Preparation method of HOF ratio fluorescent sensor and application of the same to detection of ammonia gas in breath of patient with hepatorenal disease

Abstract

The application relates to a preparation method of a HOF ratio fluorescent sensor and application of the HOF ratio fluorescent sensor in ammonia gas detection in exhaled air of patients with liver and kidney diseases. The material can detect trace ammonia gas, the lower limit of ammonia gas detection is 0.7 ppm, two opposite fluorescent response signals can accurately quantitatively detect the concentration of ammonia gas in the exhaled air of patients with liver and kidney diseases, the ammonia gas concentration obtained through testing keeps a high positive correlation with the blood ammonia concentration, specifically, the exhaled air of patients with liver and kidney diseases is collected in an aluminum foil gas collecting bag, the collected gas is introduced into an aqueous solution containing a ratio fluorescent sensing material Pt2Cu4@HOF-101, and the aqueous solution is statically placed for a period of time to complete ammonia gas detection in the exhaled air of the patient and fluorescent color development, or the patient directly blows the gas into the aqueous solution of the fluorescent sensing material to complete ammonia gas detection. Non-invasive rapid detection of liver and kidney diseases can be effectively realized, and the HOF ratio fluorescent sensor 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] This invention relates to the interdisciplinary field of nanomaterials and biochemistry, specifically to an HOF ratio fluorescence sensor and its application in detecting ammonia in the exhaled breath of patients with liver and kidney diseases. Background Technology

[0002] Breath contains a wealth of metabolic and physiological information, which can provide a basis for disease diagnosis. Ammonia (NH3), as a biomarker for liver and kidney diseases, holds promise for replacing time-consuming blood tests and enabling non-invasive diagnosis of liver and kidney patients in clinical practice through accurate detection of exhaled ammonia. Portable ammonia fluorescence sensors have attracted widespread attention from researchers in recent years due to their ease of operation and clear color switching. However, the specific identification and detection of ammonia in complex exhaled breath components remains challenging due to limitations in sensor sensitivity and selectivity. Therefore, the development of highly accurate ammonia sensing materials is of great significance for rapid screening and disease monitoring of liver and kidney diseases.

[0003] In the prior art, CN113325042B discloses a sodium-type MTW molecular sieve, its preparation method and application, and an ammonia gas sensor, its preparation method and application. It provides a sodium-type MTW molecular sieve with a silica-to-alumina ratio of 20-35. The sodium-type MTW molecular sieve contains Na in its channels and cages. + Ions, as freely mobile cations, are used to balance the negative charge of the aluminosilicate molecular sieve framework. The sodium-type MTW molecular sieve provided by this invention has a low silica-to-alumina ratio and the advantages of high ionic conductivity and low resistance. Using the sodium-type MTW molecular sieve provided by this invention as a sensitive material improves the performance and stability of the chemical resistance ammonia gas sensor. However, its detection limit is still relatively high, only reaching 5 ppm, and the detection sensitivity is greatly affected by other gas components.

[0004] Hydrogen-bonded organic frameworks (HOFs) exhibit great potential in fluorescence sensing due to their abundant specific recognition sites and strong luminescence properties. Currently, most HOF fluorescent sensing materials can be categorized into fluorescence quenching and fluorescence enhancement types. However, single-signal-response fluorescence sensors often struggle to accurately quantify trace analyte concentrations. Ratiometric fluorescence sensors, by using the ratio of the luminescence intensities of two substances, can almost eliminate interference from probe concentration, potentially enabling precise quantitative detection of analytes. However, this material has not yet seen mature applications in ammonia detection. Therefore, constructing highly controllable ratiometric fluorescent HOF materials by doping HOFs with other luminescent substances is crucial for advancing the diagnosis of liver and kidney diseases using breath tests.

[0005] Based on this, this application proposes an HOF ratio fluorescence sensor and its application in detecting ammonia in the exhaled breath of patients with liver and kidney diseases. It utilizes luminescent metal nanoclusters and π-stacked HOF structures to achieve highly controllable and precise assembly, which greatly improves their high sensitivity and selectivity for detecting trace biomarkers. Summary of the Invention

[0006] This invention provides an application of an HOF ratio fluorescence sensor for detecting ammonia in the exhaled breath of patients with liver and kidney diseases, effectively enabling the detection of ammonia in the exhaled breath of these patients.

[0007] The specific technical solution adopted in this invention is as follows:

[0008] A method for preparing an HOF ratio fluorescence sensor is characterized by comprising the following steps: dispersing Pt2Cu4L8 in anhydrous ethanol, adding 1,3,6,8-tetra(4-carboxyphenyl)pyrene, sealing the resulting mixed solution and reacting it at high temperature, and obtaining Pt2Cu4@HOF-101 ratio fluorescence sensing material after washing and drying.

[0009] The Pt2Cu4L8 is obtained according to the method disclosed in patent CN116496789A, abbreviated as Pt2Cu4, where L is the levonorgestrel ligand. The preparation method of Pt2Cu4L8 is as follows: the ligand levonorgestrel is dissolved in dichloromethane solution, H2PtCl6 methanol solution is added and stirred, triethylamine is added and stirred, then copper tetraacetonitrile hexafluorophosphate dichloromethane solution is added and stirred continuously. After the reaction is completed, the reaction solution is placed at room temperature in the dark to slowly evaporate, and crystals Pt2Cu4 MNC are precipitated. After filtration and drying at room temperature, the Pt2Cu4L8 product is obtained.

[0010] As a further improvement to the synthesis method of the present invention, the amounts of Pt2Cu4L8 and 1,3,6,8-tetra(4-carboxyphenyl)pyrene are controlled at a mass ratio of 0.5 to 4:1, the reaction temperature is controlled at 60 to 70°C, and the reaction time is 24 to 36 h.

[0011] This invention further provides the application of Pt2Cu4@HOF-101 obtained by the above-mentioned HOF ratio fluorescence sensor preparation method in the detection of ammonia in the exhaled breath of patients with liver and kidney diseases. The method is characterized by the following steps: collecting the exhaled breath of patients with liver and kidney diseases in an aluminum foil gas collection bag; passing the collected gas into an aqueous solution containing Pt2Cu4@HOF-101 and allowing it to stand for a period of time to complete the detection and fluorescence development of ammonia in the patient's exhaled breath; or, having the patient directly blow air into an aqueous solution of the fluorescent sensing material to complete the ammonia detection. A 365nm ultraviolet lamp is used for qualitative analysis of the detection 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 to the method for detecting ammonia in the exhaled breath of patients with liver and kidney diseases of the present invention, the concentration of the aqueous solution of Pt2Cu4@HOF-101 is 2-10 mg / 100 mL, and the volume used for a single test is 4-5 mL.

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

[0014] Technical effect

[0015] 1) The method for preparing the HOF ratiometric fluorescence sensor provided by this invention employs a solvothermal method. Pt2Cu4@HOF-101 ratiometric fluorescence sensing material can be prepared through simple mixing, ultrasonication, heating reaction, centrifugation, and washing. The material prepared in a single batch can be used for approximately 20 detections. By scaling up the amounts of metal nanoclusters and HOF organic ligands proportionally, gram-level products can be obtained in a single batch, enabling the mass production of fluorescence sensors.

[0016] 2) Compared with traditional single-signal HOF fluorescent sensing materials, the Pt2Cu4@HOF-101 ratiometric fluorescent sensing material prepared in this invention has a stronger detection capability for trace ammonia, a more obvious fluorescence color change, and a detection limit of 0.7 ppm. Furthermore, the reverse dual fluorescence signal largely avoids interference from probe concentration and analyte concentration, enabling more accurate quantification of trace ammonia concentration.

[0017] 3) The Pt2Cu4@HOF-101 ratiometric fluorescence sensing material prepared in this invention 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 monitoring in clinical practice. Attached Figure Description

[0018] Figure 1 The X-ray diffraction comparison diagrams of Pt2Cu4@HOF-101 and HOF-101 prepared in this invention are shown.

[0019] Figure 2 A comparison of the Fourier transform infrared spectra of Pt2Cu4@HOF-101 prepared in this invention with those of HOF-101 and Pt2Cu4.

[0020] Figure 3 X-ray diffraction patterns of Pt2Cu4@HOF-101 prepared in this invention after treatment with different acid and alkali solutions;

[0021] Figure 4 Comparison of Pt2Cu4@HOF-101 with different doping amounts prepared in this invention with high-angle annular dark-field scanning transmission images and secondary electron images of HOF-101;

[0022] Figure 5 The structure diagram of Pt2Cu4@HOF-101 obtained by three-dimensional electron diffraction analysis;

[0023] Figure 6 The excitation spectrum (A) and emission spectrum (B) of Pt2Cu4@HOF-101 and Pt2Cu4 prepared in this invention are shown.

[0024] Figure 7 Fluorescence emission spectra of X-Pt2Cu4@HOF-101 and HOF-101 with different doping amounts prepared in this invention after being placed in a low-concentration ammonia atmosphere.

[0025] Figure 8 (A) Fluorescence emission spectrum of Pt2Cu4@HOF-101 prepared for this invention after being placed in a low-concentration ammonia atmosphere and (B) Stern-Volmer equation obtained by fitting;

[0026] Figure 9 (A) Fluorescence emission spectrum of Pt2Cu4@HOF-101 after actual blood ammonia testing and (B) Comparison of ammonia concentration in patient's exhaled breath with blood ammonia concentration;

[0027] Figure 10 A comparative diagram showing the selectivity of Pt2Cu4@HOF-101 prepared in this invention for possible components of human exhaled air;

[0028] Figure 11 This is a diagram showing the application of Pt2Cu4@HOF-101 in human breath testing. Detailed Implementation

[0029] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be described in detail below with reference to specific embodiments and experimental data. Obviously, the described embodiments are only a part of the embodiments of this invention, and not all of them. Based on the embodiments of this invention, all other implementations obtained by those skilled in the art without creative effort are within the scope of protection of this invention. The invention will be further illustrated below through specific examples.

[0030] Example 1: Preparation of Pt2Cu4@HOF-101 material

[0031] Pt₂Cu₄ was synthesized according to the method reported in patent CN116496789A. 4.5 mg of Pt₂Cu₄ was dispersed in anhydrous ethanol and sonicated for 5 min. Then, 1.5 mg of 1,3,6,8-tetra(4-carboxyphenyl)pyrene was added, followed by sonication for another 5 min. The resulting mixture was transferred to a 10 ml glass bottle and reacted in a 60 °C oven for 24 h. The reacted sample was centrifuged, washed, and dried to obtain Pt₂Cu₄@HOF-101 material.

[0032] Preparation of Pt2Cu4@HOF-101 ratiometric fluorescence sensing reagent and detection of ammonia

[0033] 6 mg of Pt2Cu4@HOF-101 prepared in Example 1 was dispersed in 100 mL of ultrapure water. For a single detection, 4 mL of the Pt2Cu4@HOF-101 aqueous solution was placed in an ammonia atmosphere for 5 min. The change in fluorescence intensity after ammonia detection was observed using a 365 nm UV lamp to achieve qualitative detection of trace ammonia. Furthermore, for quantitative analysis, a standard curve needs to be established first. Specifically, 4 mL of the Pt2Cu4@HOF-101 aqueous solution was placed in ammonia atmospheres of 2, 5, 7, 10, 15, and 20 ppm, and the samples were detected using a fluorescence spectrometer. The Stern-Volmer equation for fluorescence intensity as a function of ammonia concentration was obtained by fitting the ratio of fluorescence intensity at 443 nm to 640 nm. When detecting unknown concentrations of ammonia, 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 subsequent quantitative detection of ammonia in the exhaled breath of patients with liver and kidney diseases.

[0034] Pt2Cu4@HOF-101 ratiometric fluorescence sensor applied to breath detection in patients with liver and kidney diseases.

[0035] Exhaled breath from patients with liver and kidney disease was collected in an aluminum foil gas collection bag. The collected gas was then passed into an aqueous solution containing Pt2Cu4@HOF-101 and allowed to stand for 5 minutes to detect ammonia in the exhaled breath. Alternatively, patients could blow air into a glass vial containing an aqueous solution of Pt2Cu4@HOF-101 for 3 minutes using a silicone tube with a dropper to detect ammonia in their exhaled breath. Preliminary fluorescence observation of the material was performed using a 365nm ultraviolet lamp. Immediately afterwards, the emission spectrum of the material was obtained using a fluorescence spectrometer. The ratio of fluorescence intensity at 443nm to 640nm in the emission spectrum was substituted into the fitted Stern-Volmer equation to determine the actual concentration of ammonia in the patient's exhaled breath.

[0036] To confirm the crystallinity and crystal structure of the synthesized sample, X-ray diffraction tests were performed on the sample synthesized in Example 1. The results were compared with the simulated peaks of the theoretical crystal structure of HOF-101. The comparison results are as follows: Figure 1 As shown, the simulated peaks of Pt2Cu4@HOF-101 prepared by the method of this invention are consistent with the theoretical crystal form of the original HOF-101 structure, proving the successful synthesis of Pt2Cu4@HOF-101 and suggesting that Pt2Cu4 is mainly distributed inside HOF-101 rather than on the surface. To determine whether the structure of Pt2Cu4 remains intact after being incorporated into HOF-101, Fourier transform infrared spectroscopy was performed on the sample synthesized in Example 1, and the results are as follows. Figure 2 As shown, Pt₂Cu₄ at 2013 cm⁻¹ -1 The stretching vibration peak at the point is retained in Pt2Cu4@HOF-101, proving that the structure of Pt2Cu4 remains intact in the composite material.

[0037] Since ammonia is an alkaline gas, to determine the structural stability of Pt2Cu4@HOF-101 during actual testing, the sample prepared in Example 1 was immersed in an aqueous solution with pH = 5-9 for 1 day and then subjected to X-ray diffraction testing. The results are as follows: Figure 3 As shown, the sample still maintains its original crystal structure after being soaked in solutions of different pH values ​​for 1 day, indicating that the material has good acid and alkali stability and can maintain its structural integrity during the detection of trace ammonia.

[0038] To determine the dispersion of Pt2Cu4 in HOF-101 and the specific structure of Pt2Cu4@HOF-101, the sample prepared in Example 1 was characterized by transmission electron microscopy and three-dimensional electron diffraction. Figure 4 A comparison of high-angle annular dark-field scanning transmission electron microscopy (HAADF-STEM) and secondary electron microscopy (SE-STEM) images of Pt2Cu4@HOF-101 with different doping concentrations shows that Pt2Cu4 is mainly distributed inside HOF-101 rather than on the surface, and the signal of Pt2Cu4 becomes more pronounced with increasing doping concentration. The structure of Pt2Cu4@HOF-101 obtained by three-dimensional electron diffraction analysis is shown below. Figure 5 As shown, Pt2Cu4 is located inside the HOF-101 structure and forms a close interaction with HOF-101.

[0039] To investigate whether the encapsulation of metal nanoclusters within HOF-101 affected its luminescence properties, the fluorescence spectra of Pt2Cu4 and Pt2Cu4@HOF-101 prepared in Example 1 were compared at room temperature. The results are as follows: Figure 6As shown, similar excitation and emission spectra indicate that the fluorescence properties of the metal nanoclusters in the composite material are fully preserved. Furthermore, the shift in the emission peak at 610 nm in the emission spectrum indicates the formation of the Pt₂Cu₄ / HOF-101 composite material, demonstrating a close interaction and energy transfer between the metal nanoclusters and HOF.

[0040] Considering that the doping amount of Pt2Cu4 can significantly affect the sensing performance of the material, based on the method described in Example 1, a series of Pt2Cu4@HOF-101 materials with different doping amounts were synthesized, named X-Pt2Cu4@HOF-101 (X is the percentage content of Pt2Cu4, X = 9.4%, 12.5%, 18.9%, 21.1%, 23.5%), using 0.75, 1.5, 3.0, 4.5, and 6.0 mg of Pt2Cu4. Subsequently, the prepared materials were used to prepare a ratiometric fluorescence sensing reagent according to the method described in the example, and low-concentration ammonia gas sensing tests were performed, characterized by fluorescence spectroscopy. The test results are as follows... Figure 7 As shown, 21.1%-Pt2Cu4@HOF-101 exhibits the widest color change range and the most obvious reverse fluorescence signal during ammonia detection, and is considered the best material for ammonia sensing. In other words, when the molar ratio of HOF-101 to Pt2Cu4 is 3.95:1, it ensures the strongest luminescence intensity without being interfered with by the aggregation of metal nanoclusters within HOF-101. Therefore, 21.1%-Pt2Cu4@HOF-101 was used in the following ammonia sensing tests and the detection of ammonia in the exhaled breath of patients with liver and kidney diseases.

[0041] To test the detection performance of Pt2Cu4@HOF-101 material for trace ammonia, a low-concentration ammonia fluorescence sensing test was conducted using a ratiometric fluorescence sensing reagent. The emission spectra of 21.1% Pt2Cu4@HOF-101 under trace ammonia conditions were collected using a fluorescence spectrometer. The test results are as follows: Figure 8 As shown, the composite material exhibits a significant response to ammonia concentrations as low as 2 ppm. Furthermore, within a certain range, the response signal displays a highly fitted linear relationship. Specifically, at ammonia concentrations of 2–20 ppm, the Stern-Volmer equation for fluorescence intensity as a function of ammonia concentration can be obtained by fitting the ratio of fluorescence intensity at 443 nm to 640 nm. Based on 3σ / K... SV (where σ is the standard deviation of 6 blank samples) The detection limit of this material for ammonia was calculated to be 0.7 ppm.

[0042] In actual breath tests, exhaled breath samples were collected and fluorescence spectroscopy was performed on patients with liver and kidney diseases from Shanxi Bethune Hospital. The test results are as follows: Figure 9Once exposed to exhaled air, the analyte caused a significant change in the fluorescence intensity of Pt2Cu4@HOF-101 at 443 nm and 640 nm, demonstrating the material's potential for preliminary disease screening. Substituting the ratio of fluorescence intensity at 443 nm to 640 nm into the Stern-Volmer equation yielded ammonia concentrations of 1.2, 1.4, 1.4, and 1.6 ppm in the patient's breath, corresponding to blood ammonia levels of 35.9, 39.3, 40.4, and 43.6 μmol L⁻¹. -1 The results of the breath test showed a strong positive correlation with those of the blood ammonia test, demonstrating that the breath test has the ability to diagnose diseases and is expected to replace the traditional blood ammonia test.

[0043] Selectivity is also an important factor in evaluating sensing materials. The ratiometric fluorescence sensing reagent prepared in the examples was tested for possible components in 50 ppm of human exhaled breath, and the results are as follows: Figure 10 As shown, Pt2Cu4@HOF-101 showed almost no response to other components in exhaled breath, indicating that the fluorescence signal in previous breath tests of patients with liver and kidney diseases was entirely generated by ammonia, demonstrating good sensitivity. Therefore, the Pt2Cu4@HOF-101 material prepared in this invention combines both sensitivity and selectivity, and is expected to be used in clinical diagnosis and disease monitoring of patients with liver and kidney diseases.

Claims

1. A method for fabricating a HOF ratio fluorescence sensor, characterized in that, Includes the following steps: Pt2Cu4L8 was dispersed in anhydrous ethanol, and 1,3,6,8-tetra(4-carboxyphenyl)pyrene was added. The resulting mixed solution was sealed and reacted at high temperature, with the reaction temperature controlled at 60~70℃ and the reaction time being 24~36 h. The material obtained after the reaction was washed three times by centrifugation with anhydrous ethanol and dried to obtain Pt2Cu4@HOF-101 ratiometric fluorescent sensing material. The Pt2Cu4L8, abbreviated as Pt2Cu4, where L represents the levonorgestrel ligand; the mass ratio of Pt2Cu4 to 1,3,6,8-tetra(4-carboxyphenyl)pyrene is 0.5-4:1; the preparation method of Pt2Cu4 is as follows: the ligand levonorgestrel is dissolved in dichloromethane solution, H2PtCl6 in methanol solution is added, triethylamine is added and stirred, then copper tetraacetonitrile hexafluorophosphate in dichloromethane solution is added and stirred continuously. After the reaction is completed, the reaction solution is placed at room temperature in the dark to slowly evaporate, and crystals Pt2Cu4MNC are precipitated. The crystals are filtered and dried at room temperature to obtain the Pt2Cu4 product.

2. The application of Pt2Cu4@HOF-101 obtained by the preparation method of the HOF ratio fluorescence sensor as described in claim 1 in the detection of ammonia in the exhaled breath of patients with liver and kidney diseases, characterized in that... The exhaled air of patients with liver and kidney diseases is collected in an aluminum foil gas collection bag. The collected gas is then introduced into an aqueous solution containing Pt2Cu4@HOF-101 and left to stand for a period of time to complete the detection of ammonia and fluorescence color development in the patient's exhaled air. Alternatively, ammonia can be detected by having the patient blow air directly into an aqueous solution of the fluorescent sensing material.

3. The application of the HOF ratio fluorescence sensor as described in claim 2 in the detection of ammonia in the exhaled breath of patients with liver and kidney diseases, characterized in that... A fixed probe concentration was controlled in the same batch of tests, and then the patient's exhaled breath was tested.

4. The application of the HOF ratio fluorescence sensor as described in claim 2 in the detection of ammonia in the exhaled breath of patients with liver and kidney diseases, characterized in that, The Pt2Cu4@HOF-101 aqueous solution was collected after testing and subjected to preliminary qualitative analysis under a 365 nm ultraviolet lamp. The emission spectrum was obtained by fluorescence spectroscopy at an excitation wavelength of 365 nm for quantitative analysis of ammonia concentration in exhaled breath.

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