A device for detecting aldehydes in exhaled breath and a method of making the same

Abstract

The application discloses a device for detecting aldehyde compounds in exhaled breath and a preparation method thereof, and belongs to the field of biological sample analysis and mass spectrum detection. The device for detecting aldehyde compounds in exhaled breath is used for sampling, derivatization and enrichment of carbonyl compounds in exhaled breath, and comprises a fluorine fixed phase modified paper base and fluorine tag hydrazine probes fixed on the surface of the fluorine fixed phase modified paper base. The fluorine tag hydrazine probes take a hydrazine group as a reactive group and take a perfluoroalkyl chain as a fluorine tag. The fluorine tag hydrazine probes are connected with the fluorine fixed phase modified paper base through the fluorine tag and react with target carbonyl compounds through a Schiff base condensation reaction through the reactive group. The application is applied to sampling, derivatization and enrichment of carbonyl compounds in exhaled breath, solves technical problems of insufficient sensitivity, complex enrichment methods and difficulty in high-throughput simultaneous detection of multiple components in existing detection technologies of carbonyl compounds in exhaled breath, and has the characteristics of simple operation, high sensitivity, simultaneous detection of multiple carbonyl compounds and high-throughput.

Description

Technical Field

[0001] This invention belongs to the field of biological sample analysis and mass spectrometry detection, and particularly relates to a device for detecting aldehyde compounds in exhaled breath and its preparation method. Background Technology

[0002] Traditional lung cancer screening methods, such as imaging screening, bronchoscopy screening, and liquid biopsy, have limitations such as radiation exposure, invasive procedure risks, complexity, and high cost. Exhaled breath analysis, due to its non-invasive, painless, and convenient advantages, shows great potential in the early diagnosis and large-scale screening of clinical diseases.

[0003] Studies have shown that endogenous volatile organic compounds (VOCs) in exhaled breath, especially highly reactive carbonyl compounds (such as acetone, pentanal, and benzaldehyde), can serve as potential biomarkers for various diseases, including lung cancer. However, the content of carbonyl compounds in exhaled breath is extremely low (trace level), and the high humidity of the samples poses challenges to highly sensitive detection. Existing pre-enrichment methods (such as adsorbent methods and cryogenic condensation methods) have limitations such as poor selectivity, easy sample decomposition / residue, complex equipment, high cost, and poor portability.

[0004] Chinese patent CN119269699A discloses an organic matter adsorption detection method based on MOF materials. Although this method can also be used to enrich exhaled VOCs, it uses a general-purpose MOF material (MIL-101(Cr)) and a GC-MS detection platform, resulting in indiscriminate enrichment of all VOCs. It lacks selectivity for specific categories (such as carbonyl compounds), and its desorption, extraction, and detection processes are relatively cumbersome and have low integration. In addition, traditional exhaled breath analysis techniques rely on gas collection bags or canisters to collect samples and require pretreatment such as chemical derivatization and enrichment through multiple independent devices. The operation is cumbersome and time-consuming, making it difficult to meet the needs for rapid and convenient detection. Summary of the Invention

[0005] To address the shortcomings of existing technologies, the technical problem to be solved by this invention is the insufficient sensitivity, complex enrichment methods, and difficulty in high-throughput simultaneous detection of multiple components in existing exhaled breath carbonyl compound detection technologies. This invention proposes a device for detecting exhaled breath aldehyde compounds that is easy to operate, highly sensitive, capable of simultaneously detecting multiple carbonyl compounds, and offers high throughput, along with its preparation method.

[0006] To solve the aforementioned technical problem, the technical solution adopted by the present invention is as follows: The present invention provides a device for detecting aldehyde compounds in exhaled breath, used for sampling, derivatization and enrichment of carbonyl compounds in exhaled breath, comprising a fluorinated stationary phase modified paper substrate and a fluorinated tag hydrazine probe fixed on the surface of the fluorinated stationary phase modified paper substrate; The fluorinated acylhydrazine probe uses an acylhydrazine group as a reactive group and a perfluoroalkyl chain as a fluorinated tag. The fluorinated acylhydrazine probe is connected to a fluorinated stationary phase-modified paper substrate through the fluorinated tag, and undergoes a Schiff base condensation reaction with the target carbonyl compound through the reactive group.

[0007] In some embodiments, the fluorinated stationary phase modified paper substrate is a fluorinated graphite modified paper substrate.

[0008] Another aspect of the present invention provides a method for preparing a device for detecting exhaled aldehyde compounds according to the above-mentioned technical solution, including a fluorine-modified paper substrate preparation step, wherein the fluorine-modified paper substrate preparation step includes coating solution preparation, wherein the coating solution preparation includes: dissolving polyacrylonitrile in N,N-dimethylformamide and uniformly dispersing fluorinated graphite particles to form a coating solution.

[0009] In some embodiments, the particle size of the fluorinated graphite particles is 10 μm.

[0010] In some embodiments, the preparation step of the fluorinated stationary phase modified paper substrate further includes paper substrate coating, which includes coating a coating solution onto the surface of triangular chromatographic paper and drying it to obtain a fluorinated graphite modified paper substrate.

[0011] In some embodiments, a probe immobilization step is further included, which includes: adding or immersing a fluorolabeled hydrazine probe solution onto a fluorinated stationary phase-modified paper substrate, thereby immobilizing the fluorolabeled hydrazine probe on the surface of the fluorinated stationary phase-modified paper substrate through fluorine-fluorine interaction; the concentration of the fluorolabeled hydrazine probe is 2.5 mM.

[0012] In some embodiments, a fluorolabeled hydrazine probe synthesis step is further included, the fluorolabeled hydrazine probe synthesis step comprising: 4-Mercaptobenzoic acid and perfluorohexyl iodine were reacted in the presence of sodium dithionite to give a benzoic acid derivative containing a perfluoroalkyl chain, namely compound 1. Compound 1 was reacted with oxalyl chloride to convert it into the corresponding acyl chloride, yielding compound 2. Compound 2 was reacted with hydrazine hydrate to obtain compound 3, namely the fluorolabeled hydrazine probe.

[0013] In some embodiments, compound 1 is prepared by the following steps: 4-mercaptobenzoic acid is added to N,N-dimethylformamide and stirred until homogeneous. Then, saturated sodium bicarbonate solution, perfluorohexyl iodine, and sodium dithionite are added. After stirring at room temperature and reacting completely, the mixture is diluted with saturated saline solution, adjusted to weak acidity with concentrated hydrochloric acid, extracted with ethyl acetate, and the organic phases are combined, dried over anhydrous sodium sulfate, and concentrated by rotary evaporation to obtain a yellow oily substance. After chromatographic purification, compound 1 is obtained.

[0014] In some embodiments, compound 2 is prepared by the following steps: compound 1 is dissolved in anhydrous tetrahydrofuran, a few drops of N,N-dimethylformamide are added, oxaloyl chloride is added dropwise under ice-water bath cooling, and after stirring at room temperature, the solvent is evaporated to obtain an orange oily substance, namely compound 2.

[0015] In some embodiments, compound 3 is prepared by the following steps: compound 2 is dissolved in acetonitrile, added dropwise to an acetonitrile solution containing hydrazine hydrate and triethylamine, refluxed, cooled to room temperature, extracted with ethyl acetate, the organic phases are combined, dried over anhydrous sodium sulfate, concentrated by rotary evaporation, and the crude product is purified by silica gel column chromatography to obtain white crystals, namely compound 3.

[0016] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention provides a device for detecting aldehydes in exhaled breath. It achieves ordered, high-density immobilization of the probe through "fluorine-fluorine interaction" and effectively suppresses non-specific adsorption through a fluorinated graphite modification layer, significantly improving derivatization efficiency and enrichment effect. The limits of detection (LODs) for pentanal, heptanal, octanal, benzaldehyde, and acetone can reach 1.67-7.11 pg / mL, and the limits of quantitation (LOQs) can reach 5.56-23.69 pg / mL, exhibiting high sensitivity and low detection limits. This invention provides a device for detecting aldehyde compounds in exhaled breath, capable of simultaneously detecting multiple (e.g., five or more) carbonyl compounds in a single sample, with a wide linear range (0.05-100 ng / mL) and a correlation coefficient R0. 2 >0.995, meeting the requirements for constructing characteristic expiratory molecular fingerprints of diseases, and featuring high throughput and simultaneous detection of multiple components; This invention provides a device for detecting aldehydes in exhaled breath, which integrates sampling, derivatization, and enrichment steps into a micro-device (such as a syringe). It eliminates the need for cumbersome sample pretreatment and large-scale enrichment equipment, making it easy to operate, with a short detection cycle, and featuring rapid, simple, and highly integrated operation. Attached Figure Description

[0017] Figure 1 This is a schematic diagram of the technical process provided in the embodiments of the present invention; Figure 2 This is a synthetic route diagram of the fluorolabeled hydrazine probe provided in the embodiments of the present invention; Figure 3The images provided in this embodiment of the invention are SEM and mapping images of fluorinated graphite modified paper substrates, where a is a blank paper substrate SEM image, b is a 10-micron fluorinated graphite modified paper substrate SEM image, c is an 8-micron fluorinated graphite modified SEM image, d is a C element mapping image of a 10-micron fluorinated graphite modified paper substrate, e is a 10-micron fluorinated graphite modified paper substrate O element mapping image, and f is a 10-micron fluorinated graphite modified paper substrate F element mapping image. Figure 4 The image shown is an FT-IR image of a fluorinated graphite-modified paper substrate provided in an embodiment of the present invention. Figure 5 The above are XPS spectra of fluorinated graphite-modified paper substrate provided in the embodiments of the present invention, wherein a is a Survey spectrum and b is an F1s spectrum; Figure 6 This is a comparison chart of the derivatization performance of the probe 4-PFTBH provided in the embodiments of the present invention with three commercially available probes; Figure 7 This is a comparison chart of the signal performance of derived pentanal provided in an embodiment of the present invention; Figure 8 This is a performance comparison diagram between fluorinated graphite-modified paper base and blank paper base provided in an embodiment of the present invention; Figure 9 This is a schematic diagram illustrating the effect of probe concentration on the detection signal provided in an embodiment of the present invention; Figure 10 This is a schematic diagram illustrating the effect of incubation time on the detection signal provided in an embodiment of the present invention; Figure 11 This is a schematic diagram illustrating the effect of spray voltage on the detection signal provided in an embodiment of the present invention; Figure 12 This is a schematic diagram illustrating the effect of the paper base angle on the detection signal, provided in an embodiment of the present invention. Figure 13 This is a schematic diagram illustrating the effect of the horizontal distance between the paper tip and the MS inlet on the detection signal, provided in an embodiment of the present invention. Figure 14 This is a schematic diagram illustrating the effect of the spray solvent on the detection signal provided in an embodiment of the present invention; Figure 15 Mass spectra of five carbonyl compounds detected simultaneously; Figure 16 The standard curve of pentanal; Figure 17 The standard curve of heptanal; Figure 18 This is a standard curve diagram for benzaldehyde. Figure 19 The standard curve of octanal; Figure 20 This is a standard curve for acetone. Figure 21 The results of thermogram analysis of exhaled breath samples from lung cancer patients and healthy controls; Figure 22 Box plot of benzaldehyde in exhaled breath samples from lung cancer patients and healthy controls; Figure 23 Box plot of heptanal in exhaled breath samples from lung cancer patients and healthy controls; Figure 24 Box plot of acetone in exhaled breath samples from lung cancer patients and healthy controls; Figure 25 Box plot of pentanalaldehyde in exhaled breath samples from lung cancer patients and healthy controls; Figure 26 Box plot of octanal in exhaled breath samples from lung cancer patients and healthy controls. Detailed Implementation

[0018] The technical solutions in specific embodiments of the present invention will be described in detail and completely below. Obviously, the described embodiments are only some specific implementations of the overall technical solution of the present invention, and not all implementations. Based on the overall concept of the present invention, all other embodiments obtained by those skilled in the art fall within the protection scope of the present invention.

[0019] One aspect of this invention provides a device for detecting aldehyde compounds in exhaled breath, used for sampling, derivatizing, and enriching carbonyl compounds in exhaled breath, comprising a fluorinated stationary phase-modified paper substrate and a fluorinated tag hydrazine probe fixed on the surface of the fluorinated stationary phase-modified paper substrate.

[0020] The fluorolabeled acylhydrazine probe uses the acylhydrazine group as a reactive group, which undergoes a Schiff base condensation reaction with the target carbonyl compound. The reactive group is used to initiate a highly efficient and specific Schiff base condensation reaction with the target carbonyl compound. The acylhydrazine group (-NH-NH2) has high nucleophilicity, resulting in a fast reaction rate and high efficiency (up to 99.63% derivatization efficiency for pentanal), ensuring the efficient capture of trace targets. This invention utilizes the acylhydrazine probe to undergo a specific Schiff base reaction with carbonyl compounds, achieving chemically selective labeling of target analytes.

[0021] The fluoro-tagged hydrazine probe uses a perfluoroalkyl chain as a fluorotag, which is linked to a fluoro-modified paper substrate via the fluorotag. The perfluoroalkyl chain (-C6F...) 13 The probe is connected to the aromatic ring by sulfur atoms, which generates a strong fluorine-fluorine interaction with the surface of the fluorine stationary phase, thereby achieving stable fixation of the probe on the paper substrate. The fluorine-fluorine interaction has high selectivity and strong binding energy, and its unit energy contribution is significantly higher than that of the hydrophobic interaction, which ensures the firm anchoring of the probe on the paper substrate surface, forming a high-density active layer and avoiding the masking of active sites.

[0022] In some embodiments, the fluorinated stationary phase modified paper substrate is a fluorinated graphite-modified paper substrate. The fluorinated stationary phase surface constructed from this fluorinated stationary phase modified paper substrate, rich in CF bonds, can specifically adsorb molecules with perfluoroalkyl tags through fluorine-fluorine interactions, while effectively inhibiting the non-specific adsorption of non-fluorinated endogenous interfering substances such as water and lipids, thereby greatly improving the enrichment efficiency of target analytes and the detection signal-to-noise ratio.

[0023] The above technical solution provides a detection device for carbonyl compounds in exhaled breath based on fluorine solid-phase microextraction-paper spray mass spectrometry (F-SPME-PS-MS). This device enables highly sensitive, selective, high-throughput, and rapid detection of various trace carbonyl compounds in exhaled breath, providing a new technological platform for non-invasive screening of diseases such as lung cancer. This solution addresses the technical problems of existing carbonyl compound detection technologies in exhaled breath, including insufficient sensitivity, complex / costly / inconvenient enrichment methods, cumbersome and time-consuming analysis procedures, difficulty in simultaneously detecting multiple components at high throughput, and severe interference from the paper matrix. It provides a dedicated probe, functionalized paper substrate, and integrated device based on fluorine solid-phase microextraction-paper spray mass spectrometry (F-SPME-PS-MS) to achieve highly sensitive, selective, high-throughput, rapid, simple, low-cost, and easily integrated non-invasive detection of carbonyl compounds in exhaled breath.

[0024] The aforementioned detection device integrates sampling, derivatization, enrichment, and detection. It achieves ordered, high-density probe immobilization through "fluorine-fluorine interaction" and effectively suppresses non-specific adsorption through a fluorinated graphite modification layer, significantly improving derivatization efficiency and enrichment effect. The limits of detection (LODs) for pentanal, heptanal, octanal, benzaldehyde, and acetone reach 1.67-7.11 pg / mL, and the limits of quantitation (LOQs) reach 5.56-23.69 pg / mL, exhibiting high sensitivity and low detection limits. It can simultaneously detect multiple (e.g., five or more) carbonyl compounds in a single sample, with a wide linear range (0.05-100 ng / mL) and a correlation coefficient R0. 2 >0.995, meeting the requirements for constructing characteristic expiratory molecular fingerprints of diseases, and featuring high throughput and simultaneous detection of multiple components; integrating sampling, derivatization, and enrichment steps into a micro-device (such as a syringe), eliminating the need for cumbersome sample pretreatment and large enrichment equipment, making operation simple, detection cycle short, and featuring rapid, simple, and highly integrated characteristics.

[0025] The detection device described in this invention achieved a spiked recovery rate of 87.9-113.4% in actual exhaled breath samples, with a relative standard deviation (RSD) of <11.54%, indicating high accuracy, good repeatability, and no significant matrix interference. It is suitable for the analysis of non-invasive biological samples such as exhaled breath. Preliminary clinical validation shows that it can effectively distinguish between lung cancer patients and healthy volunteers, demonstrating significant application potential in early non-invasive screening for lung cancer and research on the metabolic mechanisms of the disease.

[0026] This invention designs a bifunctional probe containing hydrazide reactive groups and perfluoroalkyl enrichment tags. Through interface construction, a fluorinated graphite-modified paper substrate is obtained, providing a fluorine-fluorine interaction interface and suppressing non-specific adsorption. In use, the functionalized paper substrate can be placed in a syringe to achieve integrated "sampling-derivation-enrichment". In addition, it can be combined with paper spray mass spectrometry to achieve in-situ desorption, ionization and minute-level rapid detection of the target analyte (data acquisition completed within 1 minute, with higher throughput).

[0027] Another aspect of the present invention provides a method for preparing a device for detecting exhaled aldehyde compounds according to the above-mentioned technical solution, including a step of preparing a fluorinated stationary phase modified paper substrate. The preparation step of the fluorinated stationary phase modified paper substrate includes the preparation of a coating solution, which includes: dissolving polyacrylonitrile (PAN) in N,N-dimethylformamide (DMF) and uniformly dispersing fluorinated graphite particles to form a coating solution. PAN, as a film-forming agent, helps fluorinated graphite form a continuous and uniform coating on the paper substrate surface.

[0028] In some embodiments, the fluorinated graphite particles have a particle size of 10 μm. 10 μm fluorinated graphite particles are more uniformly distributed than 8 μm particles, better covering fiber pores and reducing localized aggregation.

[0029] In some embodiments, the preparation step of the fluorinated stationary phase-modified paper substrate further includes paper substrate coating, which comprises: coating a coating solution onto the surface of triangular chromatographic paper (such as Whatman No. 1), and drying to obtain a fluorinated graphite-modified paper substrate (FG@Paper). This modification layer can effectively suppress the non-specific adsorption of the porous structure of the paper substrate and provide a fluorine-fluorine interaction interface. The constructed CF-rich fluorinated stationary phase surface can specifically adsorb molecules with perfluoroalkyl tags through fluorine-fluorine interactions, while effectively suppressing the non-specific adsorption of non-fluorinated endogenous interfering substances such as water and lipids, thereby greatly improving the enrichment efficiency of the target analyte and the detection signal-to-noise ratio.

[0030] In some embodiments, a probe immobilization step is further included, which comprises: adding or immersing a fluorolabeled hydrazine probe solution onto a fluorinated stationary phase-modified paper substrate, thereby immobilizing the fluorolabeled hydrazine probe on the surface of the fluorinated stationary phase-modified paper substrate through fluorine-fluorine interactions; the concentration of the fluorolabeled hydrazine probe is 2.5 mM. The probe concentration is crucial to the derivatization efficiency; too low a concentration results in insufficient reaction, while too high a concentration is wasteful and may introduce background interference. 2.5 mM is the optimized concentration that yields the highest signal-to-noise ratio.

[0031] In some embodiments, a fluorolabeled hydrazine probe synthesis step is further included, the fluorolabeled hydrazine probe synthesis step comprising: 4-Mercaptobenzoic acid and perfluorohexyl iodine were reacted in the presence of sodium dithionite to give a benzoic acid derivative containing a perfluoroalkyl chain, namely compound 1. Compound 1 was reacted with oxalyl chloride to convert it into the corresponding acyl chloride, yielding compound 2. Compound 2 was reacted with hydrazine hydrate to obtain compound 3, namely the fluorolabeled hydrazine probe.

[0032] In some embodiments, compound 1 is prepared by the following steps: 4-mercaptobenzoic acid is added to N,N-dimethylformamide and stirred until homogeneous. Then, saturated sodium bicarbonate solution, perfluorohexyl iodine, and sodium dithionite are added. After stirring at room temperature and reacting completely, the mixture is diluted with saturated saline solution, adjusted to weak acidity with concentrated hydrochloric acid, extracted with ethyl acetate, and the organic phases are combined, dried over anhydrous sodium sulfate, and concentrated by rotary evaporation to obtain a yellow oily substance. After chromatographic purification, compound 1 is obtained.

[0033] In some embodiments, compound 2 is prepared by the following steps: compound 1 is dissolved in anhydrous tetrahydrofuran, a few drops of N,N-dimethylformamide are added, oxaloyl chloride is added dropwise under ice-water bath cooling, and after stirring at room temperature, the solvent is evaporated to obtain an orange oily substance, namely compound 2.

[0034] In some embodiments, compound 3 is prepared by the following steps: compound 2 is dissolved in acetonitrile, added dropwise to an acetonitrile solution containing hydrazine hydrate and triethylamine, refluxed, cooled to room temperature, extracted with ethyl acetate, the organic phases are combined, dried over anhydrous sodium sulfate, concentrated by rotary evaporation, and the crude product is purified by silica gel column chromatography to obtain white crystals, namely compound 3.

[0035] Figure 1 The entire process, from probe immobilization and sampling / online derivatization to paper spray mass spectrometry detection, is demonstrated, showcasing the high degree of integration of the method.

[0036] The detection process using the aforementioned device for detecting aldehydes in exhaled breath includes: Sampling and online derivatization: A paper substrate with the probe immobilized is placed in a sealed cavity (such as a syringe), and a certain volume of exhaled breath or gaseous standard is drawn. The sample is incubated at room temperature (preferably 30 minutes). The hydrazide group of the probe reacts with the gaseous carbonyl compound to form a Schiff base reaction, generating a hydrazone derivative, thus achieving enrichment. Incubation time is another key parameter. Too short a time results in incomplete derivatization, while too long a time reaches equilibrium. 30 minutes is the optimal balance point for achieving efficient derivatization and rapid detection.

[0037] Paper spray mass spectrometry detection: The derivatized paper substrate is removed and fixed on a paper spray ionization source. A spray solvent (preferably methanol, optionally containing an internal standard such as perfluorooctyl quaternary ammonium salt) is added, and a high-voltage direct current (preferably 4 kV) is applied to perform paper spray ionization. The ionized material is then detected by a mass spectrometer (such as Q-Exactive Orbitrap). The spray voltage, paper substrate tip angle (preferably 30°), tip-to-inlet distance (preferably 3 mm), and solvent type have all been systematically optimized. 4 kV can form a stable Taylor cone, achieving efficient ionization; a 30° tip angle can optimize the electric field distribution, forming a stable and concentrated spray; methanol can effectively disrupt fluorine-fluorine interactions, efficiently desorb the target analyte, and has good electrospray performance.

[0038] It should be noted that, in addition to physical coating, the preparation method of the fluorinated stationary phase paper base in this invention can also employ chemical bonding methods (such as silanization reaction), utilizing the hydroxyl groups of the paper base cellulose to covalently link perfluoroalkyl chains to obtain a more stable modified layer. The reactive groups of the fluorinated tag probe, besides hydrazides, can be replaced with boric acid (for cis-diols such as catecholamines), amino groups, etc., depending on the target analyte. The integrated device, besides syringes, can also be designed as other forms of miniature sealed cavities.

[0039] To provide a clearer and more detailed description of the device for detecting aldehydes in exhaled breath and its preparation method provided in the embodiments of the present invention, specific embodiments will be described below.

[0040] Example 1: Synthesis of a fluoro-tagged hydrazine probe (4-PFTBH) according to Figure 2 The steps shown are for synthesis: Synthesis of Compound 1: 4-Mercaptobenzoic acid and perfluorohexyl iodine were reacted in the presence of sodium dithionite to obtain a benzoic acid derivative containing a perfluoroalkyl chain.

[0041] Specific steps: Add 4-mercaptobenzoic acid (1.23 g, 8.0 mmol) to 80 mL of DMF and stir. Then add saturated sodium bicarbonate solution (80 mL), perfluorohexyl iodine (3.47 mL, 16 mmol), and sodium dithionite (1.39 g, 8.0 mmol), and stir at room temperature for approximately 2 hours. After the reaction is complete (TLC control: dichloromethane / methanol = 20:1), dilute with saturated saline solution (50 mL), adjust to weakly acidic pH (pH = 6) with concentrated hydrochloric acid, and extract with ethyl acetate (3 × 50 mL). Combine the organic phases, dry to anhydrous sodium sulfate, and concentrate by rotary evaporation to obtain a yellow oil. Purify by rapid chromatography (dichloromethane / methanol = 20:1) to give compound 1 (yellow solid, 2.96 g, 78%).

[0042] Synthesis of Compound 2: Compound 1 is reacted with oxalyl chloride to convert it into the corresponding acyl chloride.

[0043] Specific steps: Compound 1 (0.47 g, 1 mmol) was dissolved in 10 mL of anhydrous tetrahydrofuran, a few drops of DMF were added, and oxalyl chloride (2 mmol) was added dropwise under ice-water bath cooling. The mixture was stirred at room temperature for 4 h, and the solvent was evaporated to obtain compound 2 (orange oil). The crude product was used directly in the next step.

[0044] Synthesis of Compound 3 (Target Probe): Compound 2 was reacted with hydrazine hydrate to obtain the final product, a fluoro-tagged hydrazine probe. The probe was analyzed by mass spectrometry ([M+H)). + m / z measured 487.0132) and nuclear magnetic resonance (NMR) 1 H NMR, 19 F NMR, 13 The structure was confirmed to be correct by C NMR.

[0045] Specific steps: Compound 2 (1 mmol) was dissolved in 80 mL of acetonitrile and added dropwise to an acetonitrile solution containing hydrazine hydrate (1.2 mmol) and triethylamine (0.5 mL). The mixture was refluxed for 3 h (monitored by TLC). After cooling to room temperature, the mixture was extracted with ethyl acetate (3 × 50 mL). The organic phases were combined, dried over anhydrous sodium sulfate, and concentrated by rotary evaporation. The crude product was purified by silica gel column chromatography (dichloromethane / methanol = 15:1) to give compound 3 (white crystals, 0.245 g, 50.4%).

[0046] Example 2: Preparation of Fluorine Stationary Phase Modified Paper Substrate Coating solution preparation: Dissolve 5g of polyacrylonitrile (PAN) in 70mL of DMF and heat to 90℃. Add 0.3g of fluorinated graphite with a particle size of 10μm and stir for 24 hours to disperse evenly.

[0047] Paper-based modification: Whatman No.1 chromatography paper was cut into triangles with a vertices of 30° and sides of 6mm × 9mm. 100μL of the coating solution was evenly added dropwise onto the triangular paper, and the paper was dried in a vacuum drying oven at 40℃ for 30 minutes to obtain FG@Paper. Characterization was performed using SEM, mapping, FT-IR, and XPS (e.g., [missing information]). Figure 3 , 4 As shown in Figure 5, the fluorinated graphite was successfully and uniformly modified onto the paper substrate surface. SEM and mapping images showed that 10 μm fluorinated graphite particles formed a continuous and uniform coating on the paper substrate surface; FT-IR and XPS spectra confirmed the successful introduction of CF bonds.

[0048] Example 3: Preparation of Fluorine Tag Probe Modified Paper Substrate The fluorine-tagged probe synthesized in Example 1 was diluted with acetonitrile to a concentration of 2.5 mmol / L. The FG@Paper prepared in Example 2 was immersed in the probe solution at room temperature for 10 minutes, then removed and dried in a vacuum drying oven at 30°C for 20 minutes to obtain the probe-functionalized paper substrate, which was stored in a refrigerator at 4°C for later use.

[0049] Example 4: Derivatization of gaseous carbonyl compounds and optimization of paper spray mass spectrometry detection conditions Using pentanal as a model molecule, gaseous derivatization was performed in a 50 mL syringe using a paper substrate prepared in Example 3.

[0050] like Figure 9-14 As shown, the optimal conditions were obtained through optimization: probe concentration: 2.5 mM; incubation time: 30 minutes (room temperature); spray voltage: 4 kV; paper tip angle: 30°; horizontal distance between paper tip and MS injection port: 3 mm; spray solvent: methanol (optionally, with the addition of internal standard perfluorooctyl quaternary ammonium salt).

[0051] Example 5: Simultaneous detection and quantitative analysis of multiple gaseous carbonyl compounds Probe performance verification: The probe 4-PFTBH of this invention was derivatized and detected for pentanal under the same conditions as three commercially available fluorine-containing probes (PFBHA, HDFD-1, HDFD-2). The results are as follows: Figure 6 As shown, the signal response value of 4-PFTBH is significantly better than that of other probes, which is attributed to the high reactivity of its hydrazide group and the optimized molecular structure, achieving the best balance between probe immobilization and target enrichment.

[0052] Small molecule validation: The probe was reacted with gaseous standards of six representative carbonyl compounds (pentanal, heptanal, octanal, benzaldehyde, p-methylbenzaldehyde, and acetone). Mass spectrometry analysis clearly observed the characteristic peaks of the corresponding hydrazone derivatives in all cases, confirming the probe's broad-spectrum derivatization ability. Comparative experiments showed that, as Figure 7 As shown, the signal response value of derivatized pentanal is about 40 times higher than that of pure pentanal directly measured, with a derivatization efficiency of up to 99.63%.

[0053] Paper-based performance comparison: The derivatization effect of probes on pentanal on blank paper and fluorinated graphite-modified paper (FG@Paper) was compared, and the results are as follows: Figure 8 As shown, the signal response value of the derivative on FG@Paper is 25 times that of the blank paper substrate. This demonstrates that the fluorinated graphite modification layer can effectively suppress non-specific adsorption and achieve ordered immobilization of the probe through fluorine-fluorine interactions, significantly improving enrichment efficiency.

[0054] Standard curve establishment: A mixed gas standard containing pentanal, heptanal, octanal, benzaldehyde, and acetone was prepared within a concentration range of 0.05-100 ng / mL. Using the paper substrate prepared in Example 3, derivatization and PS-MS detection were performed under the optimized conditions of Example 4 (incubation for 30 minutes), with perfluorooctyl quaternary ammonium salt as the internal standard. A standard curve was obtained by plotting the peak area of ​​the target analyte against the peak area of ​​the internal standard against the concentration, as shown below. Figure 16-20 As shown. The five compounds exhibit good linearity (R0). 2 >0.995), LODs ranged from 1.67 to 7.11 pg / mL, and LOQs ranged from 5.56 to 23.69 pg / mL. Mass spectrometry was also performed. Figure 15 The results showed that the five derivatives and the internal standard peaks did not interfere with each other and eluted simultaneously in the mass spectrum, demonstrating the high-throughput detection capability.

[0055] Analysis of actual exhaled breath samples: 500 mL of exhaled breath was collected from 18 lung cancer patients and 21 healthy volunteers. 50 mL was drawn using a syringe, derivatized on the paper substrate of Example 3 for 30 minutes, and then analyzed by PS-MS. Quantitative results were analyzed using heatmaps and box plots, as shown below. Figure 21-26 As shown, the levels of all five carbonyl compounds in the lung cancer patient group were significantly higher than those in the healthy control group, proving that this method can effectively distinguish between the two groups and capture lung cancer-related metabolic characteristics.

[0056] Spiked recoveries: Low, medium, and high concentrations of standards were added to real exhaled breath samples, and the recoveries were calculated. The results ranged from 87.9% to 113.4%, with an RSD of <11.54%, indicating that the method is accurate and reliable.

Claims

1. A device for detecting aldehyde compounds in exhaled breath, characterized in that, For sampling, derivatization and enrichment of carbonyl compounds in exhaled breath, including a fluorinated stationary phase modified paper substrate and a fluorinated tag hydrazine probe fixed on the surface of the fluorinated stationary phase modified paper substrate; The fluorinated acylhydrazine probe uses an acylhydrazine group as a reactive group and a perfluoroalkyl chain as a fluorinated tag. The fluorinated acylhydrazine probe is connected to the fluorinated stationary phase-modified paper substrate through the fluorinated tag, and undergoes a Schiff base condensation reaction with the target carbonyl compound through the reactive group.

2. The device for detecting exhaled aldehyde compounds according to claim 1, characterized in that, The fluorine-modified paper substrate is a fluorinated graphite-modified paper substrate.

3. The method for preparing the device for detecting aldehydes in exhaled breath according to claim 1 or 2, characterized in that, The paper substrate preparation step includes a fluorine-modified stationary phase preparation step, wherein the fluorine-modified stationary phase preparation step includes a coating solution preparation step, wherein the coating solution preparation step includes: dissolving polyacrylonitrile in N,N-dimethylformamide and uniformly dispersing fluorinated graphite particles to form a coating solution.

4. The preparation method according to claim 3, characterized in that, The fluorinated graphite particles have a particle size of 10 μm.

5. The preparation method according to claim 3, characterized in that, The preparation step of the fluorinated stationary phase modified paper substrate further includes paper substrate coating, which includes coating the coating solution onto the surface of triangular chromatographic paper and drying it to obtain a fluorinated graphite modified paper substrate.

6. The preparation method according to claim 3, characterized in that, The method also includes a probe immobilization step, which includes: adding or immersing a fluorinated tag hydrazine probe solution onto a fluorinated stationary phase-modified paper substrate, thereby immobilizing the fluorinated tag hydrazine probe on the surface of the fluorinated stationary phase-modified paper substrate through fluorine-fluorine interaction; the concentration of the fluorinated tag hydrazine probe is 2.5 mM.

7. The preparation method according to claim 3, characterized in that, It also includes a fluoro-tagged hydrazine probe synthesis step, which includes: 4-Mercaptobenzoic acid and perfluorohexyl iodine were reacted in the presence of sodium dithionite to give a benzoic acid derivative containing a perfluoroalkyl chain, namely compound 1. Compound 1 was reacted with oxalyl chloride to convert it into the corresponding acyl chloride, yielding compound 2. The compound 2 was reacted with hydrazine hydrate to obtain compound 3, which is the fluorolabeled hydrazine probe.

8. The preparation method according to claim 7, characterized in that, Compound 1 was prepared by the following steps: 4-mercaptobenzoic acid was added to N,N-dimethylformamide and stirred until homogeneous. Then, saturated sodium bicarbonate solution, perfluorohexyl iodine, and sodium dithionite were added. After stirring at room temperature and reacting completely, the mixture was diluted with saturated saline solution, adjusted to weak acidity with concentrated hydrochloric acid, extracted with ethyl acetate, and the organic phases were combined, dried over anhydrous sodium sulfate, and concentrated by rotary evaporation to obtain a yellow oily substance. The substance was purified by chromatography to obtain compound 1.

9. The preparation method according to claim 7, characterized in that, Compound 2 was prepared by the following steps: Compound 1 was dissolved in anhydrous tetrahydrofuran, a few drops of N,N-dimethylformamide were added, oxaloyl chloride was added dropwise under ice-water bath cooling, and after stirring at room temperature, the solvent was evaporated to obtain an orange oily substance, namely compound 2.

10. The preparation method according to claim 7, characterized in that, Compound 3 was prepared by the following steps: Compound 2 was dissolved in acetonitrile, added dropwise to an acetonitrile solution containing hydrazine hydrate and triethylamine, refluxed, cooled to room temperature, extracted with ethyl acetate, the organic phases were combined, dried over anhydrous sodium sulfate, concentrated by rotary evaporation, and the crude product was purified by silica gel column chromatography to obtain white crystals, namely compound 3.

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