In-situ inactivation stabilization and rapid mass spectrometry detection method for sputum sample

Abstract

The invention provides an in-situ inactivation stabilization and rapid mass spectrometry detection method for a sputum sample, which comprises the following steps: mixing the sputum sample collected in a sputum collector with a stabilizer on a sample collection site, and sealing and uniformly shaking to obtain a viscous sample mixed solution; the stabilizer is an ethanol solution with the volume percent of 75-95%; the sealed sputum collector is stored and transferred at the environment temperature of 4-30 DEG C; the transferred viscous sample mixed solution is directly fed into a neutral desorption electrospray extraction ionization ion source, the viscous sample mixed solution is atomized into neutral aerosol plume, the neutral aerosol plume and the charged electrospray extraction plume are subjected to cross collision in space, and molecules in the sample are directly desorbed and ionized under normal pressure; the method comprises the following steps: acquiring a mass spectrum fingerprint spectrogram of a sputum sample through mass spectrum detection, inputting the mass spectrum fingerprint spectrogram into a database, and classifying and distinguishing the mass spectrum fingerprint spectrogram by utilizing a preset mathematical model. The method is simple in process, low in cost and easy to standardize.

Description

Technical Field

[0001] This invention relates to a method for detecting sputum samples, specifically to an in-situ inactivation and stabilization method for sputum samples and a rapid mass spectrometry detection method, belonging to the field of microbial testing and mass spectrometry analysis technology. Background Technology

[0002] Sputum, as a secretion from the lungs and respiratory tract, contains abundant endogenous small-molecule metabolites such as amino acids, phospholipids, organic acids, and lipid mediators. These metabolites carry important information about the body's pathophysiological state and are typical biological samples for early screening of respiratory diseases such as lung cancer and for metabolomics research. However, traditional sputum sample collection, transportation, and testing processes suffer from at least the following technical deficiencies, which limit its application in the field of early disease warning.

[0003] First, the instability of small molecule metabolites and dependence on cold chain. Sputum samples are rich in various hydrolytic enzymes and microbial metabolic activity. After collection, under the influence of enzymatic reactions and potential microorganisms, thermally unstable small molecule metabolites in the sample will rapidly degrade, oxidize, or transform, leading to the loss of original metabolic fingerprint information. To ensure the integrity of metabolomics information, current procedures typically refrigerate collected samples and rely entirely on cold chain logistics systems for transportation. This not only results in high logistics costs but also limits the feasibility of transporting and analyzing samples after collection in places lacking cold chain conditions, such as medical examination centers, pharmacies, or homes.

[0004] Secondly, mass spectrometry analysis involves cumbersome pretreatment and significant metabolite loss. Sputum is a typical complex biological matrix characterized by high viscosity, high salinity, and high protein content. Traditional chromatography-mass spectrometry (GC-MS) techniques require centrifugation to remove cell debris, liquid-liquid extraction or solid-phase extraction to remove matrix interferences, and possible derivatization before analyzing small molecule metabolites in sputum. These steps are not only time-consuming and labor-intensive, but also prone to the loss of volatile metabolites or incomplete extraction of nonpolar lipids during pretreatment. Furthermore, they are highly dependent on operator experience, making standardized operation difficult.

[0005] Therefore, there is an urgent need for an integrated solution that can achieve immediate stabilization of sputum samples at the collection site, long-term preservation at room temperature, and direct mass spectrometry analysis, in order to break through the dependence of existing technologies on cold chain and complex pretreatment, and meet the needs of early rapid screening for diseases such as lung cancer. Summary of the Invention

[0006] Based on the above background, the purpose of this invention is to provide an in-situ inactivation and stabilization method for sputum samples and a rapid mass spectrometry detection method. By combining high-concentration ethanol in-situ fixation, a room-temperature transport system and open-type ionization mass spectrometry technology, the invention achieves zero cold chain storage and zero pretreatment for direct detection of sputum samples, solving the technical problems of high transport costs and cumbersome and complex mass spectrometry pretreatment in the prior art.

[0007] To achieve the above-mentioned objectives, the present invention provides the following technical solution: An in-situ inactivation and stabilization method for sputum samples followed by rapid mass spectrometry detection includes the following steps: S1. At the sample collection site, the sputum sample collected in the sputum collector is mixed with a stabilizer, sealed and shaken to obtain a viscous sample mixture; the stabilizer is an ethanol solution with a volume percentage of 75%~95%; S2. The sealed sputum collector shall be stored and transported at an ambient temperature of 4℃~30℃. S3. The viscous sample mixture after transportation is directly sent into a neutral desorption electrospray extraction ionization source to atomize the viscous sample mixture into a neutral aerosol plume, which crosses and collides with the charged electrospray extraction plume in space, directly desorbing and ionizing the molecules in the sample under normal pressure. S4. Obtain the mass spectrometry fingerprint spectrum of the sputum sample by mass spectrometry detection, input the mass spectrometry fingerprint spectrum into the database, and classify and distinguish the mass spectrometry fingerprint spectrum using a preset mathematical model.

[0008] An ethanol solution with a volume percentage of 75%–95% provides the optimal balance between biosafety, chemical stability, and mass spectrometry compatibility when used as a stabilizer. Ethanol concentrations below 75% are insufficient to completely inactivate certain ethanol-resistant pathogens and are inadequate to adequately inhibit endogenous enzyme activity. Ethanol concentrations above 95% may lead to excessive sample dehydration, causing some proteins to form overly dense precipitates that can encapsulate target metabolites, reducing the extraction efficiency of subsequent mass spectrometry analysis.

[0009] By limiting the stabilizer to a 75%-95% ethanol solution and mixing it with sputum samples immediately at the collection site, a triple technical effect can be achieved: First, ethanol in this concentration range can penetrate the cell membrane of pathogens, causing proteins to denature and coagulate instantly, achieving immediate inactivation at the biosafety level; second, it effectively inhibits the activity of endogenous enzymes in the sample, blocking enzymatic degradation reactions, thereby freezing the metabolic state of the sample at the molecular level; third, ethanol, as an organic solvent, can initially dissolve mucoproteins in sputum.

[0010] Preferably, in step S1, the volume ratio of the sputum sample to the stabilizer is 1:2~5.

[0011] Preferably, in step S3, the solvent for forming the electrospray extraction plume is a methanol-water mixture containing 0.1% to 1% formic acid by volume, wherein the volume ratio of methanol to water is 1:1.

[0012] Preferably, in step S4, the preset mathematical model is a machine learning classification model trained based on the mass spectrometry fingerprint spectrum of sputum samples with known classification.

[0013] Compared with the prior art, the present invention has the following advantages: This invention provides an in-situ inactivation and stabilization method for sputum samples and rapid mass spectrometry detection. Through the ethanol fixation in step S1, the target analyte in the viscous sample mixture can be preserved at an ambient temperature of 4°C to 30°C for at least 24 hours without affecting subsequent detection results. This completely eliminates the reliance on cold chain, allowing samples to be transported at room temperature using ordinary logistics, greatly reducing transportation costs. This invention employs neutral desorption electrospray extraction ionization technology for viscous sample mixtures containing ethanol. It eliminates the need for traditional secondary sample pretreatment methods such as centrifugation and extraction, directly atomizing the sample into a neutral aerosol, which then collides with the charged extraction plume at atmospheric pressure. Subsequently, mass spectrometry is used to directly obtain the global mass spectrometric fingerprint of the sputum sample, which is then input into a machine learning classification model trained on known classification methods. Through chemometric feature extraction from high-dimensional data, rapid, objective, and automated classification of the sample state is achieved, significantly improving detection throughput and accuracy. Attached Figure Description

[0014] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0015] Figure 1 This is a schematic flowchart of an in-situ inactivation and stabilization method for sputum samples and a rapid mass spectrometry detection method according to the present invention. Detailed Implementation

[0016] The technical solution of the present invention will be further described in detail below through specific embodiments and in conjunction with the accompanying drawings. It should be understood that the implementation of the present invention is not limited to the following embodiments, and any modifications and / or alterations made to the present invention will fall within the protection scope of the present invention.

[0017] In this invention, unless otherwise specified, all parts and percentages are by weight, and the equipment and raw materials used are commercially available or commonly used in the art. Unless otherwise specified, the methods in the following embodiments are conventional methods in the art. Unless otherwise specified, the components or equipment in the following embodiments are general standard parts or components known to those skilled in the art, and their structures and principles can be learned by those skilled in the art through technical manuals or conventional experimental methods.

[0018] The embodiments of the present invention will now be described in detail with reference to the accompanying drawings. In this detailed description, numerous specific details are set forth to facilitate explanation and provide a thorough understanding of the embodiments of the present invention. However, one or more embodiments may be practiced by those skilled in the art without these specific details.

[0019] Example 1

[0020] like Figure 1 As shown in the figure, this embodiment discloses an in-situ inactivation and stabilization method for sputum samples and a rapid mass spectrometry detection method, which includes the following steps: S1, On-site stabilization Deep cough sputum samples were collected using a disposable sputum collection device pre-laced with stabilizer, with a collection volume of approximately 3 mL. The stabilizer was a 75% (v / v) ethanol solution. The mixing volume ratio of sputum sample to stabilizer was 1:3, resulting in 9 mL of ethanol solution.

[0021] Tighten the sealing cap and shake the sputum collector to thoroughly mix the sputum with the ethanol, resulting in a homogeneous, viscous sample mixture. During this process, the high concentration of ethanol penetrates into the interior of pathogens such as bacteria and viruses, causing protein denaturation and inactivation, while simultaneously inhibiting the activity of endogenous enzymes in the sample. The mixed viscous sample mixture shows no obvious precipitation or stratification, indicating that its bioinfectiousness has been eliminated.

[0022] Step S2, Room temperature storage and transportation The sealed sputum collection device was placed at room temperature and stored and transported in a standard insulated box without ice packs. It can be stored at ambient temperatures between 4°C and 30°C for at least 24 hours without affecting subsequent test results. Therefore, the simulated transport conditions were 24 hours at room temperature (25°C), after which it was transferred to the mass spectrometry laboratory.

[0023] Step S3: Neutral desorption electrospray extraction ionization direct analysis The viscous sample mixture in the sputum collection device after transport is directly removed and injected into the nebulizer of a neutral desorption electrospray extraction ionization source. Driven by an inert gas, the viscous sample mixture is atomized into a neutral aerosol plume. Simultaneously, the extraction solvent is introduced into the electrospray needle, forming a charged electrospray extraction plume under the action of a high-voltage power supply. The extraction solvent is a methanol-water mixture containing 0.5% formic acid by volume, with a methanol to water volume ratio of 1:1.

[0024] Neutral aerosol plumes and charged electrospray extraction plumes collide at a specific angle in a spatial collision zone. During this process, target molecules in the neutral aerosols are efficiently extracted into charged droplets and undergo electrospray ionization under ambient pressure to form gaseous ions. These ions are then drawn into a mass spectrometer for analysis.

[0025] Step S4: Mass spectrometry detection and classification Mass spectrometric fingerprints of the sputum samples were acquired using high-resolution mass spectrometry. The acquisition mode was positive ion mode, with a mass-to-charge ratio scan range of m / z 50-1000 and an acquisition time of 30-60 seconds.

[0026] The mass spectrometry fingerprint spectrum is input into the database, and a pre-built mathematical model is used to classify and distinguish the mass spectrometry fingerprint spectrum. The pre-built mathematical model is a machine learning classification model trained based on the mass spectrometry fingerprint spectrum of sputum samples with known classifications, and outputs the disease category or health status of the sample.

[0027] Example 2

[0028] This embodiment discloses an in-situ inactivation and stabilization method for sputum samples and a rapid mass spectrometry detection method. The method is the same as that in Example 1, except that the stabilizer is an 85% (v / v) ethanol solution.

[0029] Example 3

[0030] This embodiment discloses an in-situ inactivation and stabilization method for sputum samples and a rapid mass spectrometry detection method. The method is the same as that in Example 1, except that the stabilizer is a 95% volume percentage ethanol solution.

[0031] Comparative Example 1 This embodiment discloses an in-situ inactivation and stabilization method for sputum samples and a rapid mass spectrometry detection method. The method is the same as that in Embodiment 1, except that no stabilizer is added to the collected sputum samples.

[0032] Comparative Example 2 This embodiment discloses an in-situ inactivation and stabilization method for sputum samples and a rapid mass spectrometry detection method. The method is the same as that in Embodiment 1, except that the stabilizer is a 50% (v / v) ethanol solution.

[0033] Comparative Example 3 This embodiment discloses an in-situ inactivation and stabilization method for sputum samples and a rapid mass spectrometry detection method. The method is the same as that in Example 1, except that the stabilizer is a 70% (v / v) ethanol solution.

[0034] Comparative Example 4 This embodiment discloses an in-situ inactivation and stabilization method for sputum samples and a rapid mass spectrometry detection method. The method is the same as that in Example 1, except that the stabilizer is a 100% volume percentage ethanol solution.

[0035] The stabilization effects of Examples 1-3 and Comparative Examples 1-4 were evaluated by monitoring the signal intensity retention rate of the characteristic metabolites phosphatidylcholine and lysophosphatidylcholine, as well as changes in the physical state of the samples. The test results are shown in Table 1.

[0036] Table 1. Comparison of stabilization effects in Examples 1-3 and Comparative Examples 1-4

[0037] Compared to Comparative Example 1, all experimental groups with added ethanol showed significant stabilization effects, confirming the necessity of adding stabilizers at the sample collection site. In Comparative Example 1, without stabilizers, the retention rate of characteristic metabolites was less than 10% after 24 hours, and the sample had spoiled and could not be used for mass spectrometry analysis.

[0038] Comparative Example 2 used a 50% (v / v) ethanol solution as a stabilizer. While it had some antibacterial effect, it could not completely inactivate ethanol-resistant pathogens and was insufficient to adequately inhibit the activity of proteases and lipases. During 24 hours of storage, residual enzyme activity led to a metabolite degradation rate of over 40%. Furthermore, the 50% ethanol solution may cause some proteins to form soluble complexes, which could actually promote enzyme-substrate contact.

[0039] Comparative Example 3 used a 70% volume percentage ethanol solution as a stabilizer. 70% ethanol is close to but lower than the 75% lower limit of the present invention. Although it has a certain protective effect on some metabolites, the retention rate of characteristic metabolites reached 76.4% after 24 hours. However, some microorganisms may still survive, and the enzyme inhibition is incomplete, resulting in high baseline noise of mass spectrometry and affecting the detection sensitivity.

[0040] Comparative Example 4 used a 100% volume ethanol solution as a stabilizer. Although anhydrous ethanol can completely inactivate pathogens, it can cause excessive dehydration of proteins in the sample, forming overly dense precipitates that are difficult to redisperse. These dense precipitates encapsulate some target metabolites, especially lipid-soluble molecules, which cannot be effectively released during subsequent nebulization, resulting in a decrease in mass spectrometry signal intensity and poor reproducibility.

[0041] In summary, the 75%-95% ethanol concentration range in the stabilizer specified in this invention denatures and precipitates pathogen proteins without excessive cross-linking, maintaining sample dispersibility. Furthermore, it effectively inhibits various endogenous enzymes such as proteases, esterases, and phosphatases, blocking degradation pathways. Ethanol within this concentration range does not affect the desorption efficiency of target molecules due to excessive dehydration.

[0042] Example 2 demonstrated the best overall performance, indicating that the selection of this concentration range was not a simple choice of antibacterial concentration, but rather a synergistic optimization result that comprehensively considered biosafety, chemical stability, and compatibility with mass spectrometry analysis.

[0043] This invention, by limiting the stabilizer to a 75%-95% (v / v) ethanol solution, allows for immediate mixing with sputum samples at the collection site. Combined with ambient temperature transport conditions of 4°C to 30°C, this achieves a metabolite retention rate exceeding 95% in the sample without relying on a cold chain, while fully meeting biosafety standards. Comparative experiments clearly demonstrate that only within the ethanol concentration range specified in this invention can an optimal balance be achieved between inactivation effect, enzyme inhibition efficiency, and applicability to mass spectrometry analysis. This solves the key technical challenges of on-site sputum sample collection and ambient temperature transport, making it particularly suitable for applications in early screening and warning of diseases such as lung cancer.

[0044] This article uses specific examples to illustrate the principles and implementation methods of the present invention. The descriptions of the above embodiments are only for the purpose of helping to understand the method and core ideas of the present invention. It should be noted that those skilled in the art can make several improvements and modifications to the present invention without departing from the principles of the present invention, and these improvements and modifications also fall within the protection scope of the claims of the present invention.

Claims

1. A method for in-situ inactivation stabilization and rapid mass spectrometry detection of sputum samples, characterized in that: The method includes the following steps: S1. At the sample collection site, the sputum sample collected in the sputum collector is mixed with a stabilizer, sealed and shaken to obtain a viscous sample mixture; the stabilizer is an ethanol solution with a volume percentage of 75%~95%; S2. The sealed sputum collector shall be stored and transported at an ambient temperature of 4℃~30℃. S3. The viscous sample mixture after transportation is directly sent into a neutral desorption electrospray extraction ionization source to atomize the viscous sample mixture into a neutral aerosol plume, which crosses and collides with the charged electrospray extraction plume in space, directly desorbing and ionizing the molecules in the sample under normal pressure. S4. Obtain the mass spectrometry fingerprint spectrum of the sputum sample by mass spectrometry detection, input the mass spectrometry fingerprint spectrum into the database, and classify and distinguish the mass spectrometry fingerprint spectrum using a preset mathematical model.

2. The method for in-situ inactivation stabilization and rapid mass spectrometry detection of sputum samples according to claim 1, characterized in that: In step S1, the volume ratio of the sputum sample to the stabilizer is 1:2~5.

3. The method for in-situ inactivation stabilization and rapid mass spectrometry detection of sputum samples according to claim 1, characterized in that: In step S3, the solvent for forming the electrospray extraction plume is a methanol-water mixed solution containing 0.1% to 1% formic acid by volume, wherein the volume ratio of methanol to water is 1:

1.

4. The method for in-situ inactivation stabilization and rapid mass spectrometry detection of sputum samples according to claim 1, characterized in that: In step S4, the preset mathematical model is a machine learning classification model trained based on the mass spectrometry fingerprint spectrum of sputum samples with known classification.

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