Large Stokes shift near-infrared fluorescent probe for detecting Hg < 2 + > as well as preparation method and bioimaging application of large Stokes shift near-infrared fluorescent probe

Abstract

The invention discloses a large Stokes shift near-infrared fluorescent probe for detecting Hg < 2 + > as well as a preparation method and bioimaging application of the large Stokes shift near-infrared fluorescent probe. According to the invention, a conjugated system is expanded by introducing a quinoline structure, the emission wavelength is red-shifted to a near-infrared region (730nm), and the Stokes shift is up to 170nm, so that the interference of background fluorescence in bioimaging application can be greatly reduced. In the process of identifying Hg < 2 + > by the probe, S atoms in a sulfo-spirolactone structure are promoted to be combined with Hg < 2 + > to form HgS by virtue of the high sulfur-loving characteristic of Hg < 2 + >, so that a spirolactone ring is opened and fluorescence is generated. The probe has the advantages of fast response time (3s), low detection limit (53nM) and specific recognition capability on Hg < 2 + >. The fluorescent probe disclosed by the invention has relatively good biocompatibility and relatively low cytotoxicity, and can be applied to evaluating the drug effect of a mercury poisoning antidote in a 4T1 cell model and monitoring the change of the Hg < 2 + > level in an acute mercury poisoning mouse model in real time.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological analysis, and particularly relates to a Hg 2+ large Stokes shift near-infrared fluorescent probe, a preparation method thereof and biological imaging application. BACKGROUND

[0002] Mercury and its compounds have extremely high toxicity and bioaccumulation, can accumulate in the ecosystem and human body, and thus pose a serious threat to the ecological environment and public health, and are regarded as one of the most toxic heavy metal pollutants. Hg 2+ is mainly in the form of inorganic mercury and organic mercury and widely distributed in nature, acute mercury poisoning is caused by the intake of a large dose of Hg 2+ in the body, Hg 2+ can enter the body through the digestive tract, respiratory tract or skin and rapidly distribute to various organs of the whole body, leading to dysfunction of important organs such as the kidney, liver and central nervous system. The typical symptoms in clinic include headache, nausea, vomiting and liver and kidney damage, etc. Therefore, closely monitoring the fluctuation of Hg 2+ is crucial for maintaining the normal function of each organ and biological system.

[0003] At present, the diagnosis of acute mercury poisoning in clinic mainly relies on the detection of mercury content in biological samples (such as urine, blood, etc.). Since acute mercury poisoning is often accompanied by nervous system damage and liver and kidney function damage, the disease can also be diagnosed by observing the changes of biochemical indicators such as serum creatinine, alanine aminotransferase, aspartate aminotransferase and catecholamine. However, these methods have certain limitations. Although biological sample detection can reflect the mercury exposure level, it cannot monitor the dynamic changes of toxicity in real time. At the same time, biochemical indicators are easily interfered by other diseases and lack specificity. Compared with these diagnostic methods, optical imaging technology has shown significant application potential in the field of disease analysis and detection due to its high sensitivity, real-time monitoring capability and non-invasive nature. In particular, near-infrared fluorescence has obvious advantages in biological imaging and treatment due to its strong tissue penetration ability and low background interference. In order to achieve early diagnosis and efficacy monitoring of acute mercury poisoning, it is of great clinical significance to design and develop a near-infrared fluorescent probe capable of specifically recognizing Hg 2+ at the cellular and in vivo levels. Although there are currently a variety of near-infrared fluorescent probes for Hg 2+ detection, there are generally problems such as small Stokes shift, low signal-to-noise ratio, etc., leading to overlapping of excitation and emission spectra and self-absorption phenomenon, and there are also problems such as sensitivity and selectivity to be further improved, which limit the application effect in deep tissue imaging. SUMMARY

[0004] In the face of the above challenges, it is urgent to develop new fluorescent probes with large stokes shift and near-infrared emission, to provide more reliable technical means for early diagnosis and dynamic monitoring of acute mercury poisoning by improving signal resolution and detection sensitivity. In order to solve the technical problems existing in the fluorescent probes for detecting Hg 2+ , the present application provides a large stokes shift near-infrared fluorescent probe for detecting Hg 2+ , a preparation method thereof and biological imaging application. Based on the high thioaffinity of Hg 2+ and the strong nucleophilicity of sulfur atom, a large stokes shift oxygen heterocyclic quinoline near-infrared fluorescent probe XQ-S is synthesized from a thiolactone structure. The probe has near-infrared luminescence characteristics, a stokes shift of 170nm, can quantitatively detect Hg 2+ , has good selectivity, high sensitivity, and a detection limit as low as 53nM, is not only successfully applied to real-time imaging of exogenous Hg 2+ in 4T1 cells, but also can realize dynamic monitoring of Hg 2+ level in a mouse acute mercury poisoning model.

[0005] In order to achieve the above purpose, the technical scheme of the present application is to provide a large stokes shift near-infrared fluorescent probe for detecting Hg 2+ , the molecular formula of the fluorescent probe is C 34 H 30 N2O2S, and the molecular structure formula is as follows:

[0006]

[0007] On the other hand, the technical scheme of the present application is to provide a preparation method of the fluorescent probe, comprising the following steps:

[0008] (1) cyclohexanone is added dropwise into concentrated sulfuric acid, 4-diethylamino ketonic acid is added under stirring condition, high chloride acid is added after reaction, a precipitate is precipitated, the precipitate is filtered, washed and dried to obtain an intermediate M1;

[0009] (2) the intermediate M1 and 8-quinoline formaldehyde are dissolved in acetic acid, heated to react, the pH is adjusted to neutral, a precipitate is precipitated, the precipitate is filtered, washed and dried to obtain an intermediate M2;

[0010] (3) the intermediate M2 is dissolved in anhydrous dichloromethane, oxalyl chloride is added, the solvent is removed after reaction, and then dissolved in anhydrous dichloromethane, saturated Na2S solution is added, separated and purified after reaction to obtain the fluorescent probe.

[0011] Further, the molar ratio of cyclohexanone to 4-diethylamino ketonic acid is 1:1-1:1.5.

[0012] Further, the molar ratio of the intermediate M1 to 8-quinoline formaldehyde is 0.95:1-0.95:1.2.

[0013] Further, the molar ratio of the intermediate M2 to oxalyl chloride is 1:3-1:5.

[0014] Further, the reaction temperature of step (1) is 90 DEG C, and the time is 2.5-3.5 h.

[0015] Further, the reaction temperature of step (2) is 80 DEG C, and the time is 2-3 h.

[0016] Further, the reaction temperature of step (3) is room temperature, and the time is 4-5 h.

[0017] In another aspect, the technical scheme of the present application provides application of the fluorescent probe in detecting Hg 2+ , wherein the minimum detection limit of Hg 2+ is 53 nM, and the concentration of the fluorescent probe is 10 μM.

[0018] Further, the fluorescent probe is applied to detection imaging of the concentration level of Hg 2+ in living cells or living bodies.

[0019] The synthesis route is as follows:

[0020]

[0021] The principle of the fluorescent probe of the present application is that: since mercury ions have strong thioaffinity, they easily attack the sulfur atom in the thiospirolactone; subsequently, the complexation of Hg 2+ causes hydrolysis and rupture of the spirolactone bond, releases the intermediate M2, and is accompanied by recovery of fluorescence, thereby realizing effective detection of Hg 2+ ions.

[0022] Compared with the prior art, the present application has the following advantages and technical effects:

[0023] 1. The present application designs and synthesizes a large Stokes shift near-infrared fluorescent probe XQ-S for detecting Hg 2+ , and the Stokes shift is as high as 170 nm, which effectively reduces background interference.

[0024] 2. The concentration of the fluorescent probe prepared in the present application is 10 μM when detecting Hg 2+ , the fluorescence intensity of the fluorescent probe at the maximum emission peak in the present application has a good linear relationship with the Hg 2+ concentration in the range of 0-48 μM, and the detection limit is as low as 53 nM through calculation, which indicates that the fluorescent probe XQ-S prepared in the present application can be used as a chemical sensor for quantitative detection of Hg2+ .

[0025] 3、The prepared fluorescent probe has a maximum emission wavelength of 730nm, is located in the near-infrared region, has strong penetrability, small damage to a biological sample, and good stability, and the fluorescence intensity is stable in a wide pH range such as acid, neutral, and alkaline.

[0026] 4、The fluorescent probe is not interfered by other competitive ROS / thiol compounds / metal ions in the detection process, so that the fluorescent probe XQ-S has high selectivity and good anti-interference ability for Hg 2+ .

[0027] 5、The fluorescent probe has a rapid response time, the fluorescence signal at 730nm gradually increases with time, and reaches an equilibrium state after 3s, which shows that the probe XQ-S has a rapid response ability for Hg 2+ .

[0028] 6、The fluorescent probe provided by the application has almost no toxicity to cells, and has been successfully applied to imaging of exogenous Hg 2+ in cells and evaluation of drug efficacy of mercury poisoning antidote. Especially importantly, XQ-S also realizes visual monitoring of Hg 2+ in a mouse body caused by a drug for acute mercury poisoning, provides a technical basis for diagnosis and treatment of Hg 2+ related diseases in a biological sample and a living biological system, and has good practicability.

[0029] 7、The preparation method is simple, raw materials are cheap and easy to obtain, is easy to popularize in technology, and has good social and economic benefits. DETAILED DESCRIPTION

[0030] The drawings constituting a part of the application are used to provide further understanding of the application, the illustrative embodiments of the application and the description thereof are used to explain the application, and do not constitute improper limitation on the application. In the drawings:

[0031] Figure 1 A molecular structure diagram of the fluorescent probe XQ-S prepared in Example 1.

[0032] Figure 2 A HNMR diagram of the fluorescent probe XQ-S prepared in Example 1. 1

[0033] Figure 3 A CNMR diagram of the fluorescent probe XQ-S prepared in Example 1. 13

[0034] Figure 4 A MS diagram of the fluorescent probe XQ-S prepared in Example 1. ​​

[0035] Figure 5 UV-Vis absorption spectra of fluorescent probe XQ-S (10 μΜ) prepared in Example 1 in MeCN / Hepes (1 :1, v / v, pH=7.4) buffer system before and after adding Hg 2+ (100 μΜ).

[0036] Figure 6 Fluorescence spectra of fluorescent probe XQ-S (10 μΜ) prepared in Example 1 in MeCN / Hepes (1 :1, v / v, pH=7.4) buffer system before and after adding Hg 2+ (100 μΜ).

[0037] Figure 7 Normalized absorption and emission spectra of fluorescent probe XQ-S (10 μΜ) prepared in Example 1 in MeCN / Hepes (1 :1, v / v, pH=7.4) buffer system after reacting with Hg 2+ (100 μΜ); Abs. represents UV, FL. represents fluorescence.

[0038] Figure 8 UV-Vis absorption spectra of fluorescent probe XQ-S (10 μΜ) prepared in Example 1 in MeCN / Hepes (1 :1, v / v, pH=7.4) buffer system after adding Hg 2+ (0-100 μΜ).

[0039] Figure 9 Fluorescence emission titration spectra of fluorescent probe XQ-S (10 μΜ) prepared in Example 1 in MeCN / Hepes (1 :1, v / v, pH=7.4) buffer system after adding Hg 2+ (0-100 μΜ).

[0040] Figure 10 Linear fitting curve between fluorescence intensity of fluorescent probe XQ-S (10 μΜ) prepared in Example 1 at 730 nm and Hg 2+ concentration.

[0041] Figure 11 Fluorescence intensity changes of fluorescent probe XQ-S (10 μΜ) prepared in Example 1 in MeCN / Hepes (1 :1, v / v, pH=7.4) buffer system after reacting with different analytes (100 μΜ).

[0042] Figure 12 Competitive test chart of fluorescent probe XQ-S (10 μΜ) prepared in Example 1.

[0043] Figure 13Time-dependent fluorescence intensity change curves of fluorescent probe XQ-S (10 μM) prepared in Example 1 in MeCN / Hepes (1:1, v / v, pH = 7.4) buffer system before and after adding Hg 2+ (100 μM).

[0044] Figure 14 Fluorescence intensity change curves of fluorescent probe XQ-S (10 μM) prepared in Example 1 in different pH environments before and after adding Hg 2+ (100 μM) at 730 nm.

[0045] Figure 15 Cytotoxicity of fluorescent probe XQ-S prepared in Example 1 to 4T1 cells.

[0046] Figure 16 Fluorescence imaging (a) and three-dimensional quantitative analysis (b) of fluorescent probe XQ-S prepared in Example 1 to different concentrations of Hg 2+ in 4T1 cells, λ ex = 568 nm.

[0047] Figure 17 Fluorescence imaging (a) and three-dimensional quantitative analysis (b) of fluorescent probe XQ-S prepared in Example 1 to Hg 2+ treated by different concentrations of Na2SeO3 in 4T1 cells, λ ex = 568 nm.

[0048] Figure 18 Fluorescence imaging of fluorescent probe XQ-S prepared in Example 1 to exogenous Hg 2+ in live mice. (a) Subcutaneous injection of probe XQ-S (100 μL, 50 μM) in the left lower limb; (b) subcutaneous injection of Hg 2+ (50 μL, 1 mM) in the left lower limb, and in situ injection of probe XQ-S (50 μL, 100 μM) after 5 min. (c) Relative fluorescence intensity of groups a and b. λ ex = 560 nm, λ em = 710 nm.

[0049] Figure 19 Fluorescence imaging of fluorescent probe XQ-S prepared in Example 1 to Hg 2+ in an acute mercury poisoning mouse model. (a) Normal mouse tail vein injection of probe XQ-S (100 μL, 200 μM); (b-f) Acute mercury poisoning mouse model, fluorescence imaging was performed at 10, 30, 40, 50, and 60 min after tail vein injection of probe XQ-S (100 μL, 200 μM). (g) Relative fluorescence intensity of groups a-f. λ ex = 560 nm, λem = 710 nm. DETAILED DESCRIPTION

[0050] As used herein, the terms "comprise", "comprising", "include", "including", "have", "having" and the like are open-ended terms that are intended to mean including, but not limited to.

[0051] The raw materials used in the embodiments of the present application are all commercially available.

[0052] The technical solutions of the present application are further illustrated by the following examples.

[0053] Example 1: Preparation of fluorescent probe XQ-S

[0054] (1) Under ice-bath conditions, cyclohexanone (9.6 mM) was added dropwise into 98% concentrated sulfuric acid (20.0 mL), and then 4-diethylamino ketonic acid (9.6 mM) was added in batches under vigorous stirring. After the addition was completed, it was first reacted at 90°C for 2.5 h, cooled and poured into crushed ice (150.0 g), and then a 70% mass fraction of a perchloric acid solution (2.0 mL) was added, immediately a red precipitate appeared, which was filtered and washed with cold water for 3 times, and then dried in air to obtain a red solid, which was intermediate M1;

[0055] (2) Intermediate M1 (5.37 mM) and 8-quinoline formaldehyde (5.64 mM) were dissolved in acetic acid (10.0 mL), and then reacted at 80°C for 2 h under reflux, the color of the solution changed from reddish brown to purple. The reaction solution was cooled to room temperature and poured into distilled water (100.0 mL). The pH was adjusted to neutral with saturated sodium bicarbonate, and a purple precipitate was precipitated under stirring, which was filtered and washed with saturated NaCl for 2 times, and then dried in air to obtain a purple solid, which was intermediate M2;

[0056] Preparation of intermediate M2 1 The HNMR spectrum information is as follows: 1H NMR (400 MHz, Chloroform-d) δ 1.15 (t, J = 7.0 Hz, 6H), 1.59 - 1.70 (m, 3H), 2.07 - 2.16 (m, 1H), 2.62 - 2.80 (m, 2H), 3.34 (q, J = 7.1 Hz, 4H), 6.34 (dd, J = 8.9, 2.5 Hz, 1H), 6.49 (d, J = 8.7 Hz, 2H), 7.23 (d, J = 7.7 Hz, 1H), 7.46 (dd, J = 8.3, 4.2 Hz, 1H), 7.55 (dt, J = 9.6, 7.6 Hz, 2H), 7.61 - 7.72 (m, 2H), 7.78 (d, J = 8.1 Hz, 1H), 7.95 (d, J = 7.7 Hz, 1H), 8.17 - 8.26 (m, 2H), 9.04 (dd, J = 4.3, 1.8 Hz, 1H).

[0057] Intermediate M2 13 CNMR spectral information is as follows: 13 C NMR (101 MHz, Chloroform-d) δ 170.20, 152.95, 152.22, 149.89, 149.23, 147.05, 136.45, 136.08, 134.47, 131.83, 130.28, 129.14, 128.42, 127.55, 127.30, 126.00, 124.82, 123.54, 122.82, 121.22, 108.67, 107.76, 104.81, 97.84, 87.01, 44.39, 27.73, 23.16, 22.54, 12.59.

[0058] (3) Intermediate M2 (1.16 mM) was dissolved in anhydrous dichloromethane (15.0 mL), and oxalyl chloride (3.49 mM) was added at room temperature. After stirring at room temperature for 5 h, the solvent was removed by a rotary evaporator. The obtained acyl chloride compound solid was redissolved in anhydrous dichloromethane (10.0 mL), and saturated Na2S aqueous solution (4.0 mL) was slowly added, and stirred at room temperature for 4 h. After the reaction was completed, the reaction solution was poured into distilled water, extracted with ethyl acetate for 3 times, and the combined organic phase was dried over anhydrous Na2SO4, filtered, and the solvent was removed by a rotary evaporator to obtain a yellow crude product. The crude product was further purified by silica gel column chromatography (petroleum ether: ethyl acetate = 30: 1 ~ 6: 1, v / v), and finally a light yellow solid was obtained, which was a large Stokes shift near-infrared fluorescent probe (XQ-S), and its molecular structural formula is shown in Figure 1 .

[0059] Intermediate M2 1 H NMR spectral information is as follows (as shown inFigure 2 shown): 1 H NMR(400MHz,Chloroform-d)δ1.15(t,J=7.0Hz,6H),1.68(dd,J=16.3,3.3Hz,3H),2.24(dt,J=12.3,6.1Hz ,1H),2.70(dq,J=15.6,8.8,8.0Hz,2H),3.32(q,J=7.1Hz,4H),6.31(dd,J=8.9,2.6Hz,1H),6.39(d,J=2.5 Hz,1H),6.59(d,J=8.9Hz,1H),7.36(d,J=7.8Hz,1H),7.44-7.50(m,2H),7.58(dt,J=15.7,7.6Hz,2H),7.6 8(d,J=7.1Hz,1H),7.77(d,J=8.1Hz,1H),7.83(d,J=7.7Hz,1H),8.12-8.22(m,2H),9.04(d,J=4.2Hz,1H).

[0060] Fluorescent probe XQ-S 13 C NMR spectrum information is as follows (such as Figure 3 shown): 13 C NMR(101MHz,Chloroform-d)δ197.61,155.98,151.84,149.87,148.43,147.04,145.81,136.46,136.23,134.06,131.94,130.21,129.48,1 28.42,128.31,127.17,126.70,126.00,122.80,121.82,121.20,110. 10,108.70,107.41,97.87,68.69,44.32,28.00,26.59,22.87,12.65.

[0061] MS spectrum information of fluorescent probe XQ-S (such as Figure 4 (shown): m / z: 531.20963[C 34 H 30 N2O2S+H] + .

[0062] Example 2: Preparation of fluorescent probe XQ-S

[0063] The method of this embodiment is basically the same as that of embodiment 1, except that:

[0064] In step (1), the molar ratio of cyclohexanone (9.6 mM) to 4-diethylamino keto acid is 1:1.5.

[0065] In step (2), the molar ratio of intermediate M1 (5.37 mM) to 8-quinoline formaldehyde was 0.95:1.2.

[0066] In step (3), the molar ratio of intermediate M2 (1.16 mM) to oxalyl chloride was 1:5.

[0067] In step (1), the reaction temperature was 90℃, and the time was 3.5 h.

[0068] In step (2), the reaction temperature was 80℃, and the time was 3 h.

[0069] In step (3), the first reaction time was 4 h, and the second reaction time was 5 h.

[0070] Control Example

[0071] An enhanced fluorescence probe FS-Hg (application publication number: CN 117720549A) that specifically recognizes mercury ions and has a similar skeleton to the skeleton of the present application was selected as a control example. The molecular formula thereof is C 34 H 35 N6O2S, and the molecular structural formula thereof is:

[0072]

[0073] An enhanced fluorescence probe RANS [reference: Journal of Molecular Structure 1254 (2022) 132312] that specifically recognizes mercury ions and has a similar skeleton to the skeleton of the present application was selected as a control example. The molecular formula thereof is C 37 H 37 N5O2S, and the molecular structural formula thereof is:

[0074]

[0075] An enhanced fluorescence probe probe 1 [reference: Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 311 (2024) 123999] that specifically recognizes mercury ions and has a similar skeleton to the skeleton of the present application was selected as a control example. The molecular structural formula thereof is:

[0076]

[0077] Performance test of fluorescence probe XQ-S:

[0078] I. Ultraviolet absorption and fluorescence emission experiment of fluorescence probe XQ-S prepared in Example 1

[0079] The fluorescent probe XQ-S mother liquor was prepared with dimethyl sulfoxide at a concentration of 2 mM, and the mercury ion mother liquor was prepared with distilled water at a concentration of 20 mM. 3 mL of acetonitrile (MeCN) / Hepes (4-hydroxyethylpiperazine ethanesulfonic acid) (1:1, v / v, pH = 7.4) buffer solution was taken in a cuvette, 15 μL of fluorescent probe mother liquor was added, so that the test final concentration of fluorescent probe XQ-S was 10 μM, and the ultraviolet-visible absorption light and fluorescence spectrum were measured. Then 15 μL of mercury ion metal mother liquor was added, so that Hg 2+ The actual test final concentration was 100 μM, and the ultraviolet-visible absorption light and fluorescence spectrum were measured after mixing well. The ultraviolet-visible absorption spectra of fluorescent probe XQ-S (10 μM) before and after adding Hg 2+ (100 μM) are shown in Figure 5 , and the fluorescence spectra before and after adding Hg 2+ (100 μM) are shown in Figure 6 , and the normalized plots of absorption and fluorescence spectra after Hg 2+ (100 μM) response are shown in Figure 7 .

[0080] 3 mL of MeCN / Hepes (1:1, v / v, pH = 7.4) buffer solution was taken in a cuvette, 15 μL of fluorescent probe mother liquor (2 mM) was added, and different concentrations of Hg 2+ (0-100 μM) were added in turn, and mixed well for ultraviolet and fluorescence test. The ultraviolet-visible absorption spectrum is shown in Figure 8 , the fluorescence emission titration spectrum is shown in Figure 9 , and the emission intensity of fluorescent probe XQ-S (10 μM) at 730 nm has a linear relationship with Hg 2+ final concentration (0-48 μM) is shown in Figure 10 .

[0081] As shown in Figures 5-10 , the emission wavelength of fluorescent probe XQ-S is in the near-infrared region, the stoke shift reaches 170 nm, and the fluorescence intensity at the maximum emission peak has a good linear relationship with Hg 2+ concentration in the range of 0-48 μM, and the detection limit is as low as 53 nM. In addition, during the reaction of probe XQ-S with Hg 2+ , the color of the solution changes from colorless to purple, which is due to the strong thiofilicity of Hg 2+ which combines with S in the structure of the probe to generate HgS, the lactam ring is opened, and the fluorescence is recovered.

[0082] The above fluorescence test uses a steady-state / transient fluorescence spectrometer to detect the probe XQ-S. The quantitative fluorescence detection parameters are: the excitation wavelength is 560 nm, the emission spectrum scanning range is 590-900 nm, and the excitation and emission slit widths are both 6 nm.

[0083] II. The fluorescence probe XQ-S prepared in Example 1 has high selectivity for Hg 2+ Fluorescence detection selectivity

[0084] In order to evaluate the specific recognition ability of the probe XQ-S for Hg 2+ , we carried out a selectivity experiment, and recorded the fluorescence intensity changes of the probe in the presence of different analytes. The selected analytes are common reactive oxygen species ROS / thiol compounds / metal ions (H2O2, HCIO, GSH, Cys, Hcy, Ni 2+ , Cu 2+ , Cr 3+ , Al 3+ , Fe 2+ , Cd 2+ , Mg 2+ , Co 2+ , Hg 2+ , K + , Zn 2+ , Pb 2+ , Fe 3+ , Mn 2+ , Ag + , Ca 2+ ). First, a 2 mM fluorescence probe XQ-S stock solution was prepared using dimethyl sulfoxide, and various metal ion, reactive oxygen species, and biological thiol aqueous solutions (stock solutions) were prepared at a concentration of 20 mM using distilled water. Next, 3 mL of MeCN / Hepes (1:1, v / v, pH=7.4) buffer solution was taken in a cuvette, and 15 μL of the fluorescence probe stock solution and 15 μL of other analyte stock solutions were added in sequence, so that the actual test concentrations of the fluorescence probe XQ-S and other analytes were 10 μM and 100 μM, respectively. The mixture was mixed well and used for fluorescence testing, and the same concentration of Hg 2+ was added as a control. As shown in Figure 11 , the fluorescence signal changes caused by other metal ions, reactive oxygen species, and biological thiol compounds are negligible. Only when Hg 2+ is added to the probe solution, a significant fluorescence enhancement phenomenon is observed, thereby verifying the high selectivity of the probe XQ-S for Hg 2+ . In addition, we used a test strip detection method. The test strip was first treated by immersing it in a buffer solution containing the probe XQ-S, and then exposed to various analytes including Hg 2+ . By visual observation, we found that only the test strip immersed in Hg 2+The color of the test strip in solution changed from purple. This further proved the specific recognition of probe XQ-S to Hg 2+ .

[0085] Three, other common active oxygen / amino acid / metal ion interference test of fluorescent probe XQ-S detection of Hg 2+

[0086] Based on the above experimental results, we continue to carry out the competition experiment. First prepare the probe XQ-S mother liquor (2mM), and mercury ion mother liquor (100μM). Take 3mL MeCN / Hepes (1:1, v / v, pH=7.4) buffer solution in a colorimetric tube, and then add the corresponding test substance (metal ion, active oxygen and biological thiol) to it, the final concentration is 100μM, then add 15μL of probe mother liquor, test, and then add mercury ion mother liquor, test. The results are shown in Figure 12 , in the presence of other analyte, the fluorescence intensity of the probe at 730nm is weak. However, after adding Hg 2+ to the reaction system, the fluorescence signal at 730nm is sharply enhanced, indicating that other coexisting analyte will not interfere with the ability of probe XQ-S to detect Hg 2+ , and the probe XQ-S shows good anti-interference ability.

[0087] Four, response time determination of fluorescent probe XQ-S to Hg 2+

[0088] The response time of the probe is one of the key parameters for evaluating its performance, which is crucial for practical application. Therefore, we investigated the response time change of probe XQ-S to Hg 2+ in MeCN / Hepes (1:1, v / v, pH=7.4) buffer system. Prepare a test solution of probe XQ-S with a concentration of 10μM. In the experiment, the excitation wavelength of probe XQ-S is determined to be 560nm, and the intensity of the emission spectrum is recorded at 730nm. Pause when the test instrument works for 10s, add mercury ion with a final concentration of 100μM to the test solution, and then turn on the test instrument to continue the spectrum scan. Then arrange the recorded emission intensity data and draw the response time spectrum diagram of probe XQ-S to mercury ion detection. The results are shown in Figure 13 , the fluorescence signal of probe XQ-S at 730nm has high stability, indicating that the probe can exist stably in the buffer solution. Subsequently, Hg 2+ is added to the probe solution (final concentration 100μM), we observe that the fluorescence signal at 730nm gradually enhances with time, and reaches equilibrium state after 3s. This phenomenon indicates that probe XQ-S has fast response ability to Hg 2+ .​​

[0089] 5. Effect of pH on Hg Detection by Fluorescent Probe XQ-S 2+ Impact

[0090] We studied the stability of the probe XQ-S under different pH conditions and its detection of Hg 2+ The ability of the probe XQ-S was studied. First, Hepes buffer and acetonitrile were fully mixed in a volume ratio of 1:1. Then, a suitable acid-base regulator was added to adjust the pH value of the mixed solution to the range of 4.0-9.0. Then, the probe XQ-S and mercury ions were added to test the reaction between the probe XQ-S (10μM) and mercury ions (100μM). The excitation wavelength was selected as 560nm, and the emission signal intensity was recorded at a wavelength of 730nm. The data was then sorted and plotted into a pH dependence graph of the probe XQ-S for mercury ion detection. The results are shown in the figure. Figure 14 As shown in the figure, the fluorescence intensity of the probe XQ-S remained relatively stable when the solution pH was in the range of 4.0-9.0. 2+ After that, the probe XQ-S showed a significant fluorescence signal enhancement phenomenon in a wide pH range (pH = 6.0-8.0). These experimental results show that the probe XQ-S can effectively detect Hg under physiological conditions. 2+ , which has the potential for direct application in the field of biology.

[0091] VI. Cytotoxicity test of fluorescent probe XQ-S on 4T1 cells

[0092] In order to verify the XQ-S probe's ability to detect Hg in living cells, 2+ To investigate the applicability of the probe for fluorescence imaging, we first evaluated the cytotoxicity of the probe in 4T1 cells using the MTT assay. 4T1 cells were incubated with different concentrations of the probe XQ-S (0-20 μM) for 24 h, and the cell survival rate was then determined at each concentration. Figure 15 As shown, even at the highest concentration of 20 μM, the cell viability was still maintained above 84.82%. This indicates that the probe XQ-S has less cytotoxicity and is suitable for Hg in cells. 2+ Perform fluorescence imaging studies.

[0093] VII. Imaging experiment of living cells using fluorescent probe XQ-S

[0094] In view of the excellent biocompatibility of the probe XQ-S, we selected 4T1 cells as an experimental model to further investigate the ability of the probe to detect and image Hg in cells. 2+ The experiment was conducted under a STELLARIS 5 laser confocal microscope using a 568nm laser. 2+The results are as follows Figure 16 As shown in Figure 3, cells treated with probe XQ-S alone only captured weak fluorescence signals. In contrast, when cells were exposed to different concentrations of exogenous Hg 2+ After the addition of the probe, the red fluorescence signal in 4T1 cells showed an obvious concentration-dependent enhancement trend, which strongly confirmed the effectiveness of the probe XQ-S for Hg in living cells. 2+ Feasibility of imaging.

[0095] Based on the above results, this study evaluated the efficacy of mercury poisoning antidote at the cellular level. 2+ Therefore, this experiment selected Na2SeO3 to alleviate the damage caused by mercury poisoning. Figure 17 As shown, when Hg is used at a concentration of 100 μM 2+ After the cells were treated with Hg solution and then treated with probe XQ-S, a strong red fluorescence signal was observed. 2+ After incubation with different concentrations of Na2SeO3 (50μM, 100μM), the probe XQ-S was added, and the fluorescence signal in the red channel was observed to decrease in a concentration-dependent manner. The above results show that sodium selenite can effectively antagonize Hg 2+ The XQ-S probe is expected to become a potential effective tool for evaluating the efficacy of antidotes for mercury poisoning.

[0096] 8. Fluorescent Probe XQ-S for Detection of Acute Mercury Poisoning

[0097] We used the probe XQ-S to conduct in-depth Hg 2+ Detection and imaging of exogenous Hg concentration in nude mice 2+ Imaging was performed on the IVIS LuminaⅢ small animal in vivo imaging system (λ ex =560nm,λ em =710 nm) were used to perform fluorescence imaging on mice aged 5 to 6 weeks. 2+ The imaging effect in mice. Figure 18 As shown in the figure, in the control group mice injected with only the probe XQ-S (100 μL, 50 μM), the intensity of the in vivo fluorescence signal was weak. In contrast, the experimental group mice were first injected with Hg 2+Solution (50 μL, 1 mM), 5 min later, the probe XQ-S (50 μL, 100 μM) was injected. By small animal in vivo imaging system, we observed that the fluorescence intensity of the left lower limb region of the experimental mice was significantly enhanced. These results demonstrate that the probe XQ-S can efficiently detect and image Hg 2+ in vivo in live mice, and its near-infrared emission characteristics can effectively avoid the interference of background fluorescence in mice.

[0098] After confirming that the probe XQ-S can effectively detect exogenous Hg 2+ in mice in vitro, to further verify its application potential in the diagnosis of acute mercury poisoning, we constructed an acute mercury poisoning mouse model, and used the model to monitor the changes in Hg 2+ concentration in mice. After the model was successfully constructed, XQ-S (100 μL, 200 μM) was injected through the tail vein, and the changes in fluorescence signal at different time points were continuously monitored using a small animal in vivo imaging system. The results are shown in Figure 19 , in the control group of mice (a) that were intragastrically administered with physiological saline, the in vivo fluorescence signal was almost invisible after intravenous injection of the probe. In contrast, in the mercury poisoning mouse group (b-f), the fluorescence signal in the abdominal region of the mice gradually increased over time. In particular, in the acute mercury poisoning mouse group (b-f), the fluorescence intensity in the abdomen of the mercury poisoning mice reached the strongest at 50 min after injection, which was about 2.6 times that of the normal mice. The above results fully demonstrate the feasibility of the probe XQ-S for detecting Hg 2+ levels in acute mercury poisoning mice.

[0099] Through the above spectral performance tests and biological experiments, the excellent performance of the fluorescent probe XQ-S of the present application can be measured, and the results are compared with those of the control example probe, as shown in Table 1.

[0100] Table 1 Comparison of the performance of the probe XQ-S of the present application and the control example probe FS-Hg

[0101]

[0102] From the test results of a series of spectral experiments, the fluorescent probe XQ-S prepared in the present application exhibits significant advantages such as fast fluorescence signal response, high sensitivity, and strong specificity. Its large Stokes shift can reduce background fluorescence interference, thereby improving imaging capability, and the spectral performance is significantly enhanced compared with the control example. Moreover, the fluorescent probe XQ-S of the present application has relatively low cytotoxicity, and can be successfully applied to monitor the fluctuation of Hg 2+ concentration levels in 4T1 cell exogenous and acute mercury poisoning mouse pathological models. This has important significance for the in vivo detection of Hg 2+ and the diagnosis of acute mercury poisoning.

Claims

1. A method for detecting Hg 2+ The large Stokes shift near-infrared fluorescent probe is characterized by The molecular formula of the fluorescent probe is C 34 H 30 N2O2S, its molecular structure is:

2. A method for preparing the fluorescent probe according to claim 1, characterized in that: The following steps are involved: (1) Cyclohexanone is added dropwise to concentrated sulfuric acid, and 4-diethylamino keto acid is added under stirring. After the reaction, perchloric acid is added to form a precipitate, which is filtered, washed, and dried to obtain intermediate M1; (2) Dissolving the intermediate M1 and 8-quinolinecarboxaldehyde in acetic acid, heating the mixture for reaction, adjusting the pH to neutral, and filtering, washing, and drying the precipitate to obtain the intermediate M2; (3) The intermediate M2 is dissolved in anhydrous dichloromethane, oxalyl chloride is added, the solvent is removed after the reaction, and the intermediate M2 is dissolved in anhydrous dichloromethane again, a saturated Na2S solution is added, and the intermediate M2 is separated and purified after the reaction to obtain the fluorescent probe.

3. The method for preparing a fluorescent probe according to claim 2, wherein: The molar ratio of the cyclohexanone to the 4-diethylamino keto acid is 1:1 to 1:1.

5.

4. The method for preparing a fluorescent probe according to claim 2, wherein: The molar ratio of the intermediate M1 to 8-quinolinecarboxaldehyde is 0.95:1 to 0.95:1.

2.

5. The method for preparing a fluorescent probe according to claim 2, wherein: The molar ratio of the intermediate M2 to oxalyl chloride is 1:3 to 1:

5.

6. The method for preparing a fluorescent probe according to claim 2, wherein: The reaction temperature of step (1) is 90° C. and the reaction time is 2.5 to 3.5 hours.

7. The method for preparing a fluorescent probe according to claim 2, wherein: The reaction temperature of step (2) is 80° C. and the reaction time is 2 to 3 hours.

8. The method for preparing a fluorescent probe according to claim 2, wherein: The reaction temperature of step (3) is room temperature and the reaction time is 4 to 5 hours.

9. The fluorescent probe according to claim 1 or the fluorescent probe prepared by any one of claims 2 to 8 is effective in detecting Hg 2+ The application is characterized in that The Hg 2+ The minimum detection limit is 53 nM, and the concentration of the fluorescent probe during detection is 10 μM.

10. The fluorescent probe according to claim 9 is used to detect Hg 2+ The application is characterized in that For Hg in living cells or living bodies 2+ Detection imaging of concentration levels.

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