Method for rapidly detecting exhaled gas by using MALDI (matrix-assisted laser desorption ionization) mass spectrometry

Abstract

The invention discloses a method for rapidly detecting exhaled gas by using MALDI (matrix-assisted laser desorption ionization) mass spectrometry, which belongs to the technical field of biomedical detection and comprises the following steps: (1) preparing a silicon nanowire chip; (2) modifying the surface of the silicon nanowire chip; (3) manufacturing an expired gas condensation collecting device; (4) assembling the silicon nanowire chip and an expired gas condensation and collection device; (5) the person to be detected exhales gas into the exhaled gas condensation and collection device; and (6) directly carrying out MALDI mass spectrometry detection on the chip collecting the exhaled gas sample through a target plate to obtain a detection result. A miniaturized expired gas condensation collection device is combined with a surface-modified silicon nanowire chip, so that rapid condensation collection of expired gas is realized, and rapid mass spectrometric detection can be directly carried out after collection. The sampling time of the exhaled air sample is greatly shortened, the requirement for the sampling amount is reduced, meanwhile, dizziness and other conditions caused by long-time continuous air blowing are avoided, and the sampling experience is greatly improved.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedical detection, and particularly relates to a method for rapid detection of exhaled breath using MALDI mass spectrometry. Background Art

[0002] Respiratory diseases have become one of the diseases with relatively high mortality rates in recent years. It includes various common and frequently-occurring diseases such as asthma, tracheitis, bronchitis, chronic obstructive pulmonary disease, tuberculosis, lung cancer, etc. With the aggravation of air pollution, the increase in the number of smokers, population aging, and other factors, the incidence and mortality of respiratory diseases are increasing year by year. Therefore, developing rapid and accurate diagnostic and health management methods for respiratory diseases has great strategic significance.

[0003] People have a history of many years of using exhaled breath for the diagnosis of respiratory diseases and other diseases. Because exhaled breath contains a large number of substances related to human metabolism and the metabolism of the body's flora, it can not only reflect the metabolite information generated in the systemic, endogenous, and physiological processes, but also reflect the information of the air inhaled from the environment, the food or beverages ingested, etc. Research shows that the information obtained from biochemical detection of exhaled breath samples is richer and more accurate than that of single nasal and pharyngeal swab samples, and can greatly reduce the false negative probability in nucleic acid detection. In addition, the collection and detection of exhaled breath have the advantages of being non-invasive, convenient, and repeatable. Compared with traditional biochemical detection methods such as blood tests, urine tests, imaging tests, tissue sections, etc., the detection of exhaled breath and its condensate has great advantages in detection frequency, sample acquisition, and detection cost.

[0004] Currently, the preferred detection method for metabolites in human exhaled breath samples is mass spectrometry. Among them, GC-MS is the most comprehensive and sensitive method for characterizing volatile components in exhaled breath, and its exploration of the fingerprint spectra of highly volatile or semi-volatile organic compounds in exhaled breath has been very in-depth. However, there are still some deficiencies in the analysis of metabolites in exhaled breath samples by GC-MS. Gas chromatography detection has certain requirements for sample processing, and it needs to go through steps such as gas sample collection and pre-concentration, as well as derivatization, desalting, etc.; gas chromatography analysis is only suitable for indirect sampling and not for real-time analysis, and the detection takes a long time (a typical GC-MS analysis may take an hour). Therefore, GC-MS is not suitable for clinical rapid detection and diagnosis. In addition, there are losses and degradations of analytes during the GC-MS detection process, especially the losses and degradations of active or heat-sensitive metabolites.

[0005] It can be seen that to perform precise instrumental analysis on the complex components of exhaled breath, it is necessary to go through pretreatment steps such as gas sample collection and sample enrichment, as well as long detection and analysis steps. These factors restrict the application of exhaled breath in clinical diagnosis and precision medicine.

[0006] Matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF MS) has significant advantages such as fast detection speed, high throughput, and low cost compared to GC-MS. As a soft ionization technique, it has unique advantages in detecting and identifying metabolites in complex biological samples and performing extensive qualitative and quantitative analysis of metabolites. The detection effect of laser desorption ionization mass spectrometry can be further improved through surface assistance, which is called surface-assisted laser desorption / ionization mass spectrometry (SALDI). The advantages of SALDI detection are reflected in that the inorganic matrix on the surface does not ionize under laser radiation, but absorbs laser energy to rapidly increase the local temperature, promoting the desorption ionization process of the molecule to be detected.

[0007] Therefore, it is an urgent problem to be solved to perform non-invasive collection and rapid mass spectrometry detection on human exhaled gas samples, shorten the collection and analysis time of gas samples, and improve the detection efficiency. Summary of the Invention

[0008] The purpose of the present invention is to provide a method for rapid detection of exhaled gas using MALDI mass spectrometry, so as to solve the problem of low detection efficiency of exhaled gas in the prior art.

[0009] The technical solution adopted by the present invention is as follows:

[0010] A method for rapid detection of exhaled gas using MALDI mass spectrometry includes the following steps:

[0011] Step S1: Cut a p-type silicon wafer into small pieces of 3 cm × 3 cm and clean them.

[0012] Step S2: Immerse the cleaned small silicon wafer in a mixed solution of 0.02 M AgNO3 and 4.8 M HF for etching to prepare a vertical silicon nanowire array.

[0013] Step S3: Wash the small silicon wafer with dilute nitric acid to remove the silver catalyst on the small silicon wafer and ensure that the silver catalyst is completely dissolved to obtain a silicon nanowire chip.

[0014] Step S4: Disperse 100 μL of fluorinated ethylene propylene copolymer in an organic solvent to prepare a fluorinated ethylene propylene copolymer modification solution. Immerse the surface of the silicon nanowire chip in the modification solution, and then remove the excess modification solution on a spin coater to obtain a uniform surface, completing the surface modification of the silicon nanowire chip.

[0015] Step S5: Fabricate an exhaled breath condensate collection device, which includes a condensation base and a detachable exhalation mask detachably mounted on the condensation base. A shallow groove for loading the chip is provided on the upper surface of the condensation base, and a placement groove is provided at the bottom of the condensation base;

[0016] Step S6: Place the silicon nanowire chip with surface modification completed in Step S4 in the shallow groove, place a condensation gasket or frozen ice in the placement groove, and install the detachable exhalation mask on the condensation base to ensure that the ventilation port is unobstructed;

[0017] Step S7: The person to be tested exhales gas into the exhaled breath condensate collection device through the ventilation port of the detachable gas mask. The exhaled gas is condensed into a liquid by the condensation gasket or frozen ice provided at the bottom of the condensation base and collected on the surface of the silicon nanowire chip. After the collection is completed, take out the dried chip;

[0018] Step S8: Directly perform MALDI mass spectrometry detection on the silicon nanowire chip collecting the exhaled breath sample through a target plate to obtain the detection result.

[0019] Furthermore, in Step S2, the etching time is not less than 15 minutes.

[0020] Furthermore, in Step S3, the washing time with dilute nitric acid is not less than 1 hour.

[0021] Furthermore, in Step S4, the time for soaking the surface of the silicon nanowire chip in the modification solution is not less than 30 minutes.

[0022] In summary, due to the adoption of the above technical solutions, the beneficial effects of the present invention are as follows:

[0023] 1. In the present invention, through the combination of a miniaturized exhaled breath condensate collection device and a surface-modified silicon nanowire chip, rapid condensation collection and efficient mass spectrometry detection of exhaled breath are achieved. It not only greatly shortens the collection time of exhaled breath samples, reduces the demand for the sampling volume, but also avoids situations such as dizziness caused by continuous blowing for a long time, and the collection experience is greatly improved.

[0024] 2. In the present invention, the exhaled gas and its condensate sample are directly collected on the surface of the silicon nanowire chip. The silicon nanowire chip has the functions of enhancing the target capture ability and assisting the desorption ionization process. It adsorbs, captures, and enriches the biochemical molecules in the exhaled breath through the nanoscale structure on the surface, avoiding steps such as sample treatment and transfer. The silicon nanowire chip collecting the exhaled breath sample is directly subjected to MALDI mass spectrometry detection through a target plate subsequently, reducing the loss and interference of the target, and simplifying the detection process. At the same time, due to the good stability and detection repeatability of the silicon nanowire chip, it thus has the application prospect of home health management products. Brief Description of the Drawings

[0025] To more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as limiting the scope. For those of ordinary skill in the art, without creative efforts, other related drawings can also be obtained based on these drawings, where:

[0026] Figure 1 is the preparation flow chart of the silicon nanowire chip of the present invention;

[0027] Figure 2 is the structural schematic diagram of the exhaled breath condensate collection device of the present invention;

[0028] Markings in the figure: 1 - condensation base, 2 - detachable exhalation mask. Detailed Embodiments

[0029] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are some, but not all, of the embodiments of the present invention. Usually, the components of the embodiments of the present invention described and shown in the drawings here can be arranged and designed in various different configurations.

[0030] Therefore, the following detailed description of the embodiments of the present invention provided in the drawings is not intended to limit the scope of the claimed invention, but merely represents the selected embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts belong to the scope of protection of the present invention.

[0031] It should be noted that: reference numerals and letters represent similar items in the following drawings. Therefore, once an item is defined in one drawing, it does not need to be further defined and explained in subsequent drawings.

[0032] In the description of the present invention, it should be noted that the orientation or positional relationship indicated by the terms "center", "upper", "lower", "left", "right", "vertical", "horizontal", "inner", "outer", etc. is based on the orientation or positional relationship shown in the drawings or the orientation or positional relationship in which the invention product is usually placed during use. It is only for the convenience of describing the present invention and is a simplified description, rather than indicating or implying that the device or element referred to must have a specific orientation, be constructed and operated in a specific orientation, and therefore should not be construed as a limitation of the present invention. In addition, the terms "first", "second", "third", etc. are only used for descriptive distinction and cannot be understood as indicating or implying relative importance.

[0033] In addition, terms such as "horizontal" and "vertical" do not require the components to be absolutely horizontal or hanging vertically, but can be slightly inclined. For example, "horizontal" only means that its direction is more horizontal relative to "vertical", and does not mean that the structure must be completely horizontal, but can be slightly inclined.

[0034] In the description of the present invention, it should also be noted that, unless otherwise clearly specified and limited, the terms "arrange", "install", "connect", and "join" should be understood in a broad sense. For example, it can be a fixed connection, a detachable connection, or an integral connection; it can be a mechanical connection or an electrical connection; it can be directly connected or indirectly connected through an intermediate medium, and it can be the communication inside two components. For those of ordinary skill in the art, the specific meanings of the above terms in the present invention can be understood according to specific situations.

[0035] Combined with the attached drawings of the specification Figure 1-2 ,

[0036] A method for rapid detection of exhaled breath using MALDI mass spectrometry includes the following steps:

[0037] Step S1: Cut a p-type silicon wafer into small pieces of 3 cm × 3 cm and clean them.

[0038] Step S2: Immerse the cleaned small silicon wafer in a mixed solution of 0.02M AgNO3 and 4.8M HF for etching. The etching time is not less than 15 minutes to prepare a vertical silicon nanowire array.

[0039] Step S3: Wash the small silicon wafer with dilute nitric acid to remove the silver catalyst on the small silicon wafer. The washing time with dilute nitric acid is not less than 1 hour to ensure that the silver catalyst is completely dissolved, and a silicon nanowire chip is obtained.

[0040] Step S4: Disperse 100 μL of fluorinated ethylene propylene copolymer in an organic solvent to prepare a fluorinated ethylene propylene copolymer modification solution. Immerse the surface of the silicon nanowire chip in the modification solution for 30 minutes, and then remove the excess modification solution on a spin coater to obtain a uniform surface, completing the surface modification of the silicon nanowire chip.

[0041] Step S5: Manufacture an exhaled breath condensate collection device. The exhaled breath condensate collection device includes a condensation base and a detachable exhalation mask detachably installed on the condensation base. A shallow groove for loading the chip is provided on the upper surface of the condensation base, and a placement groove is provided at the bottom of the condensation base.

[0042] Step S6: Place the silicon nanowire chip with the surface modification completed in step S4 in the shallow groove, place a condensation gasket or frozen ice in the placement groove, install the detachable exhalation mask on the condensation base, and ensure that the ventilation port is unobstructed.

[0043] Step S7: The person to be tested exhales gas into the exhaled breath condensate collection device through the ventilation port of the detachable air hood. The exhaled gas is condensed into a liquid by the condensation gasket or frozen ice provided at the bottom of the condensation base and collected on the surface of the silicon nanowire chip. After the collection is completed, the dried chip is taken out.

[0044] Step S8: The silicon nanowire chip collecting the exhaled breath sample is directly subjected to MALDI mass spectrometry detection through a target plate to obtain a detection result.

[0045] In the implementation process of the present invention, after the silicon nanowire chip is prepared, it is placed in the shallow groove of the condensation base. Then the detachable exhalation hood is installed on the condensation base. And a condensation gasket or frozen ice is placed at the bottom of the condensation base. The person being collected exhales gas through the detachable exhalation hood. The exhaled gas is directly condensed into a liquid by the low temperature generated by the condensation gasket or frozen ice at the bottom of the condensation base and collected on the surface of the silicon nanowire chip. After the collection is completed, the dried chip is taken out for subsequent direct MALDI mass spectrometry detection. This method can greatly shorten the collection time of exhaled breath samples, reduce the requirement for the sampling volume, improve the detection efficiency, avoid situations such as dizziness caused by continuous blowing for a long time, and greatly improve the collection experience.

[0046] The UltrafleXtreme MALDI-TOF / TOF instrument of Bruker Daltonics company is used to detect the sample to be tested, and a 355nm Nd:YAG laser beam is used. The pulse ion extraction and laser parameters are respectively set to 120ns and 4_large. The results of 500 times and 1000 times of laser bombardment are respectively superimposed in the reflection and linear modes to obtain a mass spectrometry spectrum. All detections are carried out in parallel three times. The sample spectra are compared within batches and between batches, and the mass spectrometry spectra are all consistent, showing good stability. The dried chips are stored for different times (up to 21 days) and then detected and compared again. There are no significant changes in the general distribution and number of mass spectrometry characteristic peaks, which proves that the chip has good stability and repeatability in mass spectrometry detection. Therefore, it has the application prospect of home health management products.

[0047] The above is the embodiment of the present invention. The foregoing are the various preferred embodiments of the present invention. If the preferred implementation manners in the various preferred embodiments are not obviously self-contradictory or premised on a certain preferred implementation manner, the various preferred implementation manners can be arbitrarily superimposed and combined for use. The embodiments and the specific parameters in the embodiments are only for clearly expressing the verification process of the invention, and are not used to limit the patent protection scope of the present invention. The patent protection scope of the present invention still takes its claims as the criterion. All equivalent structural changes made by using the content of the specification and drawings of the present invention should, by the same token, be included in the protection scope of the present invention.

Claims

1. A method for rapid detection of exhaled breath using MALDI mass spectrometry, characterized in that, It includes the following steps: Step S1: Cut the p-type silicon wafer into small pieces of 3 cm × 3 cm and clean them; Step S2: Immerse the cleaned small silicon wafer in a mixed solution of 0.02M AgNO3 and 4.8M HF for etching to prepare a vertical silicon nanowire array; Step S3: Wash the small silicon wafer with dilute nitric acid to remove the silver catalyst on the small silicon wafer, ensuring that the silver catalyst is completely dissolved to obtain a silicon nanowire chip; Step S4: Disperse 100 μL of fluorinated ethylene propylene copolymer in an organic solvent to prepare a fluorinated ethylene propylene copolymer modification solution. Immerse the surface of the silicon nanowire chip in the modification solution, and then remove the excess modification solution on a spin coater to obtain a uniform surface, completing the surface modification of the silicon nanowire chip; Step S5: Manufacture an exhaled breath condensate collection device, which includes a condensation base and a detachable exhalation hood detachably installed on the condensation base. A shallow groove for loading the chip is provided on the upper surface of the condensation base, and a placement groove is provided at the bottom of the condensation base; Step S6: Place the silicon nanowire chip with surface modification completed in Step S4 in the shallow groove, place a condensation gasket or frozen ice in the placement groove, and install the detachable exhalation hood on the condensation base to ensure that the ventilation port is unobstructed; Step S7: Let the person to be tested exhale gas into the exhaled breath condensate collection device through the ventilation port of the detachable gas hood. The exhaled gas is condensed into a liquid by the condensation gasket or frozen ice provided at the bottom of the condensation base and collected on the surface of the silicon nanowire chip. After the collection is completed, take out the dried chip; Step S8: Directly perform MALDI mass spectrometry detection on the silicon nanowire chip collecting the exhaled breath sample through a target plate to obtain a detection result.

2. The method for rapid detection of exhaled breath using MALDI mass spectrometry according to claim 1, characterized in that, In the said Step S2, the etching time is not less than 15 minutes.

3. A method for rapid detection of exhaled breath using MALDI mass spectrometry according to claim 1, characterized in that, In the said Step S3, the washing time with dilute nitric acid is not less than 1 hour.

4. A method for rapid detection of exhaled breath using MALDI mass spectrometry according to claim 1, characterized in that, In the said Step S4, the time for immersing the surface of the silicon nanowire chip in the modification solution is not less than 30 minutes.

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