Preparation of low-background nanoprobe for detecting ONOO- / ClO-and whole blood detection

Abstract

The invention provides preparation of a low-background nanoprobe for detecting ONOO <-> / ClO <-> and whole blood detection, a multifunctional fluorescent nanoprobe is designed based on a donor-receptor one-to-many integrated sensing strategy based on FRET and blood self-fluorescence inhibition, a donor effectively inhibits blood self-fluorescence through an inner filter effect, and by adjusting the proportion of the receptor to the donor, the self-fluorescence of the blood can be effectively inhibited, so that the ONOO <-> / ClO <-> can be detected, and the ONOO <-> / ClO <-> can be detected. FRET effect is utilized to effectively quench donor fluorescence, so that zero background of a whole blood detection system is realized. Benefited from extremely low background fluorescence, the invention successfully detects the level of endogenous ONOO- / ClO-in the whole blood of mice with sepsis and hemolytic diseases, monitors the change of the level of ONOO- / ClO-in the process of sepsis and hemolytic diseases, and proves that the concentration of ONOO- / ClO-is positively correlated with the severity of diseases. The strategy is simple to operate and does not need complex synthesis and other operations. The zero-background generalized sensing strategy in whole blood detection provides a valuable insight for designing a fluorescent probe for detecting endogenous substances in blood.

Description

Technical Field

[0001] The present invention belongs to the technical field of organic fluorescent probes and designs a method for detecting ONOO - / ClO - Preparation of low-background nanoprobes and whole blood detection. Background Art

[0002] Abnormal activity or concentration of biomarkers in the blood can lead to a range of bodily dysfunctions. Early detection of abnormal levels of biomarkers is crucial for understanding the function of biomarkers and early disease management. Analyte-activated fluorescent molecular probes are powerful tools for detecting and bioimaging biomarkers in living systems because of their unique advantages of rapid analysis, real-time, and non-invasive imaging.

[0003] Some blood diseases such as sepsis and hemolysis are often accompanied by cells with ONOO in the blood. - / ClO - The active oxygen content represented by ONOO - / ClO - Changes in concentration can effectively monitor the degree of disease progression. However, due to the complex composition of blood and the presence of substances such as porphyrin in the blood, strong background fluorescence will be generated when excited by light, which greatly limits the signal-to-background ratio of the detection and may even cover the fluorescence signal of the probe, causing a false negative problem. In addition, many probes have the disadvantage of high background fluorescence and are not suitable for complex detection systems such as blood. Therefore, it is still of great significance to design probes with low background fluorescence and can inhibit blood autofluorescence to cope with complex biological systems. At present, through strategies such as photoinduced electron transfer (PET) and the generation or change of conjugated structures, the fluorescence signal after response can be significantly enhanced or the intrinsic fluorescence of the probe can be significantly reduced, thereby improving the signal-to-background ratio and detection sensitivity. These strategies are certainly effective and have made great progress, but they require complex design procedures and are not generally applicable.

[0004] In previous work, the inventors found that the red-shifted BODIPY dye can effectively inhibit blood autofluorescence (CN2023104182652), but how to design a simple and universal strategy based on this dye to achieve low-background endogenous analyte detection in whole blood is an urgent problem to be solved. FRET ( The FRET (Resonance Energy Transfer) mechanism allows a significant change in the emission wavelength, improving the sensitivity of the detection. Therefore, FRET-based fluorescent probes have attracted increasing attention and are widely used to detect various analytes, such as ions, natural small molecules, biomacromolecules, and cell microenvironment-related parameters. FRET fluorescent probes usually consist of two fluorophores: a donor and an acceptor. When the donor and acceptor are at an optimal distance (usually ~10nm), non-radiative energy transfer occurs, resulting in a decrease in the donor fluorescence intensity and an increase in the acceptor fluorescence intensity. Moreover, previous FRET probes had an equal ratio of donors and acceptors, resulting in poor energy transfer efficiency and a high background signal of the donor. Summary of the invention

[0005] In view of the shortcomings of the prior art, the object of the present invention is to provide a method for detecting ONOO - / ClO - Preparation of low-background nanoprobes and whole blood detection.

[0006] The present invention provides a method for detecting ONOO - / ClO - The low-background nanoprobe NPBC is composed of two fluorescent dyes: a donor and an acceptor. The structural formulas of the donor and the acceptor are as follows:

[0007]

[0008] The method for detecting ONOO - / ClO - The reaction mechanism of the nanoprobe is as follows Fig.10 shown.

[0009] In the absence of ONOO - / ClO - When the donor fluorescence is quenched by the acceptor, - / ClO - After the reaction, the acceptor is destroyed, FRET disappears, and the donor fluorescence recovers.

[0010] In a preferred embodiment, the molar ratio of the donor to the acceptor is 1:(3-6), which further extinguishes the fluorescence of the donor.

[0011] The present invention also provides a method for detecting ONOO - / ClO - The method for preparing a low-background nanoprobe comprises the following steps:

[0012] (1) Ethyl bromoacetate and p-hydroxybenzaldehyde are dissolved in acetone, potassium carbonate is added, reacted at a set temperature, the mixture is concentrated under reduced pressure, and extracted and purified again to obtain compound 1;

[0013] (2) Dissolve 2,4-dimethylpyrrole in dichloromethane, add compound 1 at a predetermined temperature, add a drop of trifluoroacetic acid as a catalyst, and then add triethylamine and boron trifluoride ether solution in sequence, stir to react at room temperature, concentrate the mixture under reduced pressure, and obtain compound 2 after extraction and purification;

[0014] (3) Compound 2 and p-methoxybenzaldehyde are dissolved in anhydrous acetonitrile, acetic acid, piperidine and a small amount of molecular sieves are added, and the mixture is refluxed at a set temperature under the protection of an inert atmosphere, and the mixture is concentrated under reduced pressure. After extraction and purification, dye 1 is obtained;

[0015] (4) adding 1,1,2-trimethyl-1H-benz[e]indole and iodoethane into a pressure tube, using acetonitrile as a solvent, reacting at a predetermined temperature, and washing after the reaction to obtain compound 3;

[0016] (5) N-(3-phenylamino-2-propyleneylidene)aniline hydrochloride and compound 3 are mixed under an inert atmosphere, sodium acetate is added, and the mixture is refluxed in anhydrous acetonitrile to obtain dye 6;

[0017] (6) dissolving dye 1 and dye 6 in THF, adding F127, adding the mixed solution into water after dissolution, ultrasonically treating, and removing THF by vacuum rotary evaporation to obtain the nanoprobe NPBC;

[0018] The reaction process is as follows:

[0019]

[0020] In a preferred embodiment, in step (1), the molar ratio of ethyl bromoacetate to p-hydroxybenzaldehyde is 1:(1.1-1.3), which facilitates subsequent purification.

[0021] Furthermore, in step (1), dichloromethane is used as the eluent.

[0022] In a preferred embodiment, in step (2), the molar ratio of 2,4-dimethylpyrrole solution, compound 1, triethylamine and boron trifluoride etherate is (2-2.2):1:(3.6-4):(6.6-7).

[0023] Furthermore, in step (2), dichloromethane is used as the eluent.

[0024] In a preferred embodiment, in step (3), the molar ratio of p-anisaldehyde to compound 2 is (2.5-3):1, and the reflux reaction time is 2-3 hours.

[0025] Furthermore, in step (3), petroleum ether / ethyl acetate (5 / 1) is used as the eluent.

[0026] In a preferred embodiment, in step (4), the molar ratio of 1,1,2-trimethyl-1H-benzo[e]indole to ethyl iodide is 1:(1.2-1.5).

[0027] Furthermore, in step (4), petroleum ether / ethyl acetate (4 / 1) is used as a washing agent to wash the generated indole salt multiple times.

[0028] In a preferred embodiment, in step (5), the molar ratio of N-(3-phenylamino-2-propyleneylidene)aniline hydrochloride to compound 3 is 1:(2.2-2.5).

[0029] Furthermore, in step (5), dichloromethane / methanol (50 / 1 to 40 / 1) is used as the eluent.

[0030] In a preferred embodiment, in step (6), the molar ratio of dye 1 to dye 6 is 1:(4-6), and the mass ratio of dye 1 to F127 is 1:(200-400).

[0031] Further, dye 1 (0.08 mg), dye 6 (0.3 mg) and F127 (25 mg) were dissolved in 1 mL THF, and the mixed solution was quickly poured into 4 mL water, ultrasonicated for half an hour, and after being evenly dispersed, the THF solution was removed by decompression to obtain the mother solution of the nanoprobe NPBC. The mother solution was stored in a refrigerator at 4°C for later use.

[0032] The present invention also provides the method for detecting ONOO - / ClO - Application of low-background nanoprobes to detect ONOO in whole blood - / ClO - content.

[0033] The inventors have found that the red-shifted BODIPY dye (dye 1) has excellent optical properties, including chemical stability to reactive oxygen species (ROS), large extinction coefficient, good photostability and strong fluorescence, and its absorption wavelength matches the emission wavelength of blood, which can effectively quench blood autofluorescence. Dye 6 can adjust the absorption wavelength by extending the conjugated system, achieve the quenching effect on the fluorescence of dye 1, and have no spectral crosstalk with the background fluorescence of dye 1. At the same time, dye 6 has a strong effect on ONOO - / ClO - Unstable, suitable as a fluorescent receptor.

[0034] The present invention proposes a one-to-many donor-acceptor fluorescence sensing strategy and designs a method for detecting ONOO - / ClO -The low-background fluorescent probe NPBC was successfully developed. By adjusting the ratio of the acceptor and the donor, the donor fluorescence was effectively quenched. At the same time, the donor can effectively quench the blood autofluorescence, achieving the effect of ultra-low background fluorescence in the whole blood detection system. Thanks to this effect, we have achieved the detection of sepsis and hemolytic diseases with whole blood ONOO - / ClO - Reliable monitoring of content.

[0035] The present invention increases the ratio of acceptors to further extinguish the fluorescence of the donor, using a "one-to-many" method through nano-encapsulation without the need for a complex synthesis process. The red-shifted BODIPY dye is used as a donor, and its own fluorescence can be quenched by the acceptor, while the blood autofluorescence can also be effectively suppressed, achieving low-background fluorescence detection of whole blood.

[0036] Compared with the prior art, the present invention has the following beneficial technical effects:

[0037] 1) The nanoprobe NPBC has very low background fluorescence and is effective for ONOO - The fluorescence response of ClO - The fluorescence response was up to about 80 times.

[0038] 2) The nanoprobe NPBC can effectively inhibit blood autofluorescence, and the quenching rate of blood autofluorescence by 15 μM NPBC is about 70%.

[0039] 3) The nanoprobe NPBC monitored whole blood ONOO in sepsis and hemolytic diseases - / ClO - The experimental results showed that the content of whole blood ONOO - / ClO - The content is positively correlated with the severity of the disease. The experimental results reflect the application prospect of the present invention in clinical monitoring of the progress of blood diseases such as sepsis and hemolysis. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 Nanoprobe NPBC and ONOO - / ClO - After the response, the process goes from "zero" background fluorescence (FRET on) to high brightness (FRET off).

[0041] Figure 2 (a) is the normalized fluorescence spectrum of donor quenching of blood autofluorescence, and (b) is the quantification of the quenching efficiency.

[0042] Figure 3 (a) is the TEM image of the constructed nanoprobe NPBC, and (b) is the DLS image of NPBC.

[0043] Figure 4 (a) The nanoprobe NPBC and its effect on ONOO - (b) is the fluorescence response of NPBC to ONOO - working curve.

[0044] Figure 5 (a) The nanoprobe NPBC shows its effect on ClO - (b) is the fluorescence response of NPBC to ClO - working curve.

[0045] Figure 6 Nanoprobe NPBC for ONOO in cells - / ClO - Confocal imaging of (a) HepG2 cells under APAP stimulation and NAC inhibition. (b) Confocal imaging of RAW264.7 cells under LPS stimulation and NAC inhibition.

[0046] Figure 7 (a) is the overlapping diagram of the absorption spectrum of nanoprobe NPBC and the blood autofluorescence spectrum, and (b) is the quenching effect of the nanoprobe on blood autofluorescence.

[0047] Figure 8 Nanoprobe NPBC for ONOO in whole blood of septic mice - / ClO - Monitoring of content (a), fluorescence response diagram of nanoprobe NPBC in whole blood with different degrees of sepsis. (b), quantification of (a) and comparison with the detection results of ELISA method.

[0048] Fig. 9 Nanoprobe NPBC was used to hemolyze ONOO in whole blood of mice - / ClO - Monitoring of content. (a) Establishment of hemolytic mouse model. (b) Fluorescence response of nanoprobe NPBC in whole blood of mice with different degrees of hemolysis. (c) Quantification of (b). (d) Comparison of ELISA test results.

[0049] Fig.10 Reaction mechanism of the nanoprobe used to detect ONOO- / ClO-. DETAILED DESCRIPTION

[0050] The present invention is further described below with reference to specific examples, but the present invention is not limited to the following examples. The methods are conventional methods unless otherwise specified, and the raw materials can be obtained from public commercial channels unless otherwise specified.

[0051] Example 1 Preparation of Nanoprobe NPBC:

[0052] (1) Dye 1 was synthesized according to the method described above. The NMR and mass spectrometry characteristics of dye 1 are as follows:

[0053] 1 H NMR(500MHz,DMSO-d6)δ7.60(d,J=8.7Hz,4H),7.55(d,J=16.3Hz,2H),7.42

[0054] (d,J=16.3Hz,2H),7.36(d,J=8.6Hz,2H),7.14(d,J=8.6Hz,2H),7.06(d,J=8.8Hz,4H),6.95(s,2H) ,5.77(s,1H),4.90(s,2H),4.20(q,J=7.1Hz,2H),3.84(s,6H),1.48(s,6H),1.23(t,J=7.1Hz,3H). 13 C NMR(500MHz,DMSO-d6)δ169.04,160.87,158.74,152.57,142.01,138.22,137.03,133.33,130.18,129.37,129.33,1 27.40,118.54,116.48,115.71,115.19,65.28,61.20,55.85,14.90,14.53.MS(MALDI-TOF-MS):m / z=662.540,calcd for(C 39 H 37 BF2N2O5) + =662.405.

[0055] (2) Dye 6 was synthesized according to the method described above. The NMR and mass spectrometry characteristics of dye 6 are as follows:

[0056] 1 H NMR(500MHz,DMSO-d6)δ8.49(t,J=13.1Hz,1H),8.28(d,J=8.5Hz,1H),8.12

[0057] (d,J=8.8Hz,1H),8.09(d,J=8.2Hz,1H),7.77(d,J=8.8Hz,1H),7.71(t,J=8.0Hz,1H),7.54(t,J =7.5Hz, 1H), 6.39 (d, J = 13.9Hz, 1H), 4.31 (q, J = 7.2Hz, 2H), 1.99 (s, 6H), 1.36 (t, J = 7.2Hz, 3H). 13C NMR(500MHz,DMSO-d6)δ173.71,153.50,139.77,133.72,131.77,130.84,130.40,129.11,128.2 0,128.11,126.01,125.23,122.59,119.41,111.88,103.01,51.20,27.17,12.94.MS(ESI):Calcd for(C 37 H 39 N2) + 511.31,Found511.36.

[0059] (3) Dye 1 (0.08 mg), dye 6 (0.3 mg) and F127 (25 mg) were dissolved in 1 mL THF. The mixed solution was quickly poured into 4 mL water and ultrasonicated for half an hour. After being evenly dispersed, the THF solution was removed by decompression to obtain the mother solution of the nanoprobe NPBC. The mother solution was stored in a refrigerator at 4°C for later use.

[0060] Figure 1 Nanoprobe NPBC and ONOO in blood - / ClO - After the response, the process goes from "zero" background fluorescence (FRET on) to high brightness (FRET off).

[0061] Figure 2 (a) is the normalized fluorescence spectrum of donor quenching blood autofluorescence, and (b) is the quantification of quenching efficiency. Figure 2 It can be seen that the donor has a good inhibitory effect on blood autofluorescence.

[0062] Example 2 Probe NPBC to ONOO in a buffer system - / ClO - Detection

[0063] 5 μM probe NPBC was mixed with ONOO - / ClO - The reaction system was shaken at room temperature for 100 minutes, and then its fluorescence emission and UV-visible absorption spectra were measured. The excitation wavelength of the fluorescence instrument was set to 600nm, and the emission wavelength receiving range was set to 620-800nm. The measurement range of the absorption spectrum was set to 500-800nm. The results are as follows: Figure 3 As shown, NPBC has an effect on ONOO - / ClO - Good response, achieving high signal-to-background ratio detection.

[0064] Figure 3(a) is the TEM image of the constructed nanoprobe NPBC, (b) is the DLS image of NPBC, Figure 3 It can be shown that the nanoprobe was successfully constructed.

[0065] Figure 4 (a) The nanoprobe NPBC and its effect on ONOO - (b) is the fluorescence response of NPBC to ONOO - The working curve from Figure 4 It can be seen that the nanoprobe NPBC has a great influence on ONOO - Has good fluorescence response.

[0066] Figure 5 (a) The nanoprobe NPBC shows its effect on ClO - (b) is the fluorescence response of NPBC to ClO - The working curve from Figure 5 It can be seen that the nanoprobe NPBC has a strong effect on ClO - Has good fluorescence response.

[0067] Example 3 Probe NPBC is used for ONOO in cells - / ClO - Detection

[0068] 5 μM probe was incubated in cells, and the confocal microscope was set to an excitation wavelength of 560 nm and an emission wavelength receiving range of 595-640 nm. Figure 4 As shown, the nanoprobe NPBC can detect ONOO in cells. - / ClO - The fluctuations have a good response.

[0069] Figure 6 Nanoprobe NPBC for ONOO in cells - / ClO - Confocal imaging of (a) HepG2 cells under APAP stimulation and NAC inhibition. (b) Confocal imaging of RAW264.7 cells under LPS stimulation and NAC inhibition. Figure 6 It can be seen that the nanoprobe NPBC can be used to detect ONOO in cells under drug stimulation. - / ClO - Monitoring of content changes.

[0070] Example 4 Probe NPBC for ONOO in whole blood of septic mice - / ClO - Content detection

[0071] 15 μM NPBC was added to a 10% whole blood PBS solution, and the fluorescence instrument was set to an excitation wavelength of 600 nm and an emission wavelength receiving range of 620-800 nm. Figure 8 As shown, the probe NPBC is sensitive to ONOO in whole blood. - / ClO - There is a good response, and the fluorescence intensity of NPBC gradually increases with the increase of sepsis degree, indicating that the nanoprobe can be used to monitor the progress of sepsis.

[0072] Figure 7 (a) is the overlapped graph of the absorption spectrum of the nanoprobe NPBC and the blood autofluorescence spectrum, and (b) is the quenching effect of the nanoprobe on blood autofluorescence. Figure 7 It can be seen that the nanoprobe has a good inhibitory effect on blood autofluorescence.

[0073] Figure 8 Nanoprobe NPBC for ONOO in whole blood of septic mice - / ClO - Monitoring of content (a), fluorescence response diagram of nanoprobe NPBC in whole blood with different degrees of sepsis. (b), quantification of (a) and comparison with the detection results of ELISA method, from Figure 8 It can be seen that the nanoprobe can effectively monitor the disease progression of sepsis.

[0074] Example 5 Probe NPBC for hemolytic mouse whole blood ONOO - / ClO - Content detection

[0075] 15 μM NPBC was added to a 10% whole blood PBS solution, and the fluorescence instrument was set to an excitation wavelength of 600 nm and an emission wavelength receiving range of 620-800 nm. Fig. 9 As shown, the probe NPBC is sensitive to ONOO in whole blood. - / ClO - There is a good response, and the fluorescence intensity of NPBC gradually increases with the increase of hemolysis degree, indicating that the nanoprobe can be used to monitor the progress of hemolytic diseases.

[0076] Fig. 9 Nanoprobe NPBC was used to hemolyze ONOO in whole blood of mice - / ClO - Monitoring of the content. (a) Establishment of hemolytic mouse model. (b) Fluorescence response of nanoprobe NPBC in whole blood of mice with different degrees of hemolysis. (c) Quantification of (b). (d) Comparison of ELISA test results. Fig. 9 It can be seen that the nanoprobe can effectively monitor the progression of hemolytic diseases.

[0077] The present invention discloses a "one-to-many" integrated sensing strategy based on FRET and blood autofluorescence suppression, and designs a multifunctional whole blood detection fluorescent nanoprobe. The FRET-based probe exhibits extremely low background fluorescence and can effectively suppress blood autofluorescence, reliably detecting endogenous ONOO in whole blood. - / ClO - The donor of the FRET system can effectively suppress the autofluorescence of blood based on the inner filter effect, and by adjusting the ratio of the acceptor to the donor, the fluorescence of the donor can be effectively quenched based on the FRET effect, thereby achieving the "zero background" of the whole blood detection system. The nanoprobe NPBC has high sensitivity and signal-to-background ratio. - / ClO - Reliable monitoring of levels showed that these levels were positively correlated with disease severity, which was confirmed by ELISA results. Importantly, this method can more accurately monitor endogenous ONOO in whole blood compared to traditional plasma testing. - / ClO - The present invention proposes a simple and effective new sensing strategy for sensitive and reliable detection of whole blood, which will promote the application of fluorescent probes in blood testing and further promote blood diagnosis.

[0078] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A method for detecting ONOO - / ClO - The low-background nanoprobe is characterized by: The nanoprobe NPBC is composed of two fluorescent dyes: a donor and an acceptor. The structural formulas of the donor and the acceptor are as follows:

2. The method for detecting ONOO according to claim 1. - / ClO - The low-background nanoprobe is characterized by: The molar ratio of donor to acceptor is 1:(3-6).

3. The method for detecting ONOO according to claim 1 or 2. - / ClO - A method for preparing a low-background nanoprobe, characterized in that: The following steps are involved: (1) Ethyl bromoacetate and p-hydroxybenzaldehyde are dissolved in acetone, potassium carbonate is added, reacted at a set temperature, the mixture is concentrated under reduced pressure, and extracted and purified again to obtain compound 1; (2) Dissolve 2,4-dimethylpyrrole in dichloromethane, add compound 1 at a predetermined temperature, add a drop of trifluoroacetic acid as a catalyst, and then add triethylamine and boron trifluoride ether solution in sequence, stir to react at room temperature, concentrate the mixture under reduced pressure, and obtain compound 2 after extraction and purification; (3) Compound 2 and p-methoxybenzaldehyde are dissolved in anhydrous acetonitrile, acetic acid, piperidine and a small amount of molecular sieves are added, and the mixture is refluxed at a set temperature under the protection of an inert atmosphere, and the mixture is concentrated under reduced pressure. After extraction and purification, dye 1 is obtained; (4) adding 1,1,2-trimethyl-1H-benz[e]indole and iodoethane into a pressure tube, using acetonitrile as a solvent, reacting at a predetermined temperature, and washing after the reaction to obtain compound 3; (5) N-(3-phenylamino-2-propyleneylidene)aniline hydrochloride and compound 3 are mixed under an inert atmosphere, sodium acetate is added, and the mixture is refluxed in anhydrous acetonitrile to obtain dye 6; (6) dissolving dye 1 and dye 6 in THF, adding F127, adding the mixed solution into water after dissolution, ultrasonically treating, and removing THF by vacuum rotary evaporation to obtain the nanoprobe NPBC; The reaction process is as follows:

4. The method for detecting ONOO according to claim 3. - / ClO - A method for preparing a low-background nanoprobe, characterized in that: In step (1), the molar ratio of ethyl bromoacetate to p-hydroxybenzaldehyde is 1:(1.1-1.3), which is easy for subsequent purification.

5. The method for detecting ONOO according to claim 3. - / ClO - A method for preparing a low-background nanoprobe, characterized in that: In step (2), the molar ratio of 2,4-dimethylpyrrole solution, compound 1, triethylamine and boron trifluoride etherate is (2-2.2):1:(3.6-4):(6.6-7).

6. The method for detecting ONOO according to claim 3. - / ClO - A method for preparing a low-background nanoprobe, characterized in that: In step (3), the molar ratio of p-anisaldehyde to compound 2 is (2.5-3):1, and the reflux reaction time is 2-3 hours.

7. The method for detecting ONOO according to claim 3. - / ClO - A method for preparing a low-background nanoprobe, characterized in that: In step (4), the molar ratio of 1,1,2-trimethyl-1H-benzo[e]indole to ethyl iodide is 1:(1.2-1.5).

8. The method for detecting ONOO according to claim 3. - / ClO - A method for preparing a low-background nanoprobe, characterized in that: In step (5), the molar ratio of N-(3-phenylamino-2-propyleneylidene)aniline hydrochloride to compound 3 is 1:(2.2-2.5).

9. The method for detecting ONOO according to claim 3. - / ClO - A method for preparing a low-background nanoprobe, characterized in that: In step (6), the molar ratio of dye 1 to dye 6 is 1:(4-6), and the mass ratio of dye 1 to F127 is 1:(200-400).

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