A method for rapid detection of exhaled breath using MALDI mass spectrometry

Abstract

The application discloses a method for rapid detection of exhaled gas by using MALDI mass spectrometry, and belongs to the technical field of biomedical detection, which comprises the following steps: (1) preparation of a silicon nanowire chip; (2) surface modification of the silicon nanowire chip; (3) manufacturing of an exhaled gas condensation collection device; (4) assembly of the silicon nanowire chip and the exhaled gas condensation collection device; (5) exhalation of gas into the exhaled gas condensation collection device by a person to be detected; (6) MALDI mass spectrometry detection of the chip with the collected exhaled gas sample through a target plate to obtain a detection result. By combining the miniaturized exhaled gas condensation collection device with the surface-modified silicon nanowire chip, rapid condensation collection of the exhaled gas is realized, and the collected sample can be directly subjected to rapid mass spectrometry detection. Not only is the collection time of the exhaled gas sample greatly shortened, and the demand for the sampling amount is reduced, but also the dizziness caused by long-time continuous blowing is avoided, and the collection experience is greatly improved.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biomedical detection technology, and particularly relates to a method for rapid detection of exhaled gas using MALDI mass spectrometry. BACKGROUND

[0002] Respiratory diseases have become one of the diseases with high mortality in recent years, which include asthma, tracheitis, bronchitis, chronic obstructive pulmonary disease, tuberculosis, lung cancer and other common and frequently-occurring diseases. With the aggravation of air pollution, the increase of smoking population, population aging and other factors, the incidence and mortality of respiratory diseases are increasing year by year, therefore, it is of great strategic significance to develop rapid and accurate respiratory disease diagnosis and health management methods.

[0003] People have been diagnosing respiratory diseases and other diseases through exhaled gas for many years, because exhaled gas contains a large amount of substances related to human metabolism and body flora metabolism, which can not only reflect systemic, endogenous and metabolic information in physiological processes, but also reflect information from inhaled air, food or beverages in the environment. Studies have shown that the information obtained by biochemical detection of exhaled gas samples is more abundant and accurate than single nasal or pharyngeal swab samples, and can greatly reduce the false negative probability in nucleic acid detection. In addition, the collection and detection of exhaled gas have the advantages of non-invasiveness, convenience, repeatability, etc. Compared with traditional biochemical detection methods such as blood test, urine test, imaging detection and tissue sectioning, exhaled gas and its condensate detection have great advantages in detection frequency, sample acquisition and detection cost.

[0004] At present, the preferred detection method for metabolites in human exhaled gas samples is mass spectrometry, among which GC-MS is the most comprehensive and sensitive method for characterizing volatile components in exhaled gas. The exploration of GC-MS for the fingerprint of highly volatile or semi-volatile organic compounds in exhaled gas has been very thorough. However, GC-MS still has some shortcomings in analyzing metabolites in exhaled gas samples. Gas chromatography detection has certain requirements for sample processing, which needs to go through gas sample collection and pre-enrichment, as well as steps such as derivatization and desalination; gas chromatography analysis is only suitable for indirect sampling, but not for real-time analysis, and the detection time is long (typical GC-MS analysis may take one hour), so GC-MS is not suitable for clinical rapid detection and diagnosis. In addition, there is loss and degradation of analytes during GC-MS detection, especially for active or heat-sensitive metabolites.

[0005] It can be seen that in order to perform precise instrument analysis on the complex components of exhaled gas, it is necessary to go through sample collection, sample enrichment and other pretreatment steps, as well as a long detection and analysis step, which restricts the application of exhaled gas in clinical diagnosis and precision medicine.

[0006] Matrix assisted laser desorption / ionization-time of flight mass spectrometry (MALDI-TOF MS) has the advantages of fast detection speed, high throughput and low cost compared with GC-MS, and 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 (SALDI). The advantage of SALDI detection is that the inorganic matrix on the surface does not ionize under laser irradiation, but absorbs laser energy to quickly raise the local temperature and promote the desorption ionization process of the molecules to be detected.

[0007] Therefore, it is an urgent problem to be solved to non-invasively collect and rapidly detect human exhaled air samples by mass spectrometry, shorten the gas sample collection and analysis time, and improve the detection efficiency. SUMMARY

[0008] The purpose of the present application is to provide a method for rapid detection of exhaled air using MALDI mass spectrometry, which solves the problem of low detection efficiency of exhaled air in the prior art.

[0009] The technical scheme adopted by the present application is as follows:

[0010] A method for rapid detection of exhaled air using MALDI mass spectrometry, comprising the following steps:

[0011] Step S1, cutting a p-type silicon wafer into small pieces of 3cm x 3cm and washing them;

[0012] Step S2, immersing the washed small piece silicon wafer in a mixed solution of 0.02M AgNO3 and 4.8M HF for etching to prepare a vertical silicon nanowire array;

[0013] Step S3, washing the small piece silicon wafer with dilute nitric acid to remove the silver catalyst on the small piece silicon wafer, ensuring that the silver catalyst is completely dissolved, to obtain a silicon nanowire chip;

[0014] Step S4, dispersing 100μL of fluorinated ethylene propylene copolymer in an organic solvent to prepare a fluorinated ethylene propylene copolymer modification solution, immersing the surface of the silicon nanowire chip in the modification solution, and then removing 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, the exhaled air condensation collection device is manufactured, the exhaled air condensation collection device includes a condensation base and a detachable exhalation cover detachably installed on the condensation base, a shallow groove for loading a chip is arranged on the upper surface of the condensation base, and a placing groove is arranged at the bottom of the condensation base;

[0016] Step S6, the silicon nanowire chip with the surface modified in step S4 is placed in the shallow groove, a condensation gasket or frozen ice is placed in the placing groove, the detachable exhalation cover is installed on the condensation base, and the air inlet is ensured to be unobstructed;

[0017] Step S7, the person to be detected exhales air into the exhaled air condensation collection device through the air inlet of the detachable exhalation cover, the exhaled air is condensed into liquid by the condensation gasket or frozen ice arranged at the bottom of the condensation base, and is collected on the surface of the silicon nanowire chip, and after the collection is completed, the dried chip is taken out;

[0018] Step S8, the silicon nanowire chip with the exhaled air sample collected is directly subjected to MALDI mass spectrum detection through a target plate, and a detection result is obtained.

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

[0020] Further, in step S3, the washing time of the dilute nitric acid is not less than 1 hour.

[0021] Further, in step S4, the silicon nanowire chip is soaked in the modification solution for not less than 30 minutes.

[0022] To sum up, due to the adoption of the above technical scheme, the present application has the following beneficial effects:

[0023] 1、In the present application, the miniaturized exhaled air condensation collection device is combined with the surface-modified silicon nanowire chip, realizing the rapid condensation collection and efficient mass spectrum detection of exhaled air. Not only the collection time of the exhaled air sample is greatly shortened, and the demand for the sampling amount is reduced, but also the dizziness caused by long-time continuous blowing is avoided, and the collection experience is greatly improved.

[0024] 2、In the present application, the exhaled air and the condensate sample thereof 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, the biochemical molecules in the exhaled air are adsorbed, captured and enriched through the surface nanostructure, the sample processing and transfer steps are avoided, the silicon nanowire chip with the exhaled air sample collected is directly subjected to MALDI mass spectrum detection through a target plate, the target loss and interference are reduced, and the detection process is simplified. At the same time, the silicon nanowire chip has good stability and detection repeatability, and thus has the application prospect of home health management products. BRIEF DESCRIPTION OF DRAWINGS

[0025] In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings needed to be used in the embodiments will be briefly introduced as follows. It should be understood that the following drawings only show some embodiments of the present application, and therefore should not be considered as limiting the scope. For those skilled in the art, other related drawings can also be obtained without creative labor on the basis of these drawings, wherein:

[0026] Fig. 1 Preparation flow chart of the silicon nanowire chip of the present application;

[0027] Fig. 2 Structure schematic diagram of the exhaled air condensation and collection device of the present application;

[0028] Markings in the figure: 1-condensation base, 2-dismountable exhalation cover. DETAILED DESCRIPTION

[0029] In order to make the purpose, technical solutions and advantages of the embodiments of the present application more clear, the technical solutions in the embodiments of the present application will be described clearly and completely in combination with the drawings in the embodiments of the present application. Obviously, the described embodiments are some embodiments of the present application, but not all the embodiments. The components of the embodiments of the present application described and shown in the drawings herein can be arranged and designed in various different configurations.

[0030] Therefore, the following detailed description of the embodiments of the present application provided in the drawings is not intended to limit the scope of the claimed invention, but only represents selected embodiments of the present application. All other embodiments obtained by those skilled in the art on the basis of the embodiments in the present application without creative labor are within the scope of protection of the present application.

[0031] It should be noted that the numerals and letters represent similar items in the following drawings, and 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 application, it should be noted that the terms "center", "upper", "lower", "left", "right", "vertical", "horizontal", "inner", "outer" and the like indicate the orientation or positional relationship based on the orientation or positional relationship shown in the drawings, or the orientation or positional relationship commonly placed when the product of the present application is used, which is only a simplified description for the convenience of describing the present application, and therefore cannot be understood as indicating or implying that the indicated device or element must have a particular orientation, be constructed and operated in a particular orientation, and therefore cannot be understood as limiting the present application. In addition, the terms "first", "second", "third" and the like are only used for differentiation, and cannot be understood as indicating or implying relative importance.

[0033] Furthermore, the terms "horizontal", "vertical", and the like, do not mean that the components must be absolutely horizontal or vertical, but can be slightly inclined. For example, "horizontal" only means that it is more horizontal than "vertical", and does not mean that the structure must be completely horizontal, but can be slightly inclined.

[0034] In the description of the present application, it should also be noted that, unless otherwise explicitly specified and limited, the terms "arrangement", "installation", "connection", "connection" should be understood broadly, for example, it can be fixedly connected, or it can be detachably connected or integrally connected; it can be mechanically connected, or it can be electrically connected; it can be directly connected, or it can be indirectly connected through an intermediate medium; it can be the communication inside two elements. For those skilled in the art, the specific meaning of the above terms in the present application can be understood according to the specific circumstances.

[0035] The drawings accompanying the specification Figs. 1-2 ,

[0036] A method for rapid detection of exhaled gas using MALDI mass spectrometry, comprising the following steps:

[0037] Step S1, cutting a p-type silicon wafer into a small piece of 3cm x 3cm, and washing it;

[0038] Step S2, immerse the washed small piece of 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 piece of silicon wafer with dilute nitric acid to remove the silver catalyst on the small piece of silicon wafer, the washing time of 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, prepare a fluorinated ethylene propylene copolymer modification solution by dispersing 100 mu L of fluorinated ethylene propylene copolymer in an organic solvent, immerse the surface of the silicon nanowire chip in the modification solution for 30 minutes, then remove the excess modification solution on a spin coater to obtain a uniform surface, and complete the surface modification of the silicon nanowire chip;

[0041] Step S5, manufacture an exhaled gas condensation collection device, which comprises a condensation base and a detachable exhalation cover detachably mounted on the condensation base, the upper surface of the condensation base is provided with a shallow groove for loading the chip, and the bottom of the condensation base is provided with a placing groove;

[0042] 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 placing groove, and install the detachable exhalation cover on the condensation base to ensure that the air inlet is unobstructed;

[0043] Step S7, the person to be detected exhales air through the air outlet of the detachable air cover into the exhaled air condensation collection device, the exhaled air is condensed into liquid by the condensation pad or frozen ice arranged at the bottom of the condensation base, and is collected on the surface of the silicon nanowire chip, and after the collection is completed, the dried chip is taken out;

[0044] Step S8, the silicon nanowire chip collecting the exhaled air sample is directly subjected to MALDI mass spectrum detection through the target plate to obtain a detection result.

[0045] In the implementation process of the present application, the silicon nanowire chip is prepared and placed in the shallow groove of the condensation base. Then the detachable exhalation cover is installed on the condensation base. And the condensation pad or frozen ice is placed at the bottom of the condensation base. The collector exhales air through the detachable exhalation cover, and the exhaled air is directly condensed into liquid by the low temperature of the condensation pad or frozen ice at the bottom of the condensation base, and is 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 spectrum detection. This method can greatly shorten the collection time of the exhaled air sample, reduce the demand for sampling amount, improve the detection efficiency, avoid dizziness caused by long-term continuous blowing, 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 detected, and a 355nm Nd:YAG laser beam is used. The pulse ion extraction and laser parameters are set to 120ns and 4_large respectively. The mass spectrum spectrum is obtained by superimposing the results of 500 times and 1000 times laser bombardment in reflection and linear modes respectively. All detections are carried out in parallel for three times. The sample spectrum is compared within and between batches, and the mass spectrum is consistent, showing good stability. The dried chip is stored for different time (the longest is 21 days), and then detected and compared again. The approximate distribution and peak number of the mass spectrum characteristic peaks do not change significantly, proving that the chip has good stability and repeatability in mass spectrum detection. Thus, it has the application prospect of home health management product.

[0047] The above is the embodiment of the present application. The foregoing is each preferred embodiment of the present application, and each preferred embodiment can be arbitrarily combined and used if it is not obviously contradictory or based on a certain preferred embodiment. The embodiments and specific parameters in the embodiments are only for clearly describing the verification process of the application, and are not used to limit the patent protection scope of the application. The patent protection scope of the application is still subject to its claims, and any equivalent structural changes made by using the content of the specification and drawings of the application should also be included in the protection scope of the application.

Claims

1. A method for rapid detection of exhaled gases using MALDI mass spectrometry, characterized in that, Includes the following steps: Step S1: Cut the p-type silicon wafer into 3 cm × 3 cm pieces and clean them; Step S2: Immerse the cleaned small silicon wafers 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, and obtain silicon nanowire chips; 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, thus completing the surface modification of the silicon nanowire chip. Step S5: Manufacture an exhaled air condensation collection device. The exhaled air condensation collection device includes a condensation base and a detachable exhalation mask that can be detachably installed on the condensation base. The upper surface of the condensation base is provided with a shallow groove for loading the chip, and the bottom of the condensation base is provided with a placement groove. Step S6: Place the silicon nanowire chip with surface modification completed in step S4 into a shallow groove, place a condensation pad or frozen ice in the groove, and install the detachable exhalation mask on the condensation base to ensure that the airway is unobstructed. Step S7: The person being tested exhales gas into the exhaled gas condensation and collection device through the vent of the detachable gas mask. The exhaled gas is condensed into liquid by the condensation pad or freezing ice set 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. Step S8: The silicon nanowire chip containing the exhaled breath sample is directly subjected to MALDI mass spectrometry detection through a target plate to obtain the detection results.

2. A method for rapid detection of exhaled gases using MALDI mass spectrometry according to claim 1, characterized in that, In step S2, the etching time is no less than 15 minutes.

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

4. A method for rapid detection of exhaled gases using MALDI mass spectrometry according to claim 1, characterized in that, In step S4, the silicon nanowire chip surface is immersed in the modification solution for no less than 30 minutes.

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