Preparation method of HOF ratio fluorescence sensor and application of HOF ratio fluorescence sensor in detection of ammonia gas in expired gas of patients with hepatic nephropathy

Abstract

The invention relates to a preparation method of an HOF ratio fluorescence sensor and application of the HOF ratio fluorescence sensor in detection of ammonia gas in exhaled air of patients with hepatic nephropathy, the material can detect trace ammonia gas, the lower limit of ammonia gas detection is 0.7 ppm, and the concentration of ammonia gas in exhaled air of the patients with hepatic nephropathy can be accurately quantified through two opposite fluorescence response signals. The method comprises the following specific steps: collecting exhaled gas of a patient with hepatic nephropathy in an aluminum foil gas collection bag, introducing the collected gas into an aqueous solution containing a ratiometric fluorescent sensing material Pt2Cu4 at HOF-101, and standing for a period of time to complete the detection and fluorescent color development of the ammonia gas in the exhaled gas of the patient. Or the patient directly blows air into the fluorescent sensing material aqueous solution to complete ammonia gas detection. The method can effectively realize non-invasive rapid detection of liver and kidney diseases, and is expected to replace traditional blood ammonia detection and be applied to the fields of clinical diagnosis and disease course monitoring of liver and kidney diseases.

Description

Technical Field

[0001] The present invention relates to the intersecting field of nanomaterials and biochemistry, and in particular to an HOF ratio fluorescence sensor and its application in detecting ammonia in the breath of patients with liver and kidney diseases. Background Art

[0002] Breathing contains rich metabolic and physiological information, which can provide a basis for disease diagnosis. 3 ) As a biomarker for liver and kidney diseases, accurate detection of ammonia in exhaled breath is expected to replace time-consuming blood tests and achieve non-invasive diagnosis of liver and kidney patients in clinical practice. Thanks to its advantages such as simple operation and significant color switching, portable ammonia fluorescence sensors have received widespread attention from researchers in recent years. However, due to the limitations of sensor sensitivity and selectivity, the specific identification and detection of ammonia in complex exhaled breath components is still difficult. Therefore, the development of highly accurate ammonia sensing materials is of great significance for the rapid screening and disease course monitoring of liver and kidney diseases.

[0003] In the prior art, CN113325042B discloses a sodium-type MTW molecular sieve and a preparation method and application thereof, an ammonia gas sensor and a preparation method and application thereof, which provides a sodium-type MTW molecular sieve, wherein the silicon-aluminum ratio of the sodium-type MTW molecular sieve is 20 to 35. The pores and the Na in the cage of the sodium-type MTW molecular sieve are + Ions are freely movable cations used to balance the negative charge of the aluminosilicate molecular sieve framework. The sodium-type MTW molecular sieve provided by the invention has a low silicon-to-aluminum ratio, and has the advantages of high ionic conductivity and low resistance. The sodium-type MTW molecular sieve provided by the invention is used as a sensitive material to improve the performance and stability of the chemical resistance ammonia gas sensor, but its detection limit is still relatively high, only reaching 5ppm, and the detection sensitivity is greatly affected by other gas components.

[0004] Hydrogen-bonded organic framework materials (HOF) have shown great potential in the field of fluorescence sensing due to their abundant specific recognition sites and strong luminescence properties. At present, most HOF fluorescent sensing materials can be divided into fluorescence quenching type and fluorescence enhancement type. However, single-signal response fluorescent sensors are often difficult to accurately quantify the concentration of trace analytes. The ratiometric fluorescence sensor can almost eliminate the interference of probe concentration through the ratio of the luminescence intensity of two substances, and is expected to achieve accurate quantitative detection of analytes, but this material has not yet been maturely applied in the field of ammonia detection. Therefore, by doping other luminescent substances inside the HOF, the construction of a highly controllable ratiometric fluorescent HOF material will play an important role in promoting the development of breath diagnosis of liver and kidney diseases.

[0005] Based on this, this application proposes a HOF ratio fluorescence sensor and its application in the detection of ammonia in the exhaled breath of patients with liver and kidney diseases, using luminescent metal nanoclusters and π-stacked HOF structures to achieve highly controllable and precise assembly, greatly improving their highly sensitive and selective detection of trace biomarkers. Summary of the invention

[0006] The present invention provides a HOF ratio fluorescence sensor and an application of ammonia detection in the breath of patients with liver and kidney diseases, which effectively realizes ammonia detection in the breath of patients with liver and kidney diseases.

[0007] The specific technical solution adopted by the present invention is:

[0008] A method for preparing a HOF ratio fluorescence sensor, characterized in that it comprises the following steps: 2 Cu 4 L 8 Dispersed in anhydrous ethanol, 1,3,6,8-tetrakis(4-carboxyphenyl)pyrene was added, the resulting mixed solution was sealed and reacted at high temperature, and Pt was obtained after washing and drying. 2 Cu 4 @HOF-101 ratiometric fluorescence sensing material.

[0009] The Pt 2 Cu 4 L 8 Obtained according to the method disclosed in patent CN116496789A, abbreviated as Pt 2 Cu 4 , L is a levonorgestrel ligand; wherein Pt 2 Cu 4 L 8 The preparation method is as follows: dissolve the ligand levonorgestrel in dichloromethane solution, add H 2 PtCl 6 The methanol solution was stirred evenly, triethylamine was added and stirred, and then a dichloromethane solution of copper hexafluorophosphate tetraacetonitrile was added and stirred continuously. After the reaction was completed, the reaction solution was placed at room temperature and protected from light to evaporate slowly, and crystals of Pt were precipitated. 2 Cu 4 MNC, filtered, and dried at room temperature to obtain Pt 2 Cu 4 L 8 product;

[0010] As a further improvement of the technical solution of the synthesis method of the present invention, the above-mentioned Pt 2 Cu 4 L 8 The mass ratio of 1,3,6,8-tetrakis(4-carboxyphenyl)pyrene is controlled at 0.5-4:1, the reaction temperature is controlled at 60-70°C, and the reaction time is 24-36h.

[0011] The present invention further provides a Pt obtained by the above HOF ratio fluorescence sensor preparation method. 2 Cu 4 The application of HOF-101 in the detection of ammonia in the breath of patients with liver and kidney diseases is characterized by comprising the following steps: collecting the exhaled breath of patients with liver and kidney diseases in an aluminum foil gas sampling bag, 2 Cu 4 The collected gas is passed into the aqueous solution of @HOF-101 and allowed to stand for a period of time to complete the detection and fluorescence color development of ammonia in the patient's exhaled breath, or the patient blows directly into the aqueous solution of the fluorescent sensor material to complete the ammonia detection. A 365nm ultraviolet lamp is used to qualitatively analyze the test results, and a fluorescence spectrometer is used to collect the emission spectrum of the material for quantitative analysis of the ammonia concentration in the exhaled breath of patients with liver and kidney diseases.

[0012] As a further improvement of the method for detecting ammonia in breath of patients with hepatic and renal diseases of the present invention, the Pt 2 Cu 4 The concentration of HOF-101 aqueous solution is 2-10 mg / 100 mL, and the dosage for a single test is 4-5 mL.

[0013] As a further improvement of the method for detecting ammonia in the breath of patients with hepatic and kidney diseases of the present invention, the excitation wavelength of the fluorescence spectrometer is set to 365 nm, and the emission spectrum is detected at 400-800 nm.

[0014] Technical Effects

[0015] 1) The preparation method of the HOF ratio fluorescence sensor provided by the present invention adopts a solvothermal method, and Pt can be prepared by simple mixing ultrasound, heating reaction, centrifugal washing. 2 Cu 4 @HOF-101 ratiometric fluorescent sensing material. The material prepared once can be used for about 20 tests. By proportionally increasing the amount of metal nanoclusters and HOF organic ligands, gram-level products can be obtained at one time, which can realize the batch preparation of fluorescent sensors.

[0016] 2) Pt prepared by the present invention 2 Cu 4 Compared with the traditional single-signal HOF fluorescent sensor material, the @HOF-101 ratiometric fluorescent sensor material has a stronger ability to detect trace ammonia, a more obvious degree of fluorescence color change, and a detection limit of 0.7ppm. In addition, the reverse dual fluorescence signal largely avoids the interference of the probe concentration on the concentration of the analyte, and can more accurately quantify the concentration of trace ammonia.

[0017] 3) Pt prepared by the present invention 2 Cu 4@HOF-101 ratiometric fluorescent sensing material can realize the detection and quantitative analysis of ammonia in the exhaled breath of patients with liver and kidney diseases, and has the potential for initial diagnosis and disease course monitoring in clinical practice. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 The Pt prepared in the present invention 2 Cu 4 @X-ray diffraction comparison of HOF-101 and HOF-101;

[0019] Figure 2 The Pt prepared in the present invention 2 Cu 4 @HOF-101 and HOF-101, Pt 2 Cu 4 Fourier transform infrared spectra comparison chart;

[0020] Figure 3 The Pt prepared in the present invention 2 Cu 4 @X-ray diffraction patterns of HOF-101 after treatment with different acid and alkali solutions;

[0021] Figure 4 The Pt with different doping amounts prepared by the present invention 2 Cu 4 @HOF-101 and HOF-101 high-angle annular dark field scanning transmission image and secondary electron image comparison;

[0022] Figure 5 The Pt obtained by three-dimensional electron diffraction analysis 2 Cu 4 @HOF-101 structure diagram;

[0023] Figure 6 The Pt prepared in the present invention 2 Cu 4 @HOF-101 and Pt 2 Cu 4 (A) Excitation spectrum and (B) Emission spectrum;

[0024] Figure 7 X-Pt with different doping amounts prepared by the present invention 2 Cu 4 @ HOF-101 and the fluorescence emission spectra of HOF-101 placed in a low concentration ammonia atmosphere;

[0025] Figure 8 The Pt prepared in the present invention 2 Cu 4@HOF-101 is placed in a low concentration ammonia atmosphere and its (A) fluorescence emission spectrum and (B) the fitted Stern-Volmer equation.

[0026] Fig. 9 For the actual blood ammonia test Pt 2 Cu 4 @HOF-101's (A) fluorescence emission spectrum and (B) comparison of ammonia concentration in patients' breath and blood;

[0027] Fig.10 The Pt prepared in the present invention 2 Cu 4 @HOF-101's selective comparison chart of possible exhaled breath components of the human body;

[0028] Fig.11 Pt 2 Cu 4 @HOF-101 application diagram in human breath detection. DETAILED DESCRIPTION

[0029] In order to make the purpose, technical scheme and advantages of the present invention clearer, the present invention is described in detail below in conjunction with specific examples and experimental data. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments in the present invention, all other implementation methods obtained by ordinary technicians in the field without making creative work belong to the scope of protection of the present invention. The present invention is further described below by specific examples.

[0030] Example 1Pt 2 Cu 4 Preparation of @HOF-101 materials

[0031] Pt was synthesized according to the method reported in patent CN116496789A. 2 Cu 4 , 4.5 mg Pt 2 Cu 4 Dispersed in anhydrous ethanol, ultrasonicated for 5 min, added 1.5 mg 1,3,6,8-tetrakis(4-carboxyphenyl)pyrene and ultrasonicated for another 5 min, the resulting mixed solution was transferred to a 10 ml glass bottle and placed in a 60 ° C oven for reaction for 24 h. The sample after the reaction was centrifuged, washed, and dried to obtain Pt 2 Cu 4 @HOF-101 material.

[0032] Pt 2 Cu 4 @HOF-101 Preparation of ratiometric fluorescence sensing reagent and ammonia detection

[0033] 6 mg of Pt prepared in Example 1 2 Cu 4 @HOF-101 is dispersed in 100mL ultrapure water. Take 4mL Pt for a single test 2 Cu 4 The HOF-101 aqueous solution was placed in an ammonia atmosphere for 5 minutes, and the change in fluorescence intensity of the material after ammonia detection was observed using a 365nm ultraviolet lamp to achieve qualitative detection of trace ammonia. In addition, for quantitative analysis, a standard curve needs to be established first. The specific method is to add 4mL Pt 2 Cu 4 @HOF-101 aqueous solution was placed in an atmosphere of 2, 5, 7, 10, 15, and 20 ppm ammonia and detected using a fluorescence spectrometer. The Stern-Volmer equation of the change of fluorescence intensity with ammonia concentration was obtained by fitting the ratio of fluorescence intensity at 443 nm to 640 nm. When detecting ammonia of unknown concentration, the specific concentration was obtained by substituting the ratio of fluorescence intensity at 443 nm to 640 nm into the Stern-Volmer equation. This process is mainly used for the quantitative detection of ammonia in the exhaled breath of patients with liver and kidney diseases in the next step.

[0034] Pt 2 Cu 4 @HOF-101 ratiometric fluorescence sensor for actual breath detection in patients with liver and kidney diseases

[0035] The exhaled air of patients with liver and kidney diseases was collected in an aluminum foil air sampling bag and injected into a Pt 2 Cu 4 The collected gas is passed into the aqueous solution of @HOF-101 and allowed to stand for 5 minutes to complete the detection of ammonia in the exhaled breath; or the patient uses a silicone tube with a dropper to pass the collected gas into a solution of Pt 2 Cu 4 The ammonia in the breath was detected by blowing air into a glass vial of HOF-101 aqueous solution for 3 minutes. A 365nm ultraviolet lamp was used to conduct preliminary fluorescence observation of the material, and then the emission spectrum of the material was immediately obtained using a fluorescence spectrometer. The ratio of the fluorescence intensity at 443nm and 640nm in the emission spectrum was substituted into the fitted Stern-Volmer equation to obtain the actual concentration of ammonia in the patient's breath.

[0036] In order to confirm the crystallinity and crystalline structure of the synthesized sample, an X-ray diffraction test was performed on the sample synthesized in Example 1, and the result was compared with the simulated peak of the theoretical crystalline structure of HOF-101. The comparison results are as follows: Figure 1 As shown, the Pt prepared by the method of the present invention 2 Cu 4 @HOF-101 is consistent with the simulated peaks of the theoretical crystal form of the original structure of HOF-101, proving that Pt2 Cu 4 The successful synthesis of @HOF-101 and the possibility of inferring Pt 2 Cu 4 It is mainly distributed inside HOF-101 rather than on the surface. 2 Cu 4 After being doped into HOF-101, the structure remains intact. The synthesized sample of Example 1 was characterized by Fourier transform infrared spectroscopy. The results are as follows: Figure 2 As shown, Pt 2 Cu 4 In 2013cm -1 The stretching vibration peak at Pt 2 Cu 4 @HOF-101 is preserved, proving Pt 2 Cu 4 The structure remains intact in the composite material.

[0037] Since ammonia is an alkaline gas, in order to determine the 2 Cu 4 The structural stability of @HOF-101 in the actual testing process was tested by X-ray diffraction test after the sample prepared in Example 1 was immersed in a pH = 5-9 aqueous solution for 1 day. The results are as follows Figure 3 As shown, after the sample was immersed in different pH solutions for 1 day, it still maintained its original crystal structure, indicating that the material has good acid-base stability and can maintain structural integrity during the detection of trace ammonia.

[0038] In order to determine the Pt 2 Cu 4 The dispersion of Pt in HOF-101 2 Cu 4 The specific structure of @HOF-101 was characterized by transmission electron microscopy and three-dimensional electron diffraction on the sample prepared in Example 1. Figure 4 For different doping amounts of Pt 2 Cu 4 @HOF-101 and HOF-101 high-angle annular dark field scanning transmission image (HAADF-STEM) and secondary electron image (SE-STEM), we can see Pt 2 Cu 4 It is mainly distributed inside HOF-101 rather than on the surface, and with the increase of doping amount, Pt 2 Cu 4 The signal of Pt obtained by three-dimensional electron diffraction analysis 2 Cu 4 @HOF-101 structure is as follows Figure 5 As shown, Pt 2 Cu4 Located inside the HOF-101 structure, it forms a tight interaction with HOF-101.

[0039] In order to explore whether the metal nanoclusters have any effect on the luminescence performance after being encapsulated inside HOF-101, the Pt 2 Cu 4 and the Pt prepared in Example 1 2 Cu 4 @ HOF-101 fluorescence spectrum. The results are as follows Figure 6 As shown in Figure 2, similar excitation and emission spectra indicate that the fluorescence properties of metal nanoclusters in the composite material are fully preserved. In addition, the shift of the emission peak at 610 nm in the emission spectrum indicates that Pt 2 Cu 4 The formation of composite materials with HOF-101 demonstrates the close interaction and energy transfer between metal nanoclusters and HOF.

[0040] Considering Pt 2 Cu 4 The doping amount may greatly affect the sensing performance of the material. Based on the method described in Example 1, Pt 2 Cu 4 The addition amounts were 0.75, 1.5, 3.0, 4.5, and 6.0 mg, respectively, to synthesize a series of Pt with different doping amounts. 2 Cu 4 @HOF-101 material, named X-Pt 2 Cu 4 @HOF-101(X is Pt 2 Cu 4 The percentage of X = 9.4%, 12.5%, 18.9%, 21.1%, 23.5%). Afterwards, the prepared material was prepared according to the method described in the embodiment to obtain a ratio fluorescence sensing reagent, which was subjected to a low concentration ammonia sensing test and characterized by fluorescence spectroscopy. The test results are shown in FIG. Figure 7 As shown, 21.1%-Pt 2 Cu 4 @HOF-101 exhibits the widest color change range and the most obvious reverse fluorescence signal during ammonia detection and is considered to be the best material for ammonia sensing. 2 Cu 4 When the molar ratio is 3.95:1, it can ensure that the material has the strongest luminescence intensity, and at the same time will not be interfered by the agglomeration of metal nanoclusters in HOF-101. Therefore, the following ammonia sensing test and ammonia detection in the breath of patients with liver and kidney diseases use 21.1%-Pt 2 Cu 4@HOF-101 proceeds.

[0041] To test Pt 2 Cu 4 @HOF-101 material detects trace ammonia, and the ratio fluorescence sensing reagent is tested for low-concentration ammonia fluorescence sensing. The fluorescence spectrometer collects 21.1%-Pt 2 Cu 4 @The emission spectrum of HOF-101 under trace ammonia conditions. The test results are as follows Figure 8 As shown in the figure, the composite material can produce a significant response to ammonia as low as 2ppm. And within a certain range, the response signal shows a highly fitted linear relationship. Specifically, under the ammonia concentration of 2-20ppm, the ratio of the fluorescence intensity at 443nm and 640nm can be used to fit the Stern-Volmer equation of the change of fluorescence intensity with ammonia concentration. According to 3σ / K SV (where σ is the standard deviation of 6 blank samples) The detection limit of ammonia for this material is calculated to be 0.7 ppm.

[0042] In the actual breath test, the exhaled breath of patients with liver and kidney diseases from Shanxi Bethune Hospital was collected and tested for fluorescence spectroscopy. The test results are as follows: Fig. 9 Once the material is exposed to exhaled breath, the analyte will cause Pt 2 Cu 4 @HOF-101 has a significant change in fluorescence intensity at 443nm and 640nm, proving that the material has the potential to screen for diseases. Substituting the ratio of fluorescence intensity at 443nm to 640nm into the Stern-Volmer equation, the concentrations of ammonia in the patient's breath were 1.2, 1.4, 1.4 and 1.6ppm, respectively, and the corresponding blood ammonia test results were 35.9, 39.3, 40.4 and 43.6μmol L -1 The results of the breath test maintained a high positive correlation with the blood ammonia test, proving that the breath test has the ability to diagnose diseases and is expected to replace traditional blood ammonia testing.

[0043] Selectivity is also one of the important factors to measure the sensing material. The ratio fluorescence sensing reagent prepared in the embodiment was tested for 50ppm possible components in human breath. The results are as follows: Fig.10 As shown, Pt 2 Cu 4 @HOF-101 has almost no response to other components of exhaled breath, indicating that the fluorescence signal in the previous breath test of patients with liver and kidney diseases was completely generated by ammonia, and the sensitivity was good. 2 Cu 4@HOF-101 material has both sensitivity and selectivity, and is expected to be used in clinical diagnosis and disease course monitoring of patients with liver and kidney diseases.

Claims

1. A method for preparing a HOF ratio fluorescence sensor, characterized in that: The following steps are involved: Pt2Cu4L8 was dispersed in anhydrous ethanol, 1,3,6,8-tetrakis(4-carboxyphenyl)pyrene was added, the obtained mixed solution was sealed and reacted at high temperature, and after washing and drying, Pt2Cu4@HOF-101 ratiometric fluorescent sensing material was obtained; The Pt2Cu4L8 is obtained according to the method disclosed in patent CN116496789A, abbreviated as Pt2Cu4, where L is the levonorgestrel ligand; the mass ratio of the Pt2Cu4 to 1,3,6,8-tetrakis(4-carboxyphenyl)pyrene is 0.5-4:

1.

2. The method for preparing a HOF ratio fluorescence sensor according to claim 1, characterized in that: In the step 1, the preparation method of Pt2Cu4 is: dissolving the ligand levonorgestrel in a dichloromethane solution, adding a methanol solution of H2PtCl6, adding triethylamine and stirring, and then adding a dichloromethane solution of tetraacetonitrile copper hexafluorophosphate, stirring continuously, and after the reaction is completed, placing the reaction solution at room temperature in the dark and slowly evaporating to precipitate crystals of Pt2Cu4MNC, filtering, and drying at room temperature to obtain a Pt2Cu4 product.

3. The method for preparing a HOF ratio fluorescence sensor according to claim 1, characterized in that: The reaction temperature is controlled at 60-70° C., the reaction time is 24-36 hours, and the material obtained after the reaction is washed three times by centrifugation with anhydrous ethanol.

4. Application of Pt2Cu4@HOF-101 obtained by the preparation method of a HOF ratio fluorescence sensor as described in claims 1-3 in detecting ammonia in the breath of patients with liver and kidney diseases, characterized in that: The exhaled breath of patients with liver and kidney diseases is collected in an aluminum foil gas sampling bag, and the collected gas is passed into an aqueous solution containing Pt2Cu4@HOF-101 and left to stand for a period of time to complete the detection and fluorescence color development of ammonia in the patient's exhaled breath, or the ammonia detection is completed by the patient blowing directly into the aqueous solution of the fluorescent sensing material.

5. The use of a HOF ratio fluorescence sensor for detecting ammonia in the breath of patients with liver and kidney diseases as claimed in claim 4, characterized in that: A fixed probe concentration is controlled in the same batch of tests and then tested on the patient's exhaled breath.

6. The use of a HOF ratio fluorescence sensor for detecting ammonia in the breath of patients with liver and kidney diseases as claimed in claim 4, characterized in that: The Pt2Cu4@HOF-101 aqueous solution was collected and subjected to preliminary qualitative analysis under a 365nm ultraviolet lamp. The emission spectrum was obtained by a fluorescence spectrometer at an excitation wavelength of 365nm for quantitative analysis of ammonia concentration in exhaled breath.

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