error-prone, has poor selectivity, low sensitivity, and cannot monitor changes in target substances in real time. Patent Document No. CN109232360A discloses a two-photon fluorescent probe for detecting polysulfides. The corresponding fluorescence intensity changes significantly in the presence of polysulfides, making it suitable for polysulfide detection and significantly reducing interference from external detection conditions. However, due to its short emission wavelength, the detection has disadvantages such as low sensitivity, poor quantitative accuracy, and weak penetration ability, and is easily interfered with by biological autofluorescence.

[0004] Based on the technical defects of the above-mentioned existing polysulfide detection methods, it is necessary to establish a polysulfide detection method with good selectivity, high sensitivity and resistance to biological interference. Summary of the Invention

[0005] In response to the technical problems of poor selectivity, low sensitivity and susceptibility to biological interference in existing polysulfide detection, the present invention provides a near-infrared fluorescence and photoacoustic dual-response detection kit and its application.

[0006] The present invention utilizes near-infrared fluorescence / photoacoustic response probes to construct a near-infrared fluorescence / photoacoustic dual response detection kit, which has the characteristics of good selectivity, high sensitivity and strong anti-interference ability, and realizes accurate quantitative detection of polysulfides through near-infrared fluorescence / photoacoustic response analysis.

[0007] In order to achieve the above object, the technical solution adopted by the present invention is:

[0008] A near-infrared fluorescence-photoacoustic dual-response detection kit comprises a reagent stock solution a and a probe reagent stock solution b; the volume ratio of the reagent stock solution a to the probe reagent stock solution b is 50:1;

[0009] The probe reagent stock solution b is made of a near-infrared fluorescence / photoacoustic response probe and an organic solvent. The structural formula of the near-infrared fluorescence / photoacoustic response probe is as follows:

[0010]

[0011] It is further defined that the concentration of the near-infrared fluorescence / photoacoustic response probe in the probe reagent stock solution b is 0.01 mmol / L to 1 mmol / L.

[0012] It is further defined that the organic solvent is at least one of dimethyl sulfoxide, methanol and N,N-dimethylformamide.

[0013] It is further defined that the reagent stock solution a is composed of phosphate with a molar concentration of 5 mmol / L to 15 mmol / L; the pH value of the reagent stock solution a is 7.4±0.5.

[0014] It is further defined that the phosphate is at least one of Na2HPO4, NaH2PO4 and KH2PO4.

[0015] Application of the near-infrared fluorescence-photoacoustic dual-response detection kit in the quantitative detection of polysulfides.

[0016] Furthermore, for quantitative polysulfide detection, the fluorescence response uses an excitation wavelength of 690nm to 710nm and an emission wavelength of 764nm; the photoacoustic response uses an excitation wavelength of 735nm. This is because the photoacoustic response is a "light → heat → sound" conversion process and does not involve the re-emission of photons.

[0017] Application of the near-infrared fluorescence-photoacoustic dual-response detection kit in rapid imaging detection of polysulfides.

[0018] It is further defined that, in the rapid imaging detection of polysulfides, the excitation wavelength used to obtain the laser confocal fluorescence imaging image is 633 nm, and the emission wavelength is 680 nm.

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] 1. The detection principle of the polysulfide detection kit of the present invention is that the fluorescence of the probe reagent stock solution b itself is weak, but the fluorescence intensity is significantly enhanced after reacting with polysulfides, producing strong absorption at 740nm to 780nm and significantly enhanced fluorescence at 764nm. This fluorescence emission band is in the near-infrared light region, with little background interference and is not interfered with by other common metal cations, anions, sulfur-containing amino acids and other substances, so it has high accuracy and sensitivity.

[0021] 2. The polysulfide detection kit provided by the present invention has high detection sensitivity and selectivity, and exhibits dual fluorescence and photoacoustic response characteristics. Both fluorescence intensity and photoacoustic intensity increase with increasing polysulfide concentration. When the polysulfide concentration is between 2.5 μmol / L and 60 μmol / L, the fluorescence intensity and polysulfide concentration show a linear relationship, with a correlation R-squared exceeding 0.98. When the polysulfide concentration is between 20 μmol / L and 100 μmol / L, the photoacoustic intensity and polysulfide concentration show a linear relationship, with a correlation R-squared exceeding 0.99. It can be used for the quantitative detection of polysulfides.

[0022] 3. The polysulfide detection kit provided by the present invention adopts a near-infrared fluorescence / photoacoustic response probe with a near-infrared fluorescence emission wavelength, a fast fluorescence generation reaction speed, and stable color development within 5 minutes. During detection, the background signal is low, the noise is low, and it has an excellent photoacoustic response. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 is the fluorescence emission spectrum of the reaction between the kit and polysulfide at different concentrations;

[0024] Figure 2 This is the standard curve when the kit is used to detect polysulfide concentration;

[0025] Figure 3 This is the standard curve when the kit is used to detect the photoacoustic response of polysulfides;

[0026] Figure 4 The fluorescence emission spectra of the kit used for reactions with various interfering substances;

[0027] Figure 5 The kit is used to detect the fluorescence intensity changes of polysulfides in Hela cells;

[0028] Figure 6 The kit is used to detect the fluorescence intensity changes of polysulfides in Arabidopsis thaliana;

[0029] Figure 7 The kit is used to detect the changes in the fluorescence intensity of polysulfides in BALB\C mice;

[0030] Figure 8The kit is used to detect the changes in the photoacoustic response intensity of polysulfides in BALB\C mice. DETAILED DESCRIPTION

[0031] The present invention will be described in further detail below with reference to the accompanying drawings and examples, but the embodiments of the present invention are not limited thereto. Other methods for preparing the compounds of the present invention are considered to be within the scope of the present invention by making some conventional modifications to the reaction conditions of the present invention.

[0032] Unless otherwise defined, technical or scientific terms used in the present invention shall have the same meaning as commonly understood by one of ordinary skill in the art to which the present invention belongs.

[0033] Technologies, methods, and equipment known to those skilled in the art may not be discussed in detail, but where appropriate, such technologies, methods, and equipment should be considered part of the specification.

[0034] It should also be understood that the specific embodiments described above are only used to explain the present invention, and the scope of protection of the present invention is not limited thereto. Any technician familiar with the technical field, within the technical scope disclosed by the present invention, can make equivalent replacements or changes based on the technical solutions and inventive concepts of the present invention, which should be covered by the scope of protection of the present invention / invention.

[0035] The present invention uses the cyanine near-infrared fluorescent dye IR-780 as a skeleton to derive a sulfur-containing semi-cyanine near-infrared fluorophore with a hydroxyl group, and further synthesizes a near-infrared fluorescence / photoacoustic response probe for polysulfide detection. The probe has a fast fluorescence generation reaction speed, high accuracy and sensitivity, excellent photoacoustic response, a near-infrared fluorescence emission wavelength, and can be used to detect polysulfides by fluorescence spectroscopy.

[0036] Example 1

[0037] This embodiment provides a preparation process of a near-infrared fluorescence / photoacoustic response probe, which is detailed as follows.

[0038] S1. Preparation of compound (III)

[0039] Under alkaline conditions in the presence of sodium hydride, the compound represented by formula (V) reacts with the compound represented by formula (IV) to obtain the compound represented by formula (III).

[0040]

[0041] Compound (V) is IR-780 iodide, and compound (IV) is 3-hydroxythiophenol, both of which are directly commercially available. Compound (III) was prepared according to the following method.

[0042] In this embodiment, the preparation of compound (III) specifically includes the following steps:

[0043] S1.1. Dissolve 3-hydroxythiophenol (56.78 mg, 0.45 mmol) and sodium hydride (10.8 mg, 0.45 mmol) in N,N-dimethylformamide solution (4 mL) and stir at room temperature under nitrogen for 15 min to obtain a mixed solution. Dissolve IR-780 iodide (200 mg, 0.3 mmol) in 1 mL of N,N-dimethylformamide solution, mix well, and then add the mixture to the mixed solution. The resulting mixture is reacted at 55°C for 20 h. After the reaction is complete, the mixture is cooled to room temperature. Subsequently, the mixture is poured into cold water, and perchloric acid is added to remove the solvent to obtain a crude product.

[0044] S1.2. Purify the crude product by column chromatography using dichloromethane / ethanol (v / v, 100:1) as eluent to obtain compound (III).

[0045] S2. Preparation of the compound represented by formula (II)

[0046] In this embodiment, the preparation of the compound represented by formula (II) specifically includes the following steps:

[0047] S2.1. Sodium bicarbonate (1.63 g, 19.46 mmol) was dissolved in 25 mL of water to form a sodium bicarbonate solution; thiosalicylic acid (1 g, 6.48 mmol) was then dissolved in the sodium bicarbonate solution and cooled to 0°C. Benzoyl chloride (912 mg, 6.48 mmol) and sodium carbonate (1.37 g, 12.98 mmol) were then added, and the reaction mixture was stirred at 0°C for 30 min and then at room temperature for another 45 min.

[0048] S2.2. After the reaction is completed, the reaction mixture is acidified with concentrated hydrochloric acid to form a white precipitate. The precipitate is collected and washed with cold water to obtain the compound represented by formula (II).

[0049]

[0050] S3. Preparation of the compound represented by formula (I)

[0051] In the presence of 4-dimethylaminopyridine and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, the compound represented by formula (III) reacts with the compound represented by formula (II) to obtain the compound represented by formula (I), which is a near-infrared fluorescence / photoacoustic response probe.

[0052]

[0053] In this embodiment, the specific steps of preparing the compound represented by formula (I) include:

[0054] S3.1. Dissolve the compound of formula (II) obtained in step S2 (124.8 mg, 0.48 mmol), 4-dimethylaminopyridine (2.88 mg, 0.024 mmol), and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (34.79 mg, 0.18 mmol) in 4 mL of anhydrous dichloromethane and stir at room temperature for 15 min to obtain a mixed solution;

[0055] S3.2. Dissolve the compound represented by formula (III) (50 mg, 0.12 mmol) obtained in step S1 in 4 mL of anhydrous dichloromethane, add the obtained mixture to the mixed solution of step S3.1, and stir at room temperature for 5 h. After the reaction is completed, extract with water and dichloromethane, and then evaporate under reduced pressure to obtain a blue solid powder, which is the crude product.

[0056] S3.3. The synthesized crude product was eluted and separated with dichloromethane / methanol (v / v, 50:1) to obtain a solid powder, which is the near-infrared fluorescence / photoacoustic response probe represented by formula (I).

[0057] Example 2

[0058] This embodiment provides a near-infrared fluorescence-photoacoustic dual-response detection kit, comprising a reagent stock solution a and a probe stock solution b. The volume ratio of the reagent stock solution a to the probe reagent stock solution b is 50:1.

[0059] In this embodiment, the reagent stock solution a is composed of phosphate. The pH value of the reagent stock solution a is 7.4 (which can also be arbitrarily replaced with 7.4±0.5), and the molar concentration of the phosphate is 10mmol / L. The phosphate is made from Na2HPO4, NaH2PO4, and KH2PO4, and the mass ratio of Na2HPO4, NaH2PO4, and KH2PO4 is 1:1:1. Preferably, the reagent stock solution a has a concentration of 10mmol / L.

[0060] In this embodiment, the probe reagent stock solution b is prepared from the near-infrared fluorescence / photoacoustic response probe (prepared in Example 1) and an organic solvent.

[0061] In this embodiment, the concentration of the near-infrared fluorescence / photoacoustic response probe in the probe reagent stock solution b is 0.01 mmol / L to 1 mmol / L. Exemplarily, the concentration of the near-infrared fluorescence / photoacoustic response probe is 0.01 mmol / L, 0.02 mmol / L, 0.05 mmol / L, 0.1 mmol / L, 0.2 mmol / L, 0.5 mmol / L, or 1 mmol / L.

[0062] Preferably, the concentration of the near-infrared fluorescence / photoacoustic response probe in the probe reagent stock solution b is 1 mmol / L.

[0063] Preferably, the organic solvent is a mixture of dimethyl sulfoxide, methanol and N,N-dimethylformamide, and the mixing ratio is equal volume.

[0064] During implementation, the phosphate solution used in reagent stock solution a is HyClone TM (1X) product, phosphate solution with a pH of 7.4. The phosphate solution (PBS) is prepared using a Na2HPO4-NaH2PO4-KH2PO4 system, so the phosphate concentration refers to the total concentration of Na2HPO4, NaH2PO4, and KH2PO4. The near-infrared fluorescence / photoacoustic response probe prepared in Example 1 is then mixed with organic solvents (dimethyl sulfoxide, methanol, and N,N-dimethylformamide) to prepare probe reagent stock solution b, where the concentration of the near-infrared fluorescence / photoacoustic response probe is 1 mmol / L.

[0065] The sodium tetrasulfide required for in vitro detection of polysulfides is prepared according to conventional operating methods in the art, and the concentration of the prepared sodium tetrasulfide mother solution is 10 mmol / L.

[0066] The polysulfide detection kit is prepared by mixing the above-prepared reagent stock solution a and probe reagent stock solution b in a volume ratio of 50:1. During implementation, the two solutions are packaged separately and mixed as needed during detection.

[0067] In the above embodiment 2, the molar concentration of phosphate can be arbitrarily selected and replaced in the range of 5 mmol / L to 15 mmol / L; phosphate can be replaced by at least one of Na2HPO4, NaH2PO4 and KH2PO4; and the organic solvent can be replaced by at least one of dimethyl sulfoxide, methanol and N,N-dimethylformamide.

[0068] Example 3

[0069] This example uses an infrared fluorescence photoacoustic dual response detection kit to detect polysulfides.

[0070] The detection method provided in this embodiment is as follows:

[0071] 1) Reagent stock solution a and probe reagent stock solution b were mixed at a volume ratio of 50:1 and recorded as a blank sample. When measuring fluorescence emission spectra, the excitation wavelength was 700 nm, and the fluorescence intensity of the blank sample at an emission wavelength of 764 nm was measured.

[0072] 2) Dissolve the probe reagent stock solution b in the reagent stock solution a, and dilute the volume to 2 mL with the reagent stock solution a to form a mixed solution. At this time, the concentration of the near-infrared fluorescence / photoacoustic response probe in the mixed solution is 20 μmol / L.

[0073] 3) Then, equal amounts of the mixed solution were taken and added to a series of polysulfide standard solutions with different concentrations, wherein the concentrations of polysulfide were: 0 μmol / L, 5 μmol / L, 10 μmol / L, 15 μmol / L, 20 μmol / L, 30 μmol / L, 40 μmol / L, 50 μmol / L, 60 μmol / L, 80 μmol / L, 90 μmol / L and 100 μmol / L; the volume of the polysulfide standard solutions with different concentrations was 2 mL.

[0074] In this step, a series of polysulfide standard solutions were prepared and reacted at 37°C for 5 minutes. The fluorescence excitation spectrum and fluorescence emission spectrum were measured by a fluorescence instrument F-7000. The slit width of the excitation and emission was 10 nm. The excitation wavelength was 700 nm. The emission spectra of the polysulfide standard solutions with different concentrations were obtained as shown in the following figure. Figure 1 shown.

[0075] from Figure 1 It can be seen that in the absence of polysulfides, the fluorescence intensity at 764 nm is F0, and the probe shows weak fluorescence at 764 nm. As the concentration of polysulfides increases, the fluorescence intensity at 764 nm greatly increases, indicating that the probe has a good optical response to polysulfides.

[0076] 4) When measuring fluorescence emission spectra, the excitation wavelength is 700 nm, and the fluorescence intensity of a series of polysulfide standard solutions with different concentrations at an emission wavelength of 764 nm is measured, which is recorded as F. F has multiple sets of values. Then, the concentration C of polysulfide is used as the horizontal axis and the corresponding fluorescence intensity value F is used as the vertical axis to draw a standard curve. The standard curve is as follows: Figure 2 As shown; after linear fitting between concentration and fluorescence intensity, the regression equation of linear fitting is:

[0077] F=9.90×C+43.79(R 2 =0.9856)

[0078] Where C is the concentration of polysulfide in μmol / L.

[0079] Through the standard curve equation, it was found that the polysulfide concentration was linearly related between 2.5μmol / L and 60μmol / L, and the square of the correlation R reached above 0.98.

[0080] 5) Within the linear range, reagent stock solution a and probe reagent stock solution b at a volume ratio of 50:1 are added to the sample to be tested, and the fluorescence intensity F' of the sample to be tested at an emission wavelength of 764 nm is measured with an excitation wavelength of 700 nm. The fluorescence intensity F' is substituted into the standard curve to obtain the concentration of polysulfide in the sample to be tested.

[0081] After repeating the detection method provided in this example using conventional methods in the art, it was found that the fluorescence response detection limit (S / N=3) of the probe was 1.45 μmol / L polysulfide.

[0082] Example 4

[0083] This example uses an infrared fluorescence photoacoustic dual response detection kit to detect polysulfides.

[0084] The experimental process provided in this embodiment is:

[0085] 1) Reagent stock solution a and probe reagent stock solution b were mixed at a volume ratio of 50:1 and recorded as a blank sample. Photoacoustic response is a "light → heat → sound" conversion process, not photoluminescence (such as fluorescence or phosphorescence), and therefore does not involve photon re-emission. Photoacoustic response measurement uses 735 nm as the excitation wavelength and measures the photoacoustic intensity.

[0086] 2) Dissolve the probe reagent stock solution b in the reagent stock solution a, and dilute the volume to 2 mL with the reagent stock solution a to form a mixed solution. At this time, the concentration of the near-infrared fluorescence / photoacoustic response probe in the mixed solution is 20 μmol / L.

[0087] 3) Then, equal amounts of the mixed solution were taken and added to a series of polysulfide standard solutions with different concentrations, wherein the concentrations of polysulfide were: 20 μmol / L, 40 μmol / L, 60 μmol / L, 80 μmol / L and 100 μmol / L; the volume of the polysulfide standard solutions with different concentrations was 2 mL.

[0088] 4) When the photoacoustic response is measured, the excitation wavelength is 735 nm, and the photoacoustic intensity of a series of polysulfide standard solutions with different concentrations is measured, which is recorded as P. P has multiple sets of values. The concentration C of polysulfide is used as the horizontal axis and the corresponding photoacoustic intensity value P is used as the vertical axis to draw a standard curve. The standard curve is as follows: Figure 3 As shown; after linear fitting between concentration and photoacoustic intensity, the regression equation of linear fitting is:

[0089] P = 4.42 × C - 42.13 (R 2 =0.9950)

[0090] Where C is the concentration of polysulfide in μmol / L.

[0091] Through the standard curve equation, it was found that the polysulfide concentration was linear between 20μmol / L and 100μmol / L, and the square of the correlation R reached above 0.99.

[0092] 5) Within the linear range, reagent stock solution a and probe reagent stock solution b at a volume ratio of 50:1 are added to the sample to be tested, and the photoacoustic response intensity P' of the sample to be tested is measured at an excitation wavelength of 735 nm. The photoacoustic response intensity P' is substituted into the standard curve to obtain the concentration of polysulfide in the sample to be tested.

[0093] The detection method provided in this embodiment was subjected to repeated experiments using conventional methods in the art. The result showed that the detection limit of the photoacoustic response of the probe (S / N=3) was 3.82 μmol / L polysulfide.

[0094] Example 5

[0095] This example mainly studies the anti-biological interference ability of the near-infrared fluorescence-photoacoustic dual-response detection kit.

[0096] The near-infrared fluorescence-photoacoustic dual-response detection kit in this embodiment is provided in Example 2.

[0097] The test process in this embodiment is:

[0098] 1) Dissolve reagent stock solution b in reagent stock solution a at a volume ratio of 1:50 to prepare a mixed solution. The concentration of the near-infrared fluorescent / photoacoustic response probe in reagent stock solution b is 1 mmol / L; the concentration of the near-infrared fluorescent / photoacoustic response probe in the mixed solution is 20 μmol / L.

[0099] 2) Under the same conditions, the probe was used to detect metal cations (Na + , K + Mg 2+ 、Ga 2+ and Zn 2+ ), amino acids (Cys, Hcy and Gsh), anions (SO3 2- 、SO4 2- 、HSO3 - , ClO - 、NO2 - and S2O3 2- ) and other biochemical substances that may interfere with the detection of polysulfides, thereby evaluating the selectivity of near-infrared fluorescence / photoacoustic response probes for polysulfides.

[0100] The interfering substance was added to the reagent stock solution a with a concentration of 10 mmol / L as the solvent to obtain a set of mixed solutions. After reacting at 37°C for 5 minutes, the fluorescence emission spectra were measured using a fluorescence instrument F-7000. The fluorescence emission spectrum was measured with an excitation wavelength of 700 nm and a slit width of 10 nm for excitation and emission. The fluorescence intensity of each substance was obtained, as shown in the following figure. Figure 4 shown.

[0101] Figure 4 The kit is used for the fluorescence emission spectrum of various interfering substances. 1 to 17 are near-infrared fluorescence / photoacoustic response probes, Na + , K + Mg 2+ 、Ga 2+ 、Zn 2+ 、SO3 2- 、SO4 2- 、HSO3 - , ClO - 、NO2 - 、S 2- 、S2O3 2- , Cys, Hcy, Gsh, and sodium tetrasulfide; 1 mmol / L stock solutions of each analyte were prepared in deionized water. The metal cations and anions were then prepared in PBS buffer to a 500 μmol / L detection system, while the amino acids were prepared to a 250 μmol / L detection system. Finally, the probe was added to the reaction. The concentration of the near-infrared fluorescence / photoacoustic response probe in the detection system was 20 μmol / L. All measurements were repeated three times.

[0102] See also Figure 4 The results showed that only polysulfides could cause the near-infrared fluorescence / photoacoustic response probe to produce a significant change in the light signal response, proving that the near-infrared fluorescence / photoacoustic response probe is highly selective for polysulfides; other common inorganic salts, amino acids and metal substances had little interference effect.

[0103] Example 6

[0104] The experimental process is: HeLa cells are cultured at a rate of 1×10 4 HeLa cells were seeded at a density of 100 μg / L in a confocal laser microscope dish and incubated for 24 hours to allow attachment. HeLa cells were washed at least three times with cell culture medium without phenol red and fetal bovine serum, then incubated with the probe at 37°C. HeLa cells were infected with the near-infrared fluorescence / photoacoustic response probe (20 μmol / L) for 5 minutes as experimental groups. Concentration groups were pretreated with 20 μmol / L, 50 μmol / L, and 100 μmol / L polysulfides for 30 minutes, followed by incubation with the near-infrared fluorescence / photoacoustic response probe (20 μmol / L) for 5 minutes. In the inhibition group, cells were pretreated with 50 μmol / L and 100 μmol / L polysulfides for 30 minutes, then treated with dithiothreitol (500 μmol / L) for 30 minutes, and finally incubated with the near-infrared fluorescence / photoacoustic response probe (20 μmol / L) for 5 minutes. Dithiothreitol was selected as an inhibitor in this experiment to inhibit the production of endogenous and exogenous polysulfides in cells.

[0105] The excitation wavelength used for acquiring laser confocal fluorescence images was 633 nm and the emission wavelength was 680 nm.

[0106] The experimental results are as follows Figure 5 As shown in the figure, immediately after the addition of the near-infrared fluorescence / photoacoustic response probe, the cells showed weak fluorescence; as the polysulfide concentration increased, the fluorescence intensity of the HeLa cells gradually increased, reaching a maximum at 100 μmol / L. The fluorescence intensity was quantitatively analyzed using the normalization method within the range of 0 to 1, and the cell pixel intensity at a polysulfide concentration of 100 μmol / L was defined as 1.0. The results are shown in the figure. Figure 5 As shown in Figure 3-C, the fluorescence signal increased by approximately 66.32% and 84.20% at 20 μmol / L and 50 μmol / L, respectively. The inhibitor group decreased by approximately 8.83% and 20.74% compared to the cells treated with 50 μmol / L and 100 μmol / L.

[0107] Example 7

[0108] The experimental procedure is as follows: The Arabidopsis thaliana variety Columbia-0 was used. Seeds were soaked in a 20% (volume / volume) sodium hypochlorite solution for 8 minutes and washed four times with sterile water. The sterile seeds were then sown on 1 / 2 MS medium containing 1% (weight / volume) sucrose and 0.8% (weight / volume) agar and subjected to a cold treatment in the dark at 4°C for 3 days. Subsequently, the Arabidopsis thaliana was grown indoors at 21°C under a 16-hour light and 8-hour dark cycle. The Arabidopsis thaliana was selected for the experiment after 5 days of light exposure.

[0109] Arabidopsis thaliana was divided into three groups. The control group was directly treated with a near-infrared fluorescence / photoacoustic response probe (20 μmol / L) for 5 minutes. The concentration group was pre-treated with polysulfide (20 μmol / L, 50 μmol / L, and 100 μmol / L) for 30 minutes, and then incubated with a near-infrared fluorescence / photoacoustic response probe (20 μmol / L) for 5 minutes. The inhibition group was pre-treated with polysulfide (50 μmol / L, 100 μmol / L) for 30 minutes, then treated with dithiothreitol (500 μmol / L) for 30 minutes, and finally incubated with DOSSH (20 μmol / L) for 5 minutes. All Arabidopsis thaliana were washed three times with phosphate buffer and then imaged by laser confocal microscopy at 633 nm.

[0110] The experimental results are as follows Figure 6As shown in the figure, after the near-infrared fluorescence / photoacoustic response probe was added, the fluorescence intensity of Arabidopsis was weak; as the polysulfide concentration increased, the fluorescence intensity of Arabidopsis gradually increased, reaching a maximum at 100 μmol / L. The fluorescence intensity was quantitatively analyzed using the normalization method within the range of 0 to 1, and the pixel intensity of Arabidopsis with a polysulfide concentration of 100 μmol / L was defined as 1.0. The results are shown in the figure. Figure 6 As shown in Figure 3-C, the fluorescence signal increased by approximately 62.65% and 91.66% at 20 μmol / L and 50 μmol / L, respectively. The inhibitor group decreased by approximately 28.25% and 21.64% compared to the Arabidopsis thaliana at 50 μmol / L and 100 μmol / L.

[0111] Example 8

[0112] Female BALB / C mice aged 6-8 weeks were purchased from the Experimental Animal Center of Shaanxi Normal University for in vivo fluorescence and photoacoustic imaging experiments. Mice were fasted for 12 hours before imaging to prevent food interference. The BALB / C mice were divided into two groups.

[0113] Group 1: Mice were intraperitoneally injected with 100 μL of a 400 μmol / L polysulfide. After a certain period of time, 50 μL of a 100 μmol / L near-infrared fluorescent / photoacoustic probe was injected, and imaging was immediately performed to obtain the optimal imaging time.

[0114] Another group: Mice were intraperitoneally injected with 100 μL of polysulfide at concentrations of 20 μmol / L, 50 μmol / L, and 100 μmol / L, and then directly injected with near-infrared fluorescence / photoacoustic response probes after waiting for 5 minutes, followed by fluorescence imaging.

[0115] Mice were anesthetized with 2.5% isoflurane in an oxygen atmosphere at designated time intervals. Whole-body near-infrared (NIR) fluorescence images of mice were captured using an IVIS LuminaXR in vivo imaging system, using a 710-nm laser and a 760-nm filter for optimal imaging quality. For photoacoustic imaging, mice were depilated in the abdominal region, placed in a room-temperature water bath, and anesthetized with oxygen via a ventilator. Photoacoustic images were reconstructed using a 735-nm laser using back-projection and linear regression multispectral unmixing techniques. All experiments involving live cells and mice were in compliance with institutional animal care and use regulations.

[0116] The results showed that the time optimization experiment was carried out on mice injected with polysulfide (100 μmol / L), and the results were consistent with the in vitro detection, with the fluorescence intensity reaching a peak at 5 min (see Figure 7 -A). Figure 7 -Ab was defined as 1.0, and the fluorescence intensities (a, c, d) were approximately 94.56%, 90.45%, and 83.85%, respectively ( Figure 7 -C). Subsequently, endogenous H2S in normal mice was detected using a near-infrared fluorescence / photoacoustic response probe. n In vivo near-infrared fluorescence imaging of Figure 7 -B shows that the fluorescence intensity of the mouse abdomen is relatively low. After the injection of different concentrations of polysulfide, the fluorescence intensity gradually increases with the concentration. The intensity image at 100 μmol / L is set to 1, and the fluorescence intensity of the other images is approximately 38.45%, 63.61% and 92.48% ( Figure 7 These results indicate that the near-infrared fluorescence / photoacoustic responsive probe can be used to monitor changes in polysulfide levels in the human body via near-infrared fluorescence imaging.

[0117] Subsequently, DOSSH was used to perform photoacoustic imaging of endogenous and exogenous polysulfides in mice for further investigation. Figure 8 As shown in the figure, to eliminate the influence of hair in the detection area of mice, hair removal was performed before intraperitoneal injection, and a detector (center frequency of 7.5MHz, channel array of 128) was used to detect the photoacoustic intensity of the cross section of the mouse abdomen. To take into account individual differences between mice, a blank control was performed before each mouse test. Figure 8 As shown, the photoacoustic intensity of the control group (a, c, e) was relatively weak, but after the injection of polysulfide at different concentrations (b, d, f), the photoacoustic intensity gradually increased. Figure 8 -C represents the relative rate of change of photoacoustic intensity in the selected area of the same mouse before and after injection, showing that the higher the polysulfide concentration, the higher the relative rate of change.

[0118] It can be verified through the above Examples 6 and 7 that the near-infrared fluorescence / photoacoustic response probe detection kit can be used to intuitively determine whether the biological sample to be tested contains polysulfides by comparing the fluorescence intensity or fluorescence imaging, and the polysulfide concentration can also be quantitatively determined; Example 8 verified that the near-infrared fluorescence / photoacoustic response probe detection kit is suitable for rapid imaging detection of complex biological living samples (such as mice), and can monitor changes in polysulfide levels in the body by photoacoustic response signal imaging. In addition, in terms of being used for detecting polysulfides, the near-infrared fluorescence / photoacoustic response probe detection kit provided by the present invention is a polysulfide detection device with excellent performance and easy use, rapid response, high sensitivity, simple operation, and suitable for wide application, especially in the fields of food, living biological imaging, etc., with huge application prospects.

[0119] The above are several relatively preferred implementation methods of the preparation method of the present invention, but they cannot be used as limitations on the technical solutions protected by the present invention. Any replacement solutions obtained by ordinary technicians in this field without making creative work based on the technical ideas of the present invention should fall within the scope of protection of the present invention.

Claims

1. A near-infrared fluorescence and photoacoustic dual response detection kit, characterized in that: The method comprises a reagent stock solution a and a probe reagent stock solution b; the volume ratio of the reagent stock solution a to the probe reagent stock solution b is 50:1; The probe reagent stock solution b is made of a near-infrared fluorescence / photoacoustic response probe and an organic solvent. The structural formula of the near-infrared fluorescence / photoacoustic response probe is as follows:

2. The near-infrared fluorescence-photoacoustic dual response detection kit according to claim 1, characterized in that: In the probe reagent stock solution b, the concentration of the near-infrared fluorescence / photoacoustic response probe is 0.05 mmol / L to 1 mmol / L.

3. The near-infrared fluorescence-photoacoustic dual response detection kit according to claim 1, characterized in that: The organic solvent is at least one of dimethyl sulfoxide, methanol and N,N-dimethylformamide.

4. The near-infrared fluorescence-photoacoustic dual response detection kit according to claim 1, characterized in that: The reagent stock solution a is composed of phosphate with a molar concentration of 1 mmol / L to 10 mmol / L; the pH value of the reagent stock solution a is 7.4±0.

5.

5. The near-infrared fluorescence-photoacoustic dual response detection kit according to claim 1, characterized in that: The phosphate is at least one of Na2HPO4, NaH2PO4 and KH2PO4.

6. Use of the near-infrared fluorescence-photoacoustic dual-response detection kit as claimed in claim 1 in the quantitative detection of polysulfides.

7. The use according to claim 6, characterized in that In the quantitative detection of polysulfides, the fluorescence response uses 690nm to 710nm as the excitation wavelength and the emission wavelength is 764nm; the photoacoustic response uses 735nm as the excitation wavelength.

8. Use of the near-infrared fluorescence-photoacoustic dual-response detection kit as claimed in claim 1 in rapid imaging detection of polysulfides.

9. The use according to claim 8, characterized in that In the rapid imaging detection of polysulfides, the excitation wavelength used to obtain the laser confocal fluorescence imaging is 633 nm, and the emission wavelength is 680 nm.

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