Rapid Evaporation Ionization Mass Spectrometry and Desorption Electrospray Ionization Mass Spectrometry Analysis of Swab and Biopsy Samples

Abstract

A method is disclosed comprising providing a biological sample on a swab (900), directing a spray of charged droplets onto a surface of the swab (900) to generate a plurality of analyte ions, and analyzing the analyte ions.

Description

[0001] Related Applications

[0002] This application is a divisional application with application date of March 7, 2016, application number 201680026285.3 (PCT / GB2016 / 050621), and invention name “Rapid Evaporation Ionization Mass Spectrometry (“REIMS”) and Desorption Electrospray Ionization Mass Spectrometry (“DESI-MS”) Analysis of Swab and Biopsy Samples”.

[0003] This application claims priority to and the benefit of UK Patent Application No. 1503876.3 filed on March 6, 2015, UK Patent Application No. 1503864.9 filed on March 6, 2015, UK Patent Application No. 1518369.2 filed on October 16, 2015, UK Patent Application No. 1503877.1 filed on March 6, 2015, UK Patent Application No. 1503867.2 filed on March 6, 2015, UK Patent Application No. 1503863.1 filed on March 6, 2015, UK Patent Application No. 1503878.9 filed on March 6, 2015, UK Patent Application No. 1503879.7 filed on March 6, 2015, and UK Patent Application No. 1516003.9 filed on September 9, 2015. The entire contents of these applications are incorporated herein by reference. Technical Field

[0004] The present invention relates generally to mass spectrometry, in particular to the analysis of material by open ionisation ion sources including rapid evaporative ionisation mass spectrometry ("REIMS") ion sources and the analysis of material by desorption electrospray ionisation ("DESI") mass spectrometry. Various embodiments relate to the use of a mass spectrometer in a diagnostic method. The following embodiments are encompassed, wherein the analyte ions produced by the open ionization ion source are subsequently subjected to: (i) mass analysis by a mass analyzer, such as a quadrupole mass analyzer or a time-of-flight mass analyzer; (ii) ion mobility analysis (ion mobility analysis, IMS) and / or differential ion mobility analysis (differential ion mobility analysis, DMA) and / or field asymmetric ion mobility spectrometry (Field Asymmetric Ion Mobility Spectrometry, FAIMS) analysis; and / or (iii) the following combination: first ion mobility analysis (IMS) and / or differential ion mobility analysis (DMA) and / or field asymmetric ion mobility spectrometry (FAIMS) analysis is performed, and secondly mass analysis is then performed by a mass analyzer, such as a quadrupole mass analyzer or a time-of-flight mass analyzer (or vice versa). Various embodiments also relate to ion mobility spectrometers and / or mass analyzers and ion mobility spectrometry methods and / or mass analysis methods. Background Art

[0005] Mucosa is a protective layer responsible for trapping pathogens in the human body. Mucosa is an easily accessible and clinically highly relevant sample for diagnosing pathogens and cancer-related diseases.

[0006] It is known to use medical swabs as a standard collection device for mucous membranes.

[0007] Conventionally, the swab is placed in a sterile tube containing a buffer solution for storage, and the tube is then sent to a laboratory for analysis. The laboratory that receives the tube will swab the contents of the smear onto a culture medium, such as an agar plate. The culture medium is then incubated to grow the organisms present on the swab.

[0008] Identification of the microorganisms may then be carried out, for example, under a microscope. Any organisms present in the sample may also be identified by 16S gene sequencing and / or by using matrix-assisted laser desorption ionisation ("MALDI") mass spectrometry and then comparing the mass spectra to commercial databases.

[0009] Although easy to perform, the current approach to analyzing medical swabs for diagnostic purposes relies on culture and involves a time-consuming and costly workflow. As a result, the diagnosis and appropriate treatment of diseases such as infection or dysbiosis are greatly delayed. In addition, approximately 95% of bacteria cannot be cultured for analysis.

[0010] It would therefore be desirable to provide an improved method for mucosal analysis, such as a diagnostic method. Summary of the invention

[0011] According to one aspect, there is provided a method comprising:

[0012] Provide a sample on a swab;

[0013] directing a spray of charged droplets onto a swab surface to generate a plurality of analyte ions; and

[0014] Analyte ions are analyzed.

[0015] Desorption electrospray ionization ("DESI") is an open ionization method that involves directing a spray of (primary) charged droplets onto a surface. The electrospray mist is directed pneumatically to the sample, where the subsequent splashing (secondary) droplets carry the desorption-ionized analytes. After ionization, the resulting ions proceed through the air and into the atmospheric pressure interface of the mass spectrometer. Desorption electrospray ionization ("DESI") is a technique that allows open ionization of trace samples at atmospheric pressure without the need for sample preparation.

[0016] It is intended that a "swab" according to various embodiments be understood to include a "standard medical swab", i.e. a swab designed to be used to take biological samples such as mucous membranes. For example, the term "standard medical swab" should be understood to cover a "cotton swab" (UK) or a "cotton swab" (USA), i.e. a small ball of cotton wrapped around one or both ends of a tube. The tube can be made of plastic, rolled paper or wood.

[0017] StableFlex (RTM) fiber cores are known, which comprise a polymer coated 80 μm fused silica core. Such fiber cores are not intended to comprise a "swab" within the meaning of this application, as a material having a fiber core is not considered to comprise a "standard medical swab".

[0018] In particular, the terms "swab", "medical swab" and "standard medical swab" as used within this application are intended to exclude materials having a fused silica core.

[0019] It is known that Solid Phase Micro Extraction ("SPME") fibers can be directly analyzed using Desorption Electrospray Ionization ("DESI") Mass Spectrometry. In this technique, the SPME fiber is exposed to the headspace within a vial containing the sample. Alternatively, the SPME fiber can be immersed in the sample solution. According to the known technique, the analyte can be extracted into the liquid or gas phase SPME fiber coating, that is, the sample is no longer retained in its native state. According to the known technique, the fiber is then directly subjected to Desorption Electrospray Ionization ("DESI") Mass Spectrometry analysis by positioning the SPME fiber in the spray of the Desorption Electrospray Ionization ("DESI") Mass Spectrometry arrangement.

[0020] Therefore, this known technique requires (at least) a two-step sample preparation procedure, comprising: (i) collecting or preparing the sample, followed by (ii) extracting the analyte into the SPME fiber. Furthermore, the known specialized SPME fibers are not suitable for, for example, sampling biological material directly from a patient, such as directly sampling a mucosal membrane.

[0021] In contrast, various embodiments are based at least in part on the recognition that a sample provided on a swab, such as a standard medical swab, can be directly analyzed by desorption electrospray ionization ("DESI") mass spectrometry. Specifically, an important aspect of various embodiments is that the standard medical swab itself can be placed in the spray of a desorption electrospray ionization ("DESI") ionization source.

[0022] Various embodiments are advantageous in that they require no sample preparation steps beyond collecting the sample onto the swab.

[0023] Therefore, various embodiments provide a rapid and direct analysis method for a sample provided on a medical swab.

[0024] Various embodiments are particularly suitable and applicable for analyzing biological material, for example from a patient, such as material sampled directly from a mucosa onto a swab.

[0025] In addition, the use of medical swabs in desorption electrospray ionization ("DESI") mass spectrometry according to various embodiments opens up the possibility of performing multiple different analyses on the same sample. For example, multiple different analyses can be performed on the same swab. This approach advantageously provides multiple sets of information or data related to the same sample in a particularly convenient and efficient manner.

[0026] This ability to perform multiple different analyses on the same swab is due to the fact that desorption electrospray ionization ("DESI") mass spectrometry is a relatively non-destructive analytical technique. In addition, other commercial analytical techniques, such as culture techniques and 16S rRNA sequencing techniques, are optimized to use samples provided on (standard) medical swabs.

[0027] Thus, after a single sample is collected onto a swab, the sample on the swab can be analyzed multiple times using multiple different analytical techniques.

[0028] It will therefore be appreciated that various embodiments provide improved analytical methods, such as diagnostic methods.

[0029] Unless otherwise indicated, the terms "biological sample", "biological material" and the like are used interchangeably herein, and they may optionally include or consist of biological tissue. Biological material and the like may optionally be selected from, for example, surgical resection specimens, biopsy specimens, tissue specimens, cell specimens, smears, body fluid specimens and / or stool specimens, any of which may be sampled from a subject, such as directly sampled on a swab or removed as a biopsy, or sampled ex vivo or in vitro, such as a sample may be provided, then sampled on a swab, which may be referred to as indirect sampling on a swab, or sampled with a biopsy needle, which may be referred to as indirect sampling with a biopsy needle. The method may optionally be performed on a provided biological sample, such as a provided biopsy or a provided swab, i.e., a swab on which a sample of biological material is pre-collected.

[0030] The body fluid specimen can, for example, be optionally selected from blood, plasma, serum, sputum, lavage fluid, pus, urine, saliva, mucus, vomitus, feces, amniotic fluid, cerebrospinal fluid, pleural fluid, semen, sputum, vaginal secretions, interstitial fluid and / or lymph.

[0031] The swab may comprise a medical swab or a standard medical swab.

[0032] The swab may comprise a disposable swab.

[0033] The swabs may contain cotton, rayon, plastic, or foam.

[0034] The swab may comprise a hollow rod.

[0035] Swabs can consist of plastic, wood, or rolled paper.

[0036] The swab may comprise a swab arranged and adapted for taking a mucosal sample.

[0037] The swab can be chemically modified to enhance selectivity for the analyte.

[0038] Chemical modification can make the swab lipophilic.

[0039] Chemical modification may include forming a coating on the swab surface.

[0040] The coating may be a polymer coating.

[0041] The polymer coating may include polydivinylbenzene (DVB), a copolymer of N-vinylpyrrolidone and divinylbenzene, or polydimethylsiloxane.

[0042] Chemical modification can utilize solid phase extraction materials.

[0043] The polymer coating may contain particles of solid phase extraction material.

[0044] The solid phase extraction material may comprise polymer particles, silica particles, mixed silica particles / organic particles, carbon particles or polymer coated solid particles.

[0045] The polymer-coated solid particles may be silica particles.

[0046] The polymer-coated solid particles may comprise polydivinylbenzene (DVB), a copolymer of N-vinylpyrrolidone and divinylbenzene, or polydimethylsiloxane.

[0047] The silica particles can be surface modified by reacting with a surface modifying agent having the formula:

[0048] Z a (R') b Si—R 2 ,

[0049] Where Z=Cl、Br、I、C 1 -C 5 an alkoxy group, a dialkylamino group or a trifluoromethanesulfonate group; a and b are each an integer from 0 to 3, provided that a+b=3; R 1 It is C 1 -C 6 A linear alkyl group, a cycloalkyl group or a branched alkyl group, and R 2 It is a functional group.

[0050] R' may be selected from the group consisting of methyl, ethyl, propyl, isopropyl, butyl, tert-butyl, sec-butyl, pentyl, isopentyl, hexyl and cyclohexyl.

[0051] R 2 It may include alkyl, alkaryl, alkenyl, alkynyl, aryl, cyano, amino, diol, nitro, ester, cation exchange groups, anion exchange groups, alkyl or aryl groups containing embedded polar functional groups or chiral moieties.

[0052] R 2 Can be C1 -C 30 Alkyl, alkaryl, cyanoalkyl, diol, alkylamino, aminoalkyl or carbamate.

[0053] The silica particles may be surface modified by reaction with a compound selected from the group consisting of octyltrichlorosilane, octadecyltrichlorosilane, octyldimethylchlorosilane, and octadecyldimethylchlorosilane.

[0054] The polymer particles may comprise polydivinylbenzene or a copolymer of N-vinylpyrrolidone and divinylbenzene.

[0055] The solid phase extraction material may be an ion exchange resin.

[0056] The polymer particles may be ion exchange resin particles.

[0057] The ion exchange resin may comprise a copolymer of N-vinylpyrrolidone and divinylbenzene in which at least some of the benzene rings are sulfonated or carboxylated.

[0058] The ion exchange resin may comprise a copolymer of N-vinylpyrrolidone and divinylbenzene in which at least some of the benzene rings are replaced by imidazolium, -CH 2 -piperazinyl or quaternary ammonium substituted.

[0059] The quaternary ammonium group can be -CH 2 N + (CH 3 ) 2 (C 4 H 9 ).

[0060] The solid phase extraction material or solid particles can be attached to the swab using an adhesive.

[0061] The sample may comprise biological tissue, biological matter, bacterial colonies, fungal colonies and / or microorganisms.

[0062] The sample may be provided on the swab in its native or unmodified state.

[0063] The sample may contain: (i) mammalian cells; (ii) microorganisms; (iii) extracellular or exogenous compounds; and / or (iv) biomarkers of (i), (ii) and / or (iii).

[0064] The biomarker may be selected from fatty acids, glycerolipids, sterol lipids, sphingolipids, prenol lipids, glycolipids and / or phospholipids, and / or fingerprints of one or more thereof.

[0065] Providing the sample on a swab may comprise swiping the swab on an in vivo, in vitro or ex vivo biological sample or more generally on a target. According to one embodiment, the target may comprise an organic sample, including plastic. The target may comprise one or more bacterial colonies or one or more fungal colonies.

[0066] Providing the sample on a swab may comprise taking a mucosal sample using a swab.

[0067] The mucosa may comprise vaginal mucosa, nasal mucosa or oral mucosa.

[0068] Directing a spray of charged droplets onto the swab may include directing a spray of charged solvent droplets onto the swab.

[0069] Directing the spray of charged droplets onto the swab may include directing the spray of charged droplets onto the swab at near atmospheric pressure.

[0070] Directing the spray of charged droplets onto the swab may comprise ionising the sample using desorption electrospray ionisation ("DESI") or desorption electroflow focusing ionisation ("DEFFI").

[0071] The method may comprise directing a spray of charged droplets from a nebulizer onto a swab.

[0072] The sprayer may be positioned at a distance from the swab ranging from: (i) <0.5 mm; (ii) about 0.5-1 mm; (iii) about 1-1.5 mm; (iv) about 1.5-2 mm; (v) about 2-3 mm; (vi) about 3-4 mm; or (vii) >4 mm.

[0073] The method may comprise providing a voltage to the nebulizer within the range of: (i) <1 kV; (ii) about 1-2 kV; (iii) about 2-3 kV; (iv) about 3-4 kV; (v) about 4-5 kV; (vi) about 5-6 kV; (vii) about 6-7 kV; (viii) about 7-8 kV; (ix) about 8-9 kV; (x) about 9-10 kV; or (xi) >10 kV.

[0074] The method may comprise providing a nebulizer flow rate of the solvent in the following range: (i) <5 μL / min; (ii) about 5-7 μL / min; (iii) about 7-9 μL / min; (iv) about 9-10 μL / min; (v) about 10-11 μL / min; (vi) about 11-13 μL / min; (vii) about 13-15 μL / min; (viii) about 15-20 μL / min; or (ix) >20 μL / min.

[0075] The method may comprise providing the nebulizer with gas at a pressure in the range of: (i) <5 bar; (ii) about 5-6 bar; (iii) about 6-7 bar; (iv) about 7-8 bar; (v) about 8-9 bar; (vi) about 9-10 bar; (vii) about 10-15 bar; or (viii) >15 bar.

[0076] The gas may comprise air or nitrogen.

[0077] The method may include rotating the swab while directing the spray of charged droplets onto a surface of the swab.

[0078] The step of rotating the swab while directing the spray of charged droplets onto the swab surface may include substantially continuously rotating the swab while directing the spray of charged droplets onto the swab surface.

[0079] The method may include translating and / or vibrating the swab while directing the spray of charged droplets onto the swab surface.

[0080] The step of translating and / or vibrating the swab may comprise translating and / or vibrating the swab substantially continuously while directing the spray of charged droplets onto the swab surface.

[0081] The method may comprise translating and / or vibrating the swab substantially along the axial length of the swab.

[0082] Analyzing the analyte ions may include transferring the analyte ions through a capillary or other inlet to a mass spectrometer and / or an ion mobility spectrometer.

[0083] The inlet of the capillary or other inlet may be arranged at a distance from the swab ranging from: (i) <0.5 mm; (ii) about 0.5-1 mm; (iii) about 1-1.5 mm; (iv) about 1.5-2 mm; (v) about 2-3 mm; (vi) about 3-4 mm; or (vii) >4 mm.

[0084] Analyzing the analyte ions may include performing mass analysis and / or ion mobility analysis on the analyte ions or ions derived from the analyte ions to obtain mass spectral data and / or ion mobility data.

[0085] The following embodiments are contemplated, wherein the analyte ions produced by the open ionization ion source are subsequently subjected to: (i) mass analysis by a mass analyzer or filter, such as a quadrupole mass analyzer or a time-of-flight mass analyzer; (ii) ion mobility analysis (IMS) and / or differential ion mobility analysis (DMA) and / or field asymmetric ion mobility spectrometry (FAIMS) analysis; and / or (iii) a combination of first performing ion mobility analysis (IMS) and / or differential ion mobility analysis (DMA) and / or field asymmetric ion mobility spectrometry (FAIMS) analysis, followed by mass analysis by a mass analyzer or filter, such as a quadrupole mass analyzer or a time-of-flight mass analyzer (or vice versa). Various embodiments also relate to ion mobility spectrometers and / or mass analyzers and ion mobility spectrometry methods and / or mass analysis methods.

[0086] Analyzing the analyte ions may include determining ion mobility, collision cross sections, or interaction cross sections of the analyte ions or ions derived from the analyte ions to obtain mass spectral data and / or ion mobility data.

[0087] The method may include analyzing mass spectrometry data and / or ion mobility data to: (i) distinguish between a healthy state and a diseased state; (ii) distinguish between a potentially cancerous state and a non-cancerous state; (iii) distinguish between different types or grades of cancer; (iv) distinguish between different types or classes of samples; (v) determine whether one or more desired or undesirable substances are present in a sample; (vi) confirm the identity or authenticity of a sample; (vii) determine whether one or more impurities, illegal substances, or undesirable substances are present in a sample; (viii) determine whether a human or animal patient has an increased risk of suffering an adverse outcome; (ix) make or assist in making a diagnosis or prognosis; (x) notify a surgeon, nurse, doctor, or robot of the results of a medical procedure, surgery, or diagnosis; and / or (xi) identify and / or predict one or more diseases or clinical conditions.

[0088] The one or more diseases or clinical conditions may include (i) infection; (ii) altered microbiome; (iii) premature birth; (iv) immune disorder; (v) asthma; (vi) allergy; (vii) inflammation; (viii) cancer; (ix) necrosis; and / or (x) precancerous condition.

[0089] The method can comprise determining whether a cancerous biological tissue or tumor comprises: (i) grade I, grade II, grade III, or grade IV cancer tissue; (ii) metastatic cancer tissue; (iii) mixed grade cancer tissue; or (iv) subgrade cancer tissue.

[0090] The step of analyzing the mass spectrometry data may include performing supervised and / or unsupervised analysis on the mass spectrometry data.

[0091] The step of analyzing the mass spectrometry data and / or the ion mobility data may include using one or more of the following: univariate analysis; multivariate analysis; principal component analysis (PCA); linear discriminant analysis (LDA); maximum margin criteria (MMC); library-based analysis; soft independent modelling of classanalogy (SIMCA); factor analysis (FA); recursive partitioning (decision tree); random forest; independent component analysis (ICA); partial least squares discriminant analysis (PLS-DA); orthogonal (partial least squares) projections to latent structures (OPLS); OPLS discriminant analysis (OPLS-DA); support vector machines (SVM); (artificial) neural networks; multilayer perceptron; radial basis function (RBF); function (RBF) networks; Bayesian analysis; cluster analysis; kernelization methods; and subspace discriminant analysis.

[0092] The method may comprise analyzing the sample on the swab using one or more other different analytical methods.

[0093] The one or more other different assays may comprise a culture assay.

[0094] Culture analysis methods may include swabbing a swab on culture medium, incubating the culture medium, and examining the culture medium under a microscope.

[0095] One or more of the other various analytical methods may include a gene sequencing method.

[0096] The gene sequencing method may include a 16S rRNA gene sequencing method.

[0097] One or more of the other various analytical methods may include a matrix-assisted laser desorption ionization ("MALDI") method.

[0098] One or more of the other various analytical methods may include open ionization mass spectrometry.

[0099] One or more of the other various analytical methods may include rapid evaporative ionization mass spectrometry ("REIMS") methods.

[0100] According to one aspect, there is provided an apparatus comprising:

[0101] a first device arranged and adapted to direct a spray of charged droplets onto a swab surface to produce a plurality of analyte ions; and

[0102] A second device is arranged and adapted to analyze the analyte ions.

[0103] The first device may be arranged and adapted to direct a spray of charged solvent droplets onto the swab.

[0104] The first device may be arranged and used to direct a spray of charged droplets at near atmospheric pressure onto the swab.

[0105] The first device may comprise a desorption electrospray ionization ("DESI") ion source or a desorption current dynamic focusing ionization ("DEFFI") ion source.

[0106] The first device may comprise a sprayer arranged and adapted to direct a spray of charged droplets onto the swab.

[0107] The sprayer may be positioned at a distance from the swab ranging from: (i) <0.5 mm; (ii) about 0.5-1 mm; (iii) about 1-1.5 mm; (iv) about 1.5-2 mm; (v) about 2-3 mm; (vi) about 3-4 mm; or (vii) >4 mm.

[0108] The apparatus may comprise means arranged and adapted to provide the nebuliser with a voltage within the range of: (i) <1 kV; (ii) about 1-2 kV; (iii) about 2-3 kV; (iv) about 3-4 kV; (v) about 4-5 kV; (vi) about 5-6 kV; (vii) about 6-7 kV; (viii) about 7-8 kV; (ix) about 8-9 kV; (x) about 9-10 kV; or (xi) >10 kV.

[0109] The apparatus may comprise means arranged and adapted to provide a nebulizer flow rate of solvent in the range of: (i) <5 μL / min; (ii) about 5-7 μL / min; (iii) about 7-9 μL / min; (iv) about 9-10 μL / min; (v) about 10-11 μL / min; (vi) about 11-13 μL / min; (vii) about 13-15 μL / min; (viii) about 15-20 μL / min; or (ix) >20 μL / min.

[0110] The apparatus may comprise means arranged and adapted to provide the nebuliser with gas at a pressure in the range: (i) <5 bar; (ii) about 5-6 bar; (iii) about 6-7 bar; (iv) about 7-8 bar; (v) about 8-9 bar; (vi) about 9-10 bar; (vii) about 10-15 bar; or (viii) >15 bar.

[0111] The gas may comprise air or nitrogen.

[0112] The apparatus may comprise a third means arranged and adapted to rotate the swab while directing the spray of charged droplets onto the swab surface.

[0113] The third device may be arranged and adapted to rotate the swab substantially continuously while directing a spray of charged droplets onto the swab surface.

[0114] The apparatus may comprise fourth means arranged and adapted to translate and / or vibrate the swab while directing the spray of charged droplets onto the swab surface.

[0115] The fourth device may be arranged and adapted to substantially continuously translate and / or vibrate the swab while directing a spray of charged droplets onto the swab surface.

[0116] The fourth device may be arranged and adapted to cause the swab to translate and / or vibrate substantially along the axial length of the swab.

[0117] The apparatus may comprise a capillary or other inlet arranged and adapted to transfer the analyte ions to the second device.

[0118] The inlet of the capillary or other inlet may be arranged at a distance from the swab ranging from: (i) <0.5 mm; (ii) about 0.5-1 mm; (iii) about 1-1.5 mm; (iv) about 1.5-2 mm; (v) about 2-3 mm; (vi) about 3-4 mm; or (vii) >4 mm.

[0119] The second device may comprise a mass analyser or filter and / or an ion mobility analyser arranged and adapted to perform mass analysis and / or ion mobility analysis on the analyte ions or ions derived from the analyte ions.

[0120] The second means may comprise an ion mobility device arranged and adapted to determine ion mobility, collision cross section or interaction cross section of the analyte ions or ions derived from the analyte ions.

[0121] According to one aspect, there is provided a medical swab for use in a method as described above, wherein the swab has been chemically modified to enhance selectivity for the analyte.

[0122] The swab may be a disposable swab.

[0123] The swabs may be cotton, rayon, plastic, or foam.

[0124] Chemical modification can make the swab lipophilic.

[0125] Chemical modification may include forming a coating on the swab surface.

[0126] The coating may be a polymer coating.

[0127] The polymer coating may comprise polydivinylbenzene (DVB), a copolymer of N-vinylpyrrolidone and divinylbenzene, or polydimethylsiloxane.

[0128] Chemical modification can utilize solid phase extraction materials.

[0129] The polymer coating may contain particles of solid phase extraction material.

[0130] The solid phase extraction material may comprise polymer particles, silica particles, mixed silica particles / organic particles, carbon particles or polymer coated solid particles.

[0131] The polymer-coated solid particles may be silica particles.

[0132] The polymer-coated solid particles may comprise polydivinylbenzene (DVB), a copolymer of N-vinylpyrrolidone and divinylbenzene, or polydimethylsiloxane.

[0133] The silica particles can be surface modified by reacting with a surface modifying agent having the formula:

[0134] Z a (R') b Si—R 2 ,

[0135] Where Z=Cl、Br、I、C 1 -C5 an alkoxy group, a dialkylamino group or a trifluoromethanesulfonate group; a and b are each an integer from 0 to 3, provided that a+b=3; R 1 It is C 1 -C 6 A linear alkyl group, a cycloalkyl group or a branched alkyl group, and R 2 It is a functional group.

[0136] R' may be selected from the group consisting of methyl, ethyl, propyl, isopropyl, butyl, tert-butyl, sec-butyl, pentyl, isopentyl, hexyl and cyclohexyl.

[0137] R 2 It may include alkyl, alkaryl, alkenyl, alkynyl, aryl, cyano, amino, diol, nitro, ester, cation exchange groups, anion exchange groups, alkyl or aryl groups containing embedded polar functional groups or chiral moieties.

[0138] R 2 Can be C 1 -C 30 Alkyl, alkaryl, cyanoalkyl, diol, alkylamino, aminoalkyl or carbamate.

[0139] The silica particles may be surface modified by reaction with a compound selected from the group consisting of octyltrichlorosilane, octadecyltrichlorosilane, octyldimethylchlorosilane, and octadecyldimethylchlorosilane.

[0140] The polymer particles may comprise polydivinylbenzene or a copolymer of N-vinylpyrrolidone and divinylbenzene.

[0141] The solid phase extraction material may be an ion exchange resin.

[0142] The polymer particles may be ion exchange resin particles.

[0143] The ion exchange resin may comprise a copolymer of N-vinylpyrrolidone and divinylbenzene in which at least some of the benzene rings are sulfonated or carboxylated.

[0144] The ion exchange resin may comprise a copolymer of N-vinylpyrrolidone and divinylbenzene in which at least some of the benzene rings are replaced by imidazolium, -CH 2 -piperazinyl or quaternary ammonium substituted.

[0145] The quaternary ammonium group can be -CH 2 N + (CH 3 ) 2 (C 4 H 9 ).

[0146] The solid phase extraction material or solid particles can be attached to the swab using an adhesive.

[0147] According to one aspect, a method of chemically modifying a medical swab is provided, the method comprising attaching particles of a solid phase extraction material to a surface of the swab.

[0148] The connection may be made using an adhesive.

[0149] According to one aspect, there is provided a method for chemically modifying a medical swab, the method comprising:

[0150] (a) forming a dispersion or solution of a chemical modifier;

[0151] (b) dipping the swab into the solution or dispersion;

[0152] (c) The swab is removed from the solution or dispersion and dried.

[0153] Steps (b) and (c) may be repeated at least once.

[0154] According to one aspect, there is provided a diagnostic method comprising:

[0155] providing a sample on a swab, wherein the swab may further comprise a solid-phase extraction ("SPE") material for extracting an analyte from the liquid sample;

[0156] directing a spray of charged droplets onto a swab surface to generate a plurality of analyte ions; and

[0157] The analyte ions were mass analyzed.

[0158] The solid phase extraction material may comprise a polymer.

[0159] The solid phase extraction material may be hydrophilic.

[0160] The solid phase extraction material may comprise a reverse phase polymer.

[0161] The solid phase extraction material may comprise a cation exchange or anion exchange polymer.

[0162] The solid phase extraction material may include octadecylsilane ("ODS / C18") or polydimethylsiloxane ("PDMS").

[0163] The solid phase extraction material may comprise: (i) Oasis MAX (mixed mode cation exchange) (RTM); (ii) hydrophilic N-vinyl pyrrolidone and lipophilic divinyl benzene or Oasis hydrophilic-lipophilic-balanced (HLB) (RTM); or (iii) Oasis MCX (mixed mode cation exchange) (RTM).

[0164] The method may further include rotating the swab while directing the spray of charged droplets onto the swab surface.

[0165] The step of rotating the swab while directing the spray of charged droplets onto the swab surface may further comprise rotating the swab substantially continuously while directing the spray of charged droplets onto the swab surface.

[0166] The swabs may contain cotton, rayon, or polyester.

[0167] The swab may comprise fibers without a fused silica core.

[0168] According to one aspect, there is provided a diagnostic method comprising:

[0169] providing a sample on a swab, wherein the swab may further comprise a solid phase extraction ("SPE") material for extracting an analyte from the liquid sample;

[0170] rotating the swab and substantially simultaneously directing a spray of charged droplets onto a surface of the swab to generate a plurality of analyte ions; and

[0171] The analyte ions are subjected to mass analysis and / or ion mobility analysis.

[0172] According to one aspect, there is provided a desorption electrospray ionization ("DESI") method comprising the diagnostic method as described above.

[0173] According to one aspect, a mass spectrometry method and / or an ion mobility analysis method is provided, comprising the method as described above.

[0174] According to one aspect, there is provided an apparatus comprising:

[0175] means for directing a spray of charged droplets onto a swab surface to produce a plurality of analyte ions, wherein the swab may further comprise a solid phase extraction ("SPE") material for extracting the analyte from the liquid sample; and

[0176] A mass analyzer or filter and / or an ion mobility analyzer for performing mass analysis and / or ion mobility analysis of analyte ions.

[0177] According to one aspect, there is provided an apparatus comprising:

[0178] a first means for directing a spray of charged droplets onto a swab surface to produce a plurality of analyte ions, wherein the swab may further comprise a solid phase extraction ("SPE") material for extracting the analyte from the liquid sample;

[0179] second means for rotating the swab substantially simultaneously with the first means directing a spray of charged droplets onto the swab surface; and

[0180] A mass analyzer or filter and / or an ion mobility analyzer for performing mass analysis and / or ion mobility analysis of analyte ions.

[0181] According to an aspect, there is provided a mass analyser and / or an ion mobility analyser comprising a device as described above.

[0182] According to one aspect, there is provided a method comprising:

[0183] Provide a sample on a swab;

[0184] Wetting the swab with the first liquid;

[0185] contacting the moistened swab with the electrode to generate an aerosol, mist or vapor;

[0186] Producing multiple analyte ions from an aerosol, mist, or vapor; and

[0187] Analyte ions are analyzed.

[0188] According to one aspect, there is provided a diagnostic method comprising:

[0189] Provide a sample on a swab;

[0190] Wetting the swab with the first liquid;

[0191] contacting the moistened swab with a bipolar electrode to generate an aerosol;

[0192] generating a plurality of analyte ions from the aerosol; and

[0193] The analyte ions are subjected to mass analysis and / or ion mobility analysis.

[0194] The first liquid may comprise water.

[0195] According to one aspect, there is provided a rapid evaporative ionization mass spectrometry ("REIMS") method comprising the method as described above.

[0196] According to one aspect, there is provided an apparatus comprising:

[0197] an electrode arranged and adapted to contact a sample on a swab moistened with a first liquid so as to generate an aerosol, mist or vapor;

[0198] means arranged and adapted to produce a plurality of analyte ions from an aerosol, mist or vapor; and

[0199] An analyzer for analyzing analyte ions.

[0200] According to one aspect, there is provided an apparatus comprising:

[0201] a bipolar electrode arranged and adapted to contact a sample on a swab moistened with a first liquid to generate an aerosol;

[0202] means arranged and adapted to produce a plurality of analyte ions from the aerosol; and

[0203] A mass analyzer or filter and / or an ion mobility analyzer for performing mass analysis and / or ion mobility analysis of analyte ions.

[0204] According to one aspect, there is provided a mass spectrometer and / or an ion mobility spectrometer comprising the apparatus as described above.

[0205] According to one aspect, a swab comprising a solid phase extraction ("SPE") material for extracting an analyte from a liquid sample is provided, wherein the swab comprises a fiber without a fused silica core.

[0206] The swabs may contain cotton, rayon, or polyester.

[0207] The solid phase extraction material may be hydrophilic.

[0208] The solid phase extraction material may comprise a reverse phase polymer.

[0209] The solid phase extraction material may comprise a cation exchange or anion exchange polymer.

[0210] The solid phase extraction material may comprise octadecylsilane ("ODS / C18") or polydimethylsiloxane ("PDMS").

[0211] The solid phase extraction material may comprise: (i) Oasis MAX (mixed mode cation exchange) (RTM); (ii) hydrophilic N-vinyl pyrrolidone and lipophilic divinyl benzene or Oasis hydrophilic-lipophilic balance (HLB) (RTM); or (iii) Oasis MCX (mixed mode cation exchange) (RTM).

[0212] According to one aspect, there is provided a method comprising:

[0213] Provide a sample on a swab;

[0214] rotating the swab and substantially simultaneously directing a spray of charged droplets onto a surface of the swab to generate a plurality of analyte ions; and

[0215] Analyte ions are analyzed.

[0216] The step of rotating the swab while directing the spray of charged droplets onto the swab surface may further comprise rotating the swab substantially continuously while directing the spray of charged droplets onto the swab surface.

[0217] The method may include translating and / or vibrating the swab while directing the spray of charged droplets onto the swab surface.

[0218] According to one aspect, there is provided a method comprising:

[0219] Provide a sample on a swab;

[0220] translating and / or vibrating the swab and substantially simultaneously directing a spray of charged droplets onto a surface of the swab to generate a plurality of analyte ions; and

[0221] Analyte ions are analyzed.

[0222] The method may include substantially continuously translating and / or vibrating the swab while directing the spray of charged droplets onto the swab surface.

[0223] The method may comprise translating and / or vibrating the swab substantially along the axial length of the swab.

[0224] According to one aspect, there is provided an apparatus comprising:

[0225] a first device arranged and adapted to direct a spray of charged droplets onto a swab surface so as to produce a plurality of analyte ions;

[0226] a second means arranged and adapted to rotate the swab substantially simultaneously with the first means directing a spray of charged droplets onto the swab surface; and

[0227] An analyzer is arranged and adapted to analyze the analyte ions.

[0228] The second device may be arranged and adapted to rotate the swab substantially continuously while the first device directs the spray of charged droplets onto the swab surface.

[0229] The apparatus may comprise a third means arranged and adapted to translate and / or vibrate the swab while the first means directs the spray of charged droplets onto the swab surface.

[0230] According to one aspect, there is provided an apparatus comprising:

[0231] a first device arranged and adapted to direct a spray of charged droplets onto a swab surface so as to produce a plurality of analyte ions;

[0232] a third device arranged and adapted to translate and / or vibrate the swab while the first device directs the spray of charged droplets onto the swab surface; and

[0233] An analyzer is arranged and adapted to analyze the analyte ions.

[0234] The third device may be arranged and adapted to cause the swab to translate and / or vibrate substantially continuously while the first device directs the spray of charged droplets onto the swab surface.

[0235] The third device may be arranged and adapted to translate and / or vibrate the swab substantially along the axial length of the swab.

[0236] According to one aspect, there is provided a method comprising:

[0237] Provide a sample on a swab;

[0238] analyzing the sample on the swab using a first analytical method; and

[0239] analyzing the sample on the swab using one or more second different analytical methods;

[0240] The first analysis method may include directing a spray of charged droplets onto a swab surface to generate a plurality of analyte ions, and then analyzing the analyte ions.

[0241] Directing a spray of charged droplets onto the swab may include directing a spray of charged solvent droplets onto the swab.

[0242] Directing the spray of charged droplets onto the swab may include directing the spray of charged droplets onto the swab at near atmospheric pressure.

[0243] Directing the spray of charged droplets onto the swab may include ionizing the sample using desorption electrospray ionization ("DESI") or desorption electrodynamic focusing ionization ("DEFFI").

[0244] The one or more second assays may comprise a culture assay.

[0245] Culture analysis methods may comprise contacting the swab with culture medium, incubating the culture medium, and examining the culture medium or a sample thereof under a microscope.

[0246] The one or more second analysis methods may comprise a gene sequencing method.

[0247] The sequencing method may include a 16S rRNA gene sequencing method.

[0248] The one or more second analytical methods may comprise a matrix-assisted laser desorption ionization ("MALDI") method.

[0249] The one or more second analytical methods may comprise open ionization mass spectrometry methods.

[0250] The open ionization mass spectrometry method may be selected from the group consisting of: (i) rapid evaporation ionization mass spectrometry ("REIMS") method; (ii) laser desorption ionisation ("LDI") method; (iii) thermal desorption ionisation method; (iv) laser diode thermal desorption ("LDTD") ionisation method; (v) desorption current dynamic focusing ionisation ("DEFFI") method; (vi) dielectric barrier discharge ("DBD") plasma ionisation method; (vii) atmospheric pressure solids analysis probe ("ASAP") ionisation method; (viii) ultrasound assisted spray ionisation method; (ix) easy ambient sonic-spray ionisation ("EASI") method; (x) desorption atmospheric pressure photoionisation ("DPPI") method; (xvi) direct analysis in real time (DART) ionisation; (xvii) probe electrospray ionisation (PESI) ionisation; (xviii) solid-probe assisted electrospray ionisation (SPA-ESI) ionisation; (xix) cavitron ultrasonic surgical aspirator (CAV) ionisation; (xvii) probe electrospray ionisation (PESI) ionisation; (xviii) solid-probe assisted electrospray ionisation (SPA-ESI) ionisation; (xix) ultrasonic surgical aspirator (CAV) ionisation; (xvii) probe electrospray ionisation (PESI) ionisation; (xviii) solid-probe assisted electrospray ionisation (SPA-ESI) ionisation; (xix) aspirator, "CUSA") method; (xx) focused or unfocused ultrasound ablation method; (xxi) microwave resonance method; and (xxii) pulsed plasma RF dissection method.

[0251] The method may comprise analyzing the sample on the swab using a third different analytical method.

[0252] According to one aspect, there is provided a method comprising:

[0253] Provide a sample on a swab;

[0254] analyzing the sample on the swab in a first operating mode, wherein the first operating mode may include directing a spray of charged droplets onto the swab surface to generate a plurality of analyte ions; and

[0255] determining whether the analyte ions are likely to contain one or more ions of interest;

[0256] Wherein if it is determined that the analyte ions include one or more ions of interest, the method may further comprise:

[0257] The sample on the swab is analyzed in a second different mode of operation.

[0258] Directing a spray of charged droplets onto the swab may include directing a spray of charged solvent droplets onto the swab.

[0259] Directing the spray of charged droplets onto the swab may include directing the spray of charged droplets onto the swab at near atmospheric pressure.

[0260] Directing the spray of charged droplets onto the swab may include ionizing the sample using desorption electrospray ionization ("DESI") or desorption electrodynamic focusing ionization ("DEFFI").

[0261] The second mode of operation may include directing a spray of charged droplets onto the swab surface in a second, different mode of operation.

[0262] (i) the first operating mode may comprise a positive ion operating mode and the second operating mode may comprise a negative ion operating mode; or

[0263] (ii) The first operating mode may comprise a negative ion operating mode and the second operating mode may comprise a positive ion operating mode.

[0264] The first mode of operation may comprise directing a spray of charged droplets onto the swab surface, wherein the charged droplets comprise a first solvent or solvent composition; and

[0265] The second mode of operation may comprise directing a spray of charged droplets onto the swab surface, wherein the charged droplets comprise a second different solvent or solvent combination.

[0266] The second operating mode may comprise an optimized version of the first operating mode.

[0267] The second operating mode may include generating a plurality of analyte ions from the sample using a second different open ionization analysis method.

[0268] The second different open ionization analysis method can be selected from the group consisting of: (i) rapid evaporation ionization mass spectrometry ("REIMS") method; (ii) laser desorption ionization ("LDI") method; (iii) thermal desorption ionization method; (iv) laser diode thermal desorption ("LDTD") ionization method; (v) desorption current dynamic focusing ionization ("DEFFI") method; (vi) dielectric barrier discharge ("DBD") plasma ionization method; (vii) atmospheric pressure solid analysis probe ("ASAP") ionization method; (viii) ultrasonic assisted spray ionization method; (ix) simplified open acoustic spray ionization ("EASI") method; (x) desorption atmospheric pressure photoionization ("DAPPI") method; ( xi) paper spray ("PS") ionization method; (xii) jet desorption ionization ("JeDI") method; (xiii) touch spray ("TS") ionization method; (xiv) nanoDESI ionization method; (xv) laser ablation electrospray ("LAESI") ionization method; (xvi) direct analysis in real time ("DART") ionization method; (xvii) probe electrospray ionization ("PESI") method; (xviii) solid probe assisted electrospray ionization ("SPA-ESI") method; (xix) ultrasonic surgical aspirator ("CUSA") method; (xx) focused or unfocused ultrasound ablation method; (xxi) microwave resonance method; and (xxii) pulsed plasma RF dissection method.

[0269] The first operating mode may include analyzing analyte ions using first operating parameters; and

[0270] The second operating mode may include analyzing analyte ions from the sample using second, different operating parameters.

[0271] The first and / or second operating modes may include (i) an operating mode in which mass analysis and / or ion mobility analysis is performed on analyte ions or ions derived from analyte ions; (ii) an operating mode in which the ion mobility, collision cross section or interaction cross section of analyte ions or ions derived from analyte ions can be determined; (iii) an operating mode in which the analyte ions are further fragmented; and / or (iv) an operating mode in which the analyte ions are reacted, excited, fragmented or fractionated.

[0272] The second different operation mode may include: (i) a culture operation mode; (ii) a gene sequencing operation mode; or (iii) a matrix-assisted laser desorption ionization ("MALDI") operation mode.

[0273] The method may include selecting and / or optimizing the second operating mode based on information acquired during the first operating mode.

[0274] Methods can include:

[0275] determining whether the analyte ions analyzed in the second operating mode include one or more second ions of interest;

[0276] Wherein if it is determined that the analyte ions include one or more second ions of interest, the method may further comprise:

[0277] The sample on the swab is analyzed in a third different operating mode.

[0278] According to one aspect, there is provided an apparatus comprising:

[0279] a first device arranged and adapted to analyze a sample on the swab in a first mode of operation, wherein the first mode of operation may include directing a spray of charged droplets onto a surface of the swab to generate a plurality of analyte ions; and

[0280] A second device is arranged and adapted to determine whether the analyte ions contain one or more ions of interest and, if it is determined that the analyte ions contain the one or more ions of interest, cause the device to analyze the sample on the swab in a second, different mode of operation.

[0281] The first device may be arranged and adapted to direct a spray of charged solvent droplets onto the swab.

[0282] The first device may be arranged and adapted to direct a spray of charged droplets at near atmospheric pressure onto the swab.

[0283] The first device may comprise a desorption electrospray ionization ("DESI") device or a desorption current flow focusing ionization ("DEFFI") device.

[0284] The second device may be arranged and adapted to cause the first device to analyze the sample on the swab in a second, different mode of operation.

[0285] (i) the first operating mode may comprise a positive ion operating mode and the second operating mode may comprise a negative ion operating mode; or

[0286] (ii) The first operating mode may comprise a negative ion operating mode and the second operating mode may comprise a positive ion operating mode.

[0287] In a first mode of operation, the charged droplets may comprise a first solvent or solvent composition; and

[0288] In a second mode of operation, the charged droplets may contain a second different solvent or solvent combination.

[0289] The second operating mode may comprise an optimized version of the first operating mode.

[0290] The second device can be arranged and adjusted to be adapted to analyze a sample on a swab using a second different open ionization analysis method.

[0291] The second device can be selected from the group consisting of: (i) a rapid evaporative ionization mass spectrometry (“REIMS”) ion source; (ii) a laser desorption ionization (“LDI”) ion source; (iii) a thermal desorption ionization ion source; (iv) a laser diode thermal desorption (“LDTD”) ion source; (v) a desorption electrospray ionization flow focusing (“DEFFI”) ion source; (vi) a dielectric barrier discharge (“DBD”) plasma ion source; (vii) an atmospheric pressure solids analysis probe (“ASAP”) ion source; (viii) an ultrasonic assisted spray ion source; (ix) a simple open acoustic spray ionization (“EASI”) ion source; (x) a desorption atmospheric pressure photoionization (“DAPPI”) ion source; (xi) a paper spray (“PS”) ion source; (xii) a jet desorption ionization (“JeDI”) ion source; (xiii) a touch spray (“TS”) ion source; (xiv) a nano-DESI ion source; (xv) a laser ablation electrospray (“LAESI”) ion source; (xvi) a direct analysis in real time (“DART”) ion source; (xvii) a probe electrospray ionization (“PESI”) ion source; (xviii) a solid probe assisted electrospray ionization (“SPA-ESI”) ion source; (xix) an ultrasonic surgical aspirator (“CUSA”) device; (xx) a focused or unfocused ultrasound ablation device; (xxi) a microwave resonance device; and (xxii) a pulsed plasma RF dissection device.

[0292] The device can be arranged and adjusted to be adapted to analyze analyte ions using first operating parameters in a first operating mode; and

[0293] The device can be arranged and adjusted to be adapted to analyze analyte ions from a sample using second different operating parameters in a second operating mode.

[0294] The device can include: (i) a mass analyzer or filter and / or an ion mobility analyzer for mass analyzing and / or ion mobility analyzing analyte ions or ions derived from analyte ions in the first and / or second operating modes; (ii) an ion mobility device for determining the ion mobility, collision cross section, or interaction cross section of analyte ions or ions derived from analyte ions in the first and / or second operating modes; (iii) a fragmentation device for fragmenting analyte ions in the first and / or second operating modes; and / or (iv) one or more devices for reacting, exciting, fragmenting, and / or fractionating analyte ions in the first and / or second operating modes.

[0295] According to one aspect, there is provided a method comprising:

[0296] automatically analyzing a plurality of swabs by directing a spray of charged droplets onto a surface of each swab to generate a plurality of analyte ions; and

[0297] Analyte ions from each swab were analyzed.

[0298] A different sample may be provided on each of the plurality of swabs.

[0299] The step of automatically analyzing the plurality of swabs may comprise automatically analyzing the plurality of swabs substantially sequentially.

[0300] The step of automatically analyzing the plurality of swabs may comprise automatically analyzing two or more of the plurality of swabs substantially simultaneously or in parallel.

[0301] The method may include rotating, vibrating, and / or translating each swab and substantially simultaneously directing a spray of charged droplets onto a surface of each swab to generate a plurality of analyte ions.

[0302] According to one aspect, there is provided an apparatus comprising:

[0303] a first device arranged and adapted to automatically analyze a plurality of swabs by directing a spray of charged droplets onto a surface of each swab to generate a plurality of analyte ions; and

[0304] A second device is arranged and adapted to analyze analyte ions from each swab.

[0305] A different sample may be provided on each of the plurality of swabs.

[0306] The first device may be arranged and adapted to automatically analyse a plurality of swabs substantially sequentially.

[0307] The first device may be arranged and adapted to automatically analyze two or more of the plurality of swabs substantially simultaneously or in parallel.

[0308] The apparatus may comprise a third means arranged and adapted to rotate, vibrate and / or translate each swab whilst the first means directs a spray of charged droplets onto the surface of each swab.

[0309] According to one aspect, there is provided a method comprising:

[0310] Transfer multiple samples to different locations on a roller, sheet, belt, or substrate;

[0311] analyzing a first sample on a roll, sheet, tape, or substrate using an open ion source;

[0312] advancing a roll, sheet, belt or substrate; and

[0313] A second sample on a roll, sheet, tape or substrate is analyzed using an open ion source.

[0314] Methods can include:

[0315] (i) advancing a roll, sheet, belt or substrate;

[0316] (ii) analyzing one or more other samples on a roll, sheet, tape or substrate using an open ion source; and

[0317] (iii) optionally repeating steps (i) and (ii) one or more times.

[0318] The open ion source may comprise a desorption electrospray ionization ("DESI") ion source or a desorption current focusing ionization ("DEFFI") ion source.

[0319] The method may comprise identifying the first sample and / or the second sample and / or one or more other samples.

[0320] According to one aspect, there is provided a method comprising:

[0321] directing a spray of charged droplets onto a surface of a swab having a vaginal mucosal sample from a human or non-human animal provided thereon to produce a plurality of analyte ions;

[0322] analyzing the analyte ions to obtain mass spectral data and / or ion mobility data; and

[0323] Determining from the mass spectrometry data and / or ion mobility data: (i) whether the human or non-human animal is pregnant; (ii) the pregnancy stage or status of the human or non-human animal; (iii) whether the human or non-human animal has an increased risk of adverse pregnancy outcomes; and / or (iv) whether the human or non-human animal has an increased risk of preterm delivery or premature delivery.

[0324] The method may comprise identifying the presence of one or more microorganisms in a vaginal mucosal sample, wherein the microorganisms are optionally bacteria.

[0325] The one or more microorganisms may be selected from the group consisting of: (i) Candida albicans; (ii) Pseudomonas montelli; (iii) Staphylococcus epidermis; (iv) Moraxella catarrhalis; (v) Klebsiella pneumonia; and (vi) Lactobacillus sp.

[0326] According to one aspect, a pregnancy test or pregnancy monitoring method is provided, comprising the method as described above.

[0327] The method may comprise repeating the method periodically to monitor the progress of pregnancy in the human or non-human animal.

[0328] According to one aspect, there is provided a method of diagnosis or prognosis, comprising the method as described above.

[0329] According to one aspect, there is provided a method comprising:

[0330] providing a stool sample on an absorbent or other surface;

[0331] generating a plurality of analyte ions from a stool sample using an open ionization source; and

[0332] Analyte ions are analyzed.

[0333] The step of generating a plurality of analyte ions using an open ionization source may include directing a spray of charged droplets onto an absorbent or other surface.

[0334] The step of generating a plurality of analyte ions using an open ionization source may include generating an aerosol, mist, or vapor from the stool sample.

[0335] The method may comprise ionizing the aerosol, mist or vapor to produce analyte ions.

[0336] The absorbent surface may comprise toilet paper, tissue, a nappy or diaper or an incontinence pad or pants.

[0337] The method can include determining whether the stool sample contains blood, human blood, non-human animal blood, hemoglobin, pathogens, undesirable material, non-human material, parasitic material, or fecal or other waste products from parasites based on the analysis.

[0338] The method may include determining whether a human or non-human animal suffers from or has an anal fissure, diverticular disease, inflammatory disease, angiodysplasia and / or polyps of their colon, intestines, or other part of their body, or suffers from or has another medical disease or condition based on determining whether the stool sample contains blood, human blood, non-human animal blood, or hemoglobin.

[0339] The method may comprise analyzing the amount and / or composition of bile present in the stool sample.

[0340] The method may comprise determining whether a human or non-human animal suffers from or has liver disease, kidney disease, or other medical disease or condition from analysis of the amount and / or composition of bile present in a stool sample.

[0341] The method may comprise analyzing the composition of the gastrointestinal microbiome of a human or non-human animal.

[0342] The method may comprise determining or assessing the effect of an antibiotic or probiotic on a human or non-human animal based on analysis of the gastrointestinal microbiome in a fecal sample of the human or non-human animal.

[0343] The method can include determining, based on the analysis, whether the stool sample is likely to contain: (i) one or more specific microorganisms; (ii) one or more pathogens; (iii) one or more parasites; and / or (iv) one or more metabolites.

[0344] According to an aspect, there is provided the use of a swab as described above in a method as described above.

[0345] According to one aspect, there is provided a desorption electrospray ionization ("DESI") method comprising the method as described above.

[0346] According to one aspect, there is provided a mass spectrometry method comprising the method as described above.

[0347] According to one aspect, there is provided a desorption electrospray ionization ("DESI") ion source comprising an apparatus as described above.

[0348] According to one aspect, there is provided a mass spectrometer and / or an ion mobility spectrometer comprising the apparatus as described above.

[0349] According to one aspect, there is provided a mass spectrometer and / or an ion mobility spectrometer, comprising:

[0350] a first device arranged and adapted to receive a biopsy sample;

[0351] a second device arranged and adapted to produce analyte ions from the biopsy sample within the first device, wherein the second device may be arranged and adapted to produce first analyte ions from a first location on the biopsy sample at a first time and second analyte ions from a second, different location on the biopsy sample at a second, different time; and

[0352] An analyzer is arranged and adapted to analyze the analyte ions.

[0353] A biopsy sample can comprise a tissue sample having a longitudinal length.

[0354] The composition of the tissue sample may vary or change along the longitudinal length.

[0355] The longitudinal length may correspond to the depth within the tissue.

[0356] The biopsy sample may comprise a biopsy core or cylinder.

[0357] The first device may include a channel arranged and adapted to receive a biopsy core.

[0358] The second device may be arranged and adapted to produce first analyte ions from a first location along the longitudinal length of the biopsy sample at a first time and second analyte ions from a second different location along the longitudinal length of the biopsy sample at a second different time.

[0359] The second device may be arranged and adapted to scan at least a portion of the longitudinal length of the biopsy sample to generate analyte ions from a plurality of locations along the longitudinal length of the biopsy sample.

[0360] The second device may comprise an open ionization ion source.

[0361] The open ionization ion source may comprise an ion source selected from the group consisting of: (i) a rapid evaporation ionization mass spectrometry ("REIMS") ion source; (ii) a desorption electrospray ionization ("DESI") ion source; (iii) a laser desorption ionization ("LDI") ion source; (iv) a thermal desorption ion source; (v) a laser diode thermal desorption ("LDTD") ion source; (vi) a desorption current dynamic focusing ("DEFFI") ion source; (vii) a dielectric barrier discharge ("DBD") plasma ion source; (viii) an atmospheric pressure solid analysis probe ("ASAP") ion source; (ix) an ultrasound assisted spray ionization ion source; (x) an easy open acoustic spray ionization ("EASI") ion source; (xi) a desorption atmospheric pressure photoionization ("DPPI") ion source. (xvi) laser ablation electrospray ("LAESI") ion source; (xvii) direct analysis in real time ("DART") ion source; (xviii) probe electrospray ionization ("PESI") ion source; (xix) solid probe assisted electrospray ionization ("SPA-ESI") ion source; (xx) ultrasonic surgical aspirator ("CUSA") device; (xxi) focused or unfocused ultrasound ablation device; (xxii) microwave resonant device; and (xxiii) pulsed plasma RF dissection device.

[0362] The second device may be arranged and adapted to generate an aerosol, mist or vapor from the biopsy sample and to ionize the aerosol, mist or vapor to generate analyte ions.

[0363] The second device may include one or more electrodes arranged and adapted to contact the biopsy sample to generate an aerosol, mist or vapor.

[0364] The one or more electrodes may comprise a bipolar device or a monopolar device.

[0365] The one or more electrodes may comprise any of: (i) a monopolar device, wherein the device optionally further comprises an independent return electrode; (ii) a bipolar device; or (iii) a multi-phase RF device, wherein the device optionally further comprises one or more independent return electrodes.

[0366] One or more electrodes may comprise a rapid evaporative ionization mass spectrometry ("REIMS") device.

[0367] The mass spectrometer and / or ion mobility spectrometer may comprise means arranged and adapted to apply an AC or RF voltage to one or more electrodes in order to generate an aerosol, mist or vapour.

[0368] The means for applying an AC or RF voltage to the one or more electrodes may be arranged and adapted to apply one or more pulses of AC or RF voltage to the one or more electrodes.

[0369] Application of an AC or RF voltage to one or more electrodes can cause heat to be dissipated into the sample.

[0370] The second device may comprise a laser for illuminating the sample.

[0371] The second device may be arranged and adapted to generate an aerosol, mist or vapour from the sample by evaporating or vaporising sample material directly from the sample by Joule heating or diathermy.

[0372] The second device may be arranged and adapted to direct ultrasonic energy into the sample.

[0373] The second device may be arranged and adapted to direct a spray of charged droplets onto the biopsy sample to produce analyte ions.

[0374] The open ionization ion source may comprise a desorption electrospray ionization ("DESI") ion source or a desorption current flow focusing ionization ("DEFFI") ion source.

[0375] The desorption electrospray ionization ("DESI") ion source or the desorption current flow focusing ionization ("DEFFI") ion source may include a gradient desorption electrospray ionization ("DESI") ion source or a gradient desorption current flow focusing ionization ("DEFFI") ion source, wherein the composition of the solvent supplied to the desorption electrospray ionization ("DESI") ion source or the desorption current flow focusing ionization ("DEFFI") ion source and / or the composition of the solvent discharged therefrom changes over time.

[0376] A desorption electrospray ionization ("DESI") ion source or a desorption current focusing ionization ("DEFFI") ion source may be arranged and adapted for gradient desorption electrospray ionization analysis of a biopsy sample.

[0377] A desorption electrospray ionization ("DESI") ion source or a desorption current focused ionization ("DEFFI") ion source may be arranged and adjusted to be suitable for gradient desorption electrospray ionization analysis along the length of a biopsy sample, wherein the composition of the solvent supplied to and / or exhausted from the desorption electrospray ionization ("DESI") ion source or the desorption current focused ionization ("DEFFI") ion source varies with position along the length of the biopsy sample.

[0378] The analyzer may include: (i) a mass analyzer or filter and / or an ion mobility analyzer for performing mass analysis and / or ion mobility analysis on analyte ions and / or ions derived from the analyte ions; (ii) an ion mobility device for determining the ion mobility, collision cross section or interaction cross section of the analyte ions and / or ions derived from the analyte ions; and / or (iii) one or more fragmentation, collision or reaction devices for fragmenting or reacting the analyte ions.

[0379] According to one aspect, there is provided a mass spectrometry method comprising:

[0380] provide a biopsy sample;

[0381] generating a first analyte ion from a first location on the biopsy sample at a first time and generating a second analyte ion from a second, different location on the biopsy sample at a second, different time; and

[0382] Analyte ions are analyzed.

[0383] A biopsy sample can comprise a tissue sample having a longitudinal length.

[0384] The composition of the tissue sample may vary or change along the longitudinal length.

[0385] The longitudinal length may correspond to the depth within the tissue.

[0386] The biopsy sample may comprise a biopsy core or cylinder.

[0387] The method may include receiving a biopsy sample in the channel.

[0388] The method may include generating first analyte ions from a first location along a longitudinal length of the biopsy sample at a first time, and generating second analyte ions from a second different location along the longitudinal length of the biopsy sample at a second different time.

[0389] The method can include scanning at least a portion of the longitudinal length of the biopsy sample to generate analyte ions from a plurality of locations along the longitudinal length of the biopsy sample.

[0390] The method may include generating analyte ions using an open ionization ion source.

[0391] The open ionization ion source may comprise an ion source selected from the group consisting of: (i) a rapid evaporation ionization mass spectrometry ("REIMS") ion source; (ii) a desorption electrospray ionization ("DESI") ion source; (iii) a laser desorption ionization ("LDI") ion source; (iv) a thermal desorption ion source; (v) a laser diode thermal desorption ("LDTD") ion source; (vi) a desorption current dynamic focusing ("DEFFI") ion source; (vii) a dielectric barrier discharge ("DBD") plasma ion source; (viii) an atmospheric pressure solid analysis probe ("ASAP") ion source; (ix) an ultrasound assisted spray ionization ion source; (x) an easy open acoustic spray ionization ("EASI") ion source; (xi) a desorption atmospheric pressure photoionization ("DPPI") ion source. (xvi) laser ablation electrospray ("LAESI") ion source; (xvii) direct analysis in real time ("DART") ion source; (xviii) probe electrospray ionization ("PESI") ion source; (xix) solid probe assisted electrospray ionization ("SPA-ESI") ion source; (xx) ultrasonic surgical aspirator ("CUSA") device; (xxi) focused or unfocused ultrasound ablation device; (xxii) microwave resonant device; and (xxiii) pulsed plasma RF dissection device.

[0392] The method may comprise generating an aerosol, mist or vapor from the biopsy sample and ionizing the aerosol, mist or vapor to generate analyte ions.

[0393] The method may comprise contacting the biopsy sample with one or more electrodes to generate an aerosol, mist, or vapor.

[0394] The one or more electrodes may comprise a bipolar device or a monopolar device.

[0395] The one or more electrodes may comprise any of: (i) a monopolar device, wherein the device optionally further comprises an independent return electrode; (ii) a bipolar device; or (iii) a multi-phase RF device, wherein the device optionally further comprises one or more independent return electrodes.

[0396] One or more electrodes may comprise a rapid evaporative ionization mass spectrometry ("REIMS") device.

[0397] The method may comprise applying an AC or RF voltage to one or more electrodes to generate an aerosol, mist or vapor.

[0398] The step of applying an AC or RF voltage to the one or more electrodes may comprise applying one or more pulses of the AC or RF voltage to the one or more electrodes.

[0399] Application of an AC or RF voltage to one or more electrodes can cause heat to be dissipated into the sample.

[0400] The method may include irradiating the sample with a laser to produce analyte ions.

[0401] The method may comprise directing ultrasonic energy into the sample.

[0402] The method may include directing a spray of charged droplets onto a biopsy sample to produce analyte ions.

[0403] The step of directing the spray of charged droplets onto the biopsy sample may include generating analyte ions using a desorption electrospray ionization ("DESI") ion source or a desorption current flow focusing ionization ("DEFFI") ion source.

[0404] The method may comprise varying the composition of solvent supplied to and / or exhausted from a desorption electrospray ionization ("DESI") ion source or a desorption current focusing ionization ("DEFFI") ion source over time.

[0405] The method may comprise performing gradient desorption electrospray ionization analysis on the biopsy sample.

[0406] The method may comprise varying the composition of solvent supplied to and / or exhausted from a desorption electrospray ionization ("DESI") ion source or a desorption current focusing ionization ("DEFFI") ion source as a function of position along the length of the biopsy sample.

[0407] The method may comprise: (i) performing mass analysis and / or ion mobility analysis on analyte ions and / or ions derived from the analyte ions; (ii) determining the ion mobility, collision cross section or interaction cross section of the analyte ions and / or ions derived from the analyte ions; and / or (iii) fragmenting or reacting the analyte ions.

[0408] The method may comprise analyzing a disease.

[0409] The method may comprise determining the presence, location, margins and / or size of a tumor.

[0410] The method can include characterizing a tumor based on: (i) the aggressiveness of the tumor; (ii) the susceptibility of the tumor to treatment; (iii) whether and / or to what extent the tumor can be surgically removed; and / or (iv) whether and / or to what extent the tumor can be removed based on the location of the tumor.

[0411] According to one aspect, there is provided a method comprising:

[0412] taking a tissue sample using a biopsy needle to produce a first biopsy sample and a second biopsy sample;

[0413] analyzing the first biopsy sample in a first operating mode, wherein the first operating mode may include generating analyte ions from the first biopsy sample and analyzing the analyte ions; and

[0414] A second biopsy sample is analyzed in a second different mode of operation.

[0415] The first operating mode can include generating analyte ions from a first biopsy sample using a first open ionization analysis method.

[0416] The first open ionization analysis method can be selected from the group consisting of: (i) rapid evaporation ionization mass spectrometry ("REIMS") method; (ii) desorption electrospray ionization ("DESI") ionization method; (iii) laser desorption ionization ("LDI") method; (iv) thermal desorption ionization method; (v) laser diode thermal desorption ("LDTD") ionization method; (vi) desorption current dynamic focusing ionization ("DEFFI") method; (vii) dielectric barrier discharge ("DBD") plasma ionization method; (viii) atmospheric pressure solid analysis probe ("ASAP") ionization method; (ix) ultrasound assisted spray ionization method; (x) simplified open acoustic spray ionization ("EASI") method; (xi) desorption atmospheric pressure photoionization (DAP (xvi) laser ablation electrospray ("LAESI") ionization method; (xvii) direct analysis in real time ("DART") ionization method; (xviii) probe electrospray ionization ("PESI") method; (xix) solid probe assisted electrospray ionization ("SPA-ESI") method; (xx) ultrasonic surgical aspirator ("CUSA") method; (xxi) focused or unfocused ultrasound ablation method; (xxii) microwave resonance method; and (xxiii) pulsed plasma RF dissection method.

[0417] The step of generating analyte ions from the first biopsy sample may include directing a spray of charged droplets onto the first biopsy sample to generate analyte ions.

[0418] Directing a spray of charged droplets onto the first biopsy sample may include directing a spray of charged solvent droplets onto the first biopsy sample.

[0419] Directing the spray of charged droplets onto the first biopsy sample can include directing the spray of charged droplets onto the first biopsy sample at nearly atmospheric pressure.

[0420] Directing the spray of charged droplets onto the first biopsy sample can include ionizing the sample using desorption electrospray ionization ("DESI") or desorption current flow focusing ionization ("DEFFI").

[0421] The step of generating analyte ions from the first biopsy sample may include generating an aerosol, mist, or vapor from the first biopsy sample, and ionizing the aerosol, mist, or vapor to generate the analyte ions.

[0422] The step of generating analyte ions from the first biopsy sample may include contacting one or more electrodes with the biopsy sample to generate an aerosol, mist, or vapor.

[0423] The one or more electrodes may comprise a bipolar device or a monopolar device.

[0424] The one or more electrodes may comprise any of: (i) a monopolar device, wherein the device optionally further comprises an independent return electrode; (ii) a bipolar device; or (iii) a multi-phase RF device, wherein the device optionally further comprises one or more independent return electrodes.

[0425] One or more electrodes may comprise a rapid evaporative ionization mass spectrometry ("REIMS") device.

[0426] The method may comprise applying an AC or RF voltage to one or more electrodes to generate an aerosol, mist or vapor.

[0427] The step of applying an AC or RF voltage to the one or more electrodes may comprise applying one or more pulses of the AC or RF voltage to the one or more electrodes.

[0428] Application of an AC or RF voltage to one or more electrodes can cause heat to be dissipated into the sample.

[0429] The step of generating analyte ions from the first biopsy sample may include directing a laser beam onto the first biopsy sample to generate analyte ions.

[0430] The second mode of operation may include generating analyte ions from a second biopsy sample and analyzing the analyte ions in a second, different mode of operation.

[0431] The second operating mode can include generating analyte ions from a second biopsy sample using the first open ionization analysis method in a second, different operating mode.

[0432] (i) the first operating mode may comprise a positive ion operating mode and the second operating mode may comprise a negative ion operating mode; or

[0433] (ii) The first operating mode may comprise a negative ion operating mode and the second operating mode may comprise a positive ion operating mode.

[0434] The first mode of operation may comprise generating analyte ions from a first biopsy sample using a first solvent or solvent combination; and

[0435] The second mode of operation can include generating analyte ions from a second biopsy sample using a second different solvent or solvent combination.

[0436] The second operating mode may comprise an optimized version of the first operating mode.

[0437] The second mode of operation can include generating a plurality of analyte ions from a second biopsy sample using a second different open ionization analysis method.

[0438] The second different open ionization analysis method can be selected from the group consisting of: (i) rapid evaporation ionization mass spectrometry ("REIMS") method; (ii) desorption electrospray ionization ("DESI") ionization method; (iii) laser desorption ionization ("LDI") method; (iv) thermal desorption ionization method; (v) laser diode thermal desorption ("LDTD") ionization method; (vi) desorption current dynamic focusing ionization ("DEFFI") method; (vii) dielectric barrier discharge ("DBD") plasma ionization method; (viii) atmospheric pressure solid analysis probe ("ASAP") ionization method; (ix) ultrasound assisted spray ionization method; (x) simplified open acoustic spray ionization ("EASI") method; (xi) desorption atmospheric pressure photoionization (DPPI) method; (xiii) solid probe assisted electrospray ionization ("SPA-ESI") method; (xx) ultrasonic surgical aspirator ("CUSA") method; (xxi) focused or unfocused ultrasound ablation method; (xxii) microwave resonance method; and (xxiii) pulsed plasma RF dissection method.

[0439] The first operating mode may include analyzing analyte ions from a first biopsy sample using first operating parameters; and

[0440] The second operating mode can include analyzing analyte ions from a second biopsy sample using second, different operating parameters.

[0441] The first and / or second operating modes may include: (i) an operating mode for performing mass analysis and / or ion mobility analysis on analyte ions or ions derived from analyte ions; (ii) an operating mode for determining ion mobility, collision cross section or interaction cross section of analyte ions or ions derived from analyte ions; (iii) an operating mode for fragmenting analyte ions; and / or (iv) an operating mode for reacting, exciting, fragmenting or fractionating analyte ions.

[0442] The second different operating mode may include: (i) a gene sequencing operating mode; (ii) a matrix-assisted laser desorption ionization ("MALDI") operating mode; and / or (iii) a histopathology operating mode.

[0443] The method may include selecting and / or optimizing the second operating mode based on information acquired during the first operating mode.

[0444] The first biopsy sample can include a first portion of the tissue having a first longitudinal length and / or the second biopsy sample can include a second portion of the tissue having a second longitudinal length.

[0445] The composition of the first biopsy sample can vary or change along the first longitudinal length and / or the composition of the second biopsy sample can vary or change along the second longitudinal length.

[0446] The first longitudinal length may correspond to a depth inside the tissue and / or the second longitudinal length may correspond to a depth inside the tissue.

[0447] The first biopsy sample can comprise a biopsy core or cylinder and / or the second biopsy sample can comprise a biopsy core or cylinder.

[0448] The first biopsy sample may comprise a first portion of tissue and the second biopsy sample may comprise a second portion of tissue; and

[0449] The first portion of tissue may be adjacent to and / or connected to the second portion of tissue.

[0450] The first portion of tissue and the second portion of tissue may be adjacent and / or connected along a portion, a majority, or the entirety of the axial length of the first and / or second portion of tissue.

[0451] Removing a tissue sample using the biopsy needle can include producing the first biopsy sample and the second biopsy sample substantially simultaneously.

[0452] Removing a tissue sample using a biopsy needle may include inserting the biopsy needle into the tissue once to produce the first and second samples.

[0453] The biopsy needle may include a needle comprising a first hollow tube or barrel and a second hollow tube or barrel.

[0454] The first hollow tube or cylinder and the second hollow tube or cylinder may be connected together.

[0455] The first hollow tube or cylinder and the second hollow tube or cylinder may be connected together along a portion, a majority, or the entirety of the axial length of the first hollow tube or cylinder and / or the axial length of the second hollow tube or cylinder.

[0456] According to one aspect, a biopsy needle arranged and adapted to produce a first biopsy sample and a second biopsy sample when taking a tissue sample is provided.

[0457] The first biopsy sample can include a first portion of the tissue having a first longitudinal length and / or the second biopsy sample can include a second portion of the tissue having a second longitudinal length.

[0458] The composition of the first biopsy sample can vary or change along the first longitudinal length and / or the composition of the second biopsy sample can vary or change along the second longitudinal length.

[0459] The first longitudinal length may correspond to a depth inside the tissue and / or the second longitudinal length may correspond to a depth inside the tissue.

[0460] The first biopsy sample can comprise a biopsy core or cylinder and / or the second biopsy sample can comprise a biopsy core or cylinder.

[0461] The first biopsy sample may comprise a first portion of tissue and the second biopsy sample may comprise a second portion of tissue;

[0462] The first portion of tissue may be adjacent to and / or connected to the second portion of tissue.

[0463] The first portion of tissue and the second portion of tissue may be adjacent and / or connected along a portion, a majority, or the entirety of the axial length of the first and / or second portion of tissue.

[0464] The biopsy needle may be arranged and adapted to produce the first and second samples substantially simultaneously.

[0465] The biopsy needle may be arranged and adapted to produce the first and second samples when inserted once into the tissue.

[0466] The biopsy needle may include a needle comprising a first hollow tube or barrel and a second hollow tube or barrel.

[0467] The first hollow tube or cylinder and the second hollow tube or cylinder may be connected together.

[0468] The first hollow tube or cylinder and the second hollow tube or cylinder may be connected together along a portion, a majority, or the entirety of the axial length of the first hollow tube or cylinder and / or the axial length of the second hollow tube or cylinder.

[0469] According to one aspect, there is provided an apparatus comprising:

[0470] a biopsy needle, which contains one or more open ionization devices;

[0471] a control system arranged and adapted to actuate the one or more open ionization devices to generate an aerosol, mist or vapor from a biopsy sample within the biopsy needle; and

[0472] Analyzers are used to analyze aerosols, smoke, or vapors.

[0473] The one or more open ionization devices may include one or more electrodes arranged and adapted to contact the biopsy sample to generate an aerosol, mist, or vapor.

[0474] The one or more electrodes may comprise a bipolar device or a monopolar device.

[0475] The one or more electrodes may comprise any of: (i) a monopolar device, wherein the device optionally further comprises an independent return electrode; (ii) a bipolar device; or (iii) a multi-phase RF device, wherein the device optionally further comprises one or more independent return electrodes.

[0476] One or more electrodes may comprise a rapid evaporative ionization mass spectrometry ("REIMS") device.

[0477] The control system may be arranged and adapted to apply an AC or RF voltage to one or more electrodes in order to generate an aerosol, mist or vapor.

[0478] The control system may be arranged and adapted to apply one or more pulses of AC or RF voltage to one or more electrodes.

[0479] Application of an AC or RF voltage to one or more electrodes can cause heat to be dissipated into the sample.

[0480] One or more open ionization devices may contain a laser for irradiating the sample.

[0481] One or more open ionization devices may be arranged and adapted to evaporate or vaporize sample material directly from the sample by Joule heating or diathermy to generate an aerosol, mist or vapor from the sample.

[0482] The analyser may comprise a collision surface, and wherein the apparatus may be arranged and adapted to cause at least some of the aerosol, mist and / or vapour to impact the collision surface so as to form analyte ions.

[0483] The analyzer may include: (i) a mass analyzer or filter and / or an ion mobility analyzer for performing mass analysis and / or ion mobility analysis of aerosol, smoke, vapor or analyte ions and / or ions derived from aerosol, smoke, vapor, analyte ions; (ii) an ion mobility device for determining the ion mobility, collision cross section or interaction cross section of aerosol, smoke, vapor or analyte ions and / or ions derived from aerosol, smoke, vapor, analyte ions; and / or (iii) one or more fragmentation, collision or reaction devices for fragmenting or reacting aerosol, smoke, vapor or analyte ions.

[0484] According to one aspect, a biopsy needle is provided that includes one or more open ionization devices.

[0485] The one or more open ionization devices may include one or more electrodes arranged and adapted to contact a biopsy sample within a biopsy needle to generate an aerosol, mist, or vapor.

[0486] The one or more electrodes may comprise a bipolar device or a monopolar device.

[0487] The one or more electrodes may comprise any of: (i) a monopolar device, wherein the device optionally further comprises an independent return electrode; (ii) a bipolar device; or (iii) a multi-phase RF device, wherein the device optionally further comprises one or more independent return electrodes.

[0488] One or more electrodes may comprise a rapid evaporative ionization mass spectrometry ("REIMS") device.

[0489] One or more open ionization devices may include a laser for irradiating a biopsy sample within the biopsy needle.

[0490] According to one aspect, there is provided a method comprising:

[0491] Providing a biopsy needle comprising one or more open ionization devices;

[0492] energizing one or more open ionization devices to generate an aerosol, mist, or vapor from a biopsy sample within the biopsy needle; and

[0493] Analyze aerosols, fumes or vapors.

[0494] The one or more open ionization devices may include one or more electrodes arranged and adapted to contact the biopsy sample to generate an aerosol, mist, or vapor.

[0495] The one or more electrodes may comprise a bipolar device or a monopolar device.

[0496] The one or more electrodes may comprise any of: (i) a monopolar device, wherein the device optionally further comprises an independent return electrode; (ii) a bipolar device; or (iii) a multi-phase RF device, wherein the device optionally further comprises one or more independent return electrodes.

[0497] One or more electrodes may comprise a rapid evaporative ionization mass spectrometry ("REIMS") device.

[0498] Energizing one or more open ionization devices may include applying an AC or RF voltage to one or more electrodes to generate an aerosol, mist, or vapor.

[0499] Applying the AC or RF voltage to the one or more electrodes may include applying one or more pulses of the AC or RF voltage to the one or more electrodes.

[0500] Application of an AC or RF voltage to one or more electrodes can cause heat to be dissipated into the sample.

[0501] One or more open ionization devices may contain a laser for irradiating the sample.

[0502] The method may comprise evaporating or vaporizing sample material directly from the sample by Joule heating or diathermy, generating an aerosol, mist or vapor from the sample.

[0503] The method may include causing at least some of the aerosol, mist, and / or vapor to impact a collision surface so as to form analyte ions.

[0504] Analyzing aerosols, smoke or vapor may include: (i) performing mass analysis and / or ion mobility analysis on aerosols, smoke, vapor or analyte ions and / or ions derived from aerosols, smoke, vapor or analyte ions; (ii) determining the ion mobility, collision cross section or interaction cross section of aerosols, smoke, vapor or analyte ions and / or ions derived from aerosols, smoke, vapor or analyte ions; and / or (iii) fragmenting or reacting aerosols, smoke, vapor or analyte ions.

[0505] The method may comprise inserting a biopsy needle into tissue to provide a biopsy sample within the biopsy needle.

[0506] The method may include energizing one or more open ionization devices while inserting the biopsy sample into the tissue.

[0507] According to one aspect, there is provided a method comprising:

[0508] removing tissue samples to produce one or more biopsy samples;

[0509] analyzing one or more biopsy samples; and

[0510] Performing a diagnostic or surgical procedure using the first device may include generating analyte ions from tissue and analyzing the analyte ions, wherein one or more operating parameters of the first device are calibrated, optimized, or modified based on analysis of the one or more biopsy samples.

[0511] One or more of the one or more biopsy samples may comprise a tissue sample having a longitudinal length.

[0512] The composition of the tissue sample may vary or change along the longitudinal length.

[0513] The longitudinal length may correspond to the depth within the tissue.

[0514] One or more of the one or more biopsy samples may comprise a biopsy core or cylinder.

[0515] Analyzing the one or more biopsy samples can include generating analyte ions from the one or more biopsy samples and analyzing the analyte ions.

[0516] Generating analyte ions from the one or more biopsy samples may include generating analyte ions from the one or more biopsy samples using an open ionization ion source.

[0517] The open ionization ion source may comprise an ion source selected from the group consisting of: (i) a rapid evaporation ionization mass spectrometry ("REIMS") ion source; (ii) a desorption electrospray ionization ("DESI") ion source; (iii) a laser desorption ionization ("LDI") ion source; (iv) a thermal desorption ion source; (v) a laser diode thermal desorption ("LDTD") ion source; (vi) a desorption current dynamic focusing ("DEFFI") ion source; (vii) a dielectric barrier discharge ("DBD") plasma ion source; (viii) an atmospheric pressure solid analysis probe ("ASAP") ion source; (ix) an ultrasound assisted spray ionization ion source; (x) an easy open acoustic spray ionization ("EASI") ion source; (xi) a desorption atmospheric pressure photoionization ("DPPI") ion source. (xvi) laser ablation electrospray ("LAESI") ion source; (xvii) direct analysis in real time ("DART") ion source; (xviii) probe electrospray ionization ("PESI") ion source; (xix) solid probe assisted electrospray ionization ("SPA-ESI") ion source; (xx) ultrasonic surgical aspirator ("CUSA") device; (xxi) focused or unfocused ultrasound ablation device; (xxii) microwave resonant device; and (xxiii) pulsed plasma RF dissection device.

[0518] The step of generating analyte ions from the one or more biopsy samples may comprise generating an aerosol, mist or vapor from the one or more biopsy samples, and ionizing the aerosol, mist or vapor to generate the analyte ions.

[0519] The method may include causing at least some of the aerosol, mist, and / or vapor to impact a collision surface so as to produce analyte ions.

[0520] The step of generating analyte ions from the one or more biopsy samples may include contacting one or more electrodes with the one or more biopsy samples to generate an aerosol, mist, or vapor.

[0521] The one or more electrodes may comprise a bipolar device or a monopolar device.

[0522] The one or more electrodes may comprise any of: (i) a monopolar device, wherein the device optionally further comprises an independent return electrode; (ii) a bipolar device; or (iii) a multi-phase RF device, wherein the device optionally further comprises one or more independent return electrodes.

[0523] One or more electrodes may comprise a rapid evaporative ionization mass spectrometry ("REIMS") device.

[0524] The method may comprise applying an AC or RF voltage to one or more electrodes to generate an aerosol, mist or vapor.

[0525] The step of applying an AC or RF voltage to the one or more electrodes may comprise applying one or more pulses of the AC or RF voltage to the one or more electrodes.

[0526] Application of an AC or RF voltage to one or more electrodes can cause heat to be dissipated into the sample.

[0527] The step of generating analyte ions from the one or more biopsy samples may include directing a laser beam onto the one or more biopsy samples to generate analyte ions.

[0528] Analyzing the analyte ions may include: (i) performing mass analysis and / or ion mobility analysis on the analyte ions and / or ions derived from the analyte ions; (ii) determining the ion mobility, collision cross section or interaction cross section of the analyte ions and / or ions derived from the analyte ions; and / or (iii) fragmenting or reacting the analyte ions.

[0529] Analyzing the one or more biopsy samples may include analyzing the one or more biopsy samples using a first device.

[0530] The first device may include an open ionization ion source.

[0531] The open ionization ion source may comprise an ion source selected from the group consisting of: (i) a rapid evaporation ionization mass spectrometry ("REIMS") ion source; (ii) a desorption electrospray ionization ("DESI") ion source; (iii) a laser desorption ionization ("LDI") ion source; (iv) a thermal desorption ion source; (v) a laser diode thermal desorption ("LDTD") ion source; (vi) a desorption current dynamic focusing ("DEFFI") ion source; (vii) a dielectric barrier discharge ("DBD") plasma ion source; (viii) an atmospheric pressure solid analysis probe ("ASAP") ion source; (ix) an ultrasound assisted spray ionization ion source; (x) an easy open acoustic spray ionization ("EASI") ion source; (xi) a desorption atmospheric pressure photoionization ("DPPI") ion source. (xvi) laser ablation electrospray ("LAESI") ion source; (xvii) direct analysis in real time ("DART") ion source; (xviii) probe electrospray ionization ("PESI") ion source; (xix) solid probe assisted electrospray ionization ("SPA-ESI") ion source; (xx) ultrasonic surgical aspirator ("CUSA") device; (xxi) focused or unfocused ultrasound ablation device; (xxii) microwave resonant device; and (xxiii) pulsed plasma RF dissection device.

[0532] The step of performing a diagnostic or surgical procedure may include generating an aerosol, mist or vapor from the tissue using a first device, and ionizing the aerosol, mist or vapor to generate analyte ions.

[0533] The method may include causing at least some of the aerosol, mist or vapor to impact the collision surface so as to produce analyte ions.

[0534] Generating an aerosol, mist, or vapor from the tissue using the first device may include contacting the tissue with one or more electrodes to generate the aerosol, mist, or vapor.

[0535] The one or more electrodes may comprise a bipolar device or a monopolar device.

[0536] The one or more electrodes may comprise any of: (i) a monopolar device, wherein the device optionally further comprises an independent return electrode; (ii) a bipolar device; or (iii) a multi-phase RF device, wherein the device optionally further comprises one or more independent return electrodes.

[0537] The method may comprise applying an AC or RF voltage to one or more electrodes to generate an aerosol, mist or vapor.

[0538] Applying the AC or RF voltage to the one or more electrodes may include applying one or more pulses of the AC or RF voltage to the one or more electrodes.

[0539] Application of an AC or RF voltage to one or more electrodes can cause heat to be dissipated into the sample.

[0540] The first device and / or one or more electrodes may comprise a rapid evaporative ionization mass spectrometry ("REIMS") device.

[0541] The first device may comprise a laser for illuminating the sample.

[0542] Analyzing the analyte ions may include: (i) performing mass analysis and / or ion mobility analysis on the analyte ions and / or ions derived from the analyte ions; (ii) determining the ion mobility, collision cross section or interaction cross section of the analyte ions and / or ions derived from the analyte ions; and / or (iii) fragmenting or reacting the analyte ions.

[0543] The first device may include an electrosurgical tool.

[0544] The first device may include an electrosurgical device, a diathermy device, an ultrasonic device, a hybrid ultrasonic electrosurgical device, a surgical water jet device, a hybrid electrosurgery device, an argon plasma coagulation device, a hybrid argon plasma coagulation device, a water jet device, and / or a laser device.

[0545] Analyzing the one or more biopsy samples may include determining spatially resolved information about the one or more biopsy samples and / or tissues; and

[0546] One or more operating parameters may be calibrated, optimized or modified based on the spatially resolved information.

[0547] One or more operating parameters may be calibrated, optimized, or altered depending on the position of the first device during a diagnostic or surgical procedure.

[0548] Analyzing the one or more biopsy samples may include determining one or more tissue types of the one or more biopsy samples and / or tissues; and

[0549] One or more operating parameters may be calibrated, optimized, or modified based on the determined tissue type.

[0550] One or more operating parameters may be calibrated, optimized, or modified depending on the type of tissue being analyzed by the first device during a diagnostic or surgical procedure.

[0551] The one or more tissue types can be selected from the group consisting of: (i) healthy tissue; (ii) diseased tissue or tumor tissue; (iii) tissue containing healthy cells and diseased cells, wherein the diseased cells are optionally cancer cells; (iv) a type or grade of diseased tissue or tumor tissue; (v) tissue in the border area of ​​an organ and / or tumor; and / or (vi) tissue away from the border area of ​​an organ and / or tumor.

[0552] The one or more operating parameters of the first device may include: (i) the magnitude and / or frequency of the voltage supplied to the first device; (ii) the temperature of the first device; (iii) the composition of the matrix added to the aerosol, smoke or vapor generated by the first device; (iv) the temperature of the collision surface impacted by the aerosol, smoke or vapor generated by the first device; (v) the voltage applied to the collision surface impacted by the aerosol, smoke or vapor generated by the first device; (vi) one or more operating parameters of a mass analyzer or filter for mass analysis of analyte ions and / or ions derived from the analyte ions; (vii) one or more operating parameters of an ion mobility device for determining the ion mobility, collision cross section or interaction cross section of the analyte ions and / or ions derived from the analyte ions; and / or (viii) one or more operating parameters of a collision, reaction or fragmentation device for fragmenting or reacting the analyte ions.

[0553] The method may comprise generating or updating a library or database based on said analysis, wherein the library or database is used for calibrating or optimizing the first device during a diagnostic or surgical procedure.

[0554] According to one aspect, there is provided an apparatus comprising:

[0555] A first device for performing a diagnostic or surgical procedure, wherein the first device is arranged and adapted to generate analyte ions from tissue and to analyze the analyte ions; and

[0556] A control system arranged and adapted to calibrate, optimize or modify one or more operating parameters of a first device used for diagnosis or surgery or during a procedure based on analysis of one or more biopsy samples taken from tissue.

[0557] One or more of the one or more biopsy samples may comprise a tissue sample having a longitudinal length.

[0558] The composition of the tissue sample may vary or change along the longitudinal length.

[0559] The longitudinal length may correspond to the depth within the tissue.

[0560] One or more of the one or more biopsy samples may comprise a biopsy core or cylinder.

[0561] Analysis of the one or more biopsy samples may include analyzing the one or more biopsy samples using a first device.

[0562] The first device may include an open ionization ion source.

[0563] The open ionization ion source may comprise an ion source selected from the group consisting of: (i) a rapid evaporation ionization mass spectrometry ("REIMS") ion source; (ii) a desorption electrospray ionization ("DESI") ion source; (iii) a laser desorption ionization ("LDI") ion source; (iv) a thermal desorption ion source; (v) a laser diode thermal desorption ("LDTD") ion source; (vi) a desorption current dynamic focusing ("DEFFI") ion source; (vii) a dielectric barrier discharge ("DBD") plasma ion source; (viii) an atmospheric pressure solid analysis probe ("ASAP") ion source; (ix) an ultrasound assisted spray ionization ion source; (x) an easy open acoustic spray ionization ("EASI") ion source; (xi) a desorption atmospheric pressure photoionization ("DPPI") ion source. (xvi) laser ablation electrospray ("LAESI") ion source; (xvii) direct analysis in real time ("DART") ion source; (xviii) probe electrospray ionization ("PESI") ion source; (xix) solid probe assisted electrospray ionization ("SPA-ESI") ion source; (xx) ultrasonic surgical aspirator ("CUSA") device; (xxi) focused or unfocused ultrasound ablation device; (xxii) microwave resonant device; and (xxiii) pulsed plasma RF dissection device.

[0564] The first device may be arranged and adapted to generate an aerosol, mist or vapor from the tissue and to ionize the aerosol, mist or vapor to generate analyte ions.

[0565] The device may comprise a collision surface, wherein the device may be arranged and adapted to cause at least some of the aerosol, mist or vapor to impact the collision surface so as to produce analyte ions.

[0566] The first device may include one or more electrodes arranged and adapted to contact tissue so as to generate an aerosol, mist or vapor.

[0567] The one or more electrodes may comprise a bipolar device or a monopolar device.

[0568] The one or more electrodes may comprise any of: (i) a monopolar device, wherein the device optionally further comprises an independent return electrode; (ii) a bipolar device; or (iii) a multi-phase RF device, wherein the device optionally further comprises one or more independent return electrodes.

[0569] The apparatus may comprise means arranged and adapted to apply an AC or RF voltage to one or more electrodes so as to generate an aerosol, mist or vapour.

[0570] The means for applying an AC or RF voltage to the one or more electrodes may be arranged and adapted to apply one or more pulses of AC or RF voltage to the one or more electrodes.

[0571] Application of an AC or RF voltage to one or more electrodes can cause heat to be dissipated into the sample.

[0572] The first device and / or one or more electrodes may comprise a rapid evaporative ionization mass spectrometry ("REIMS") device.

[0573] The first device may comprise a laser for illuminating the sample.

[0574] The apparatus may comprise: (i) a mass analyser or filter and / or an ion mobility analyser for performing mass analysis and / or ion mobility analysis on analyte ions and / or ions derived from the analyte ions; (ii) an ion mobility device for determining the ion mobility, collision cross section or interaction cross section of the analyte ions and / or ions derived from the analyte ions; and / or (iii) a fragmentation, reaction or collision device for fragmenting or reacting the analyte ions.

[0575] The first device may include an electrosurgical tool.

[0576] The first device may include an electrosurgical device, a diathermy device, an ultrasonic device, a hybrid ultrasonic electrosurgical device, a surgical water jet device, a hybrid electrosurgery device, an argon plasma coagulation device, a hybrid argon plasma coagulation device, a water jet device, and / or a laser device.

[0577] The control system may be arranged and adapted to calibrate, optimize or modify one or more operating parameters based on spatially resolved information determined from analysis of one or more biopsy samples.

[0578] The control system may be arranged and adapted to calibrate, optimize or modify one or more operating parameters depending on the position of the first device during a diagnostic or surgical procedure.

[0579] The control system may be arranged and adapted to calibrate, optimize or modify one or more operating parameters based on one or more tissue types determined by analysis of one or more biopsy samples.

[0580] The control system may be arranged and adapted to calibrate, optimize or modify one or more operating parameters depending on the type of tissue analyzed by the first device during a diagnostic or surgical procedure.

[0581] The one or more tissue types can be selected from the group consisting of: (i) healthy tissue; (ii) diseased tissue or tumor tissue; (iii) tissue containing healthy cells and diseased cells, wherein the diseased cells are optionally cancer cells; (iv) a type or grade of diseased tissue or tumor tissue; (v) tissue in the border area of ​​an organ and / or tumor; and / or (vi) tissue away from the border area of ​​an organ and / or tumor.

[0582] The one or more operating parameters of the first device may include: (i) the magnitude and / or frequency of the voltage supplied to the first device; (ii) the temperature of the first device; (iii) the composition of the matrix added to the aerosol, smoke or vapor generated by the first device; (iv) the temperature of the collision surface impacted by the aerosol, smoke or vapor generated by the first device; (v) the voltage applied to the collision surface impacted by the aerosol, smoke or vapor generated by the first device; (vi) one or more operating parameters of a mass analyzer or filter for mass analysis of analyte ions and / or ions derived from the analyte ions; (vii) one or more operating parameters of an ion mobility device for determining the ion mobility, collision cross section or interaction cross section of the analyte ions and / or ions derived from the analyte ions; and / or (viii) one or more operating parameters of a collision, reaction or fragmentation device for fragmenting or reacting the analyte ions.

[0583] The control system may be arranged and adapted to generate or update a library or database based on said analysis and to use the library or database to calibrate or optimize the first device during a diagnostic or surgical procedure.

[0584] In any of the various aspects and embodiments described herein, analysis of analyte ions may generate spectral data and / or ion mobility data, which may then be analyzed.

[0585] Analysis of the spectral data and / or ion mobility data may include analyzing one or more sample spectra to classify the sample.

[0586] Analyzing one or more sample spectra to classify the sample may include unsupervised analysis of one or more sample spectra (eg, performing dimensionality reduction) and / or supervised analysis of one or more sample spectra (eg, performing classification).

[0587] Analyzing one or more sample spectra may include first an unsupervised analysis (eg, performing dimensionality reduction) followed by a supervised analysis (eg, performing classification).

[0588] Analyzing one or more sample spectra may include using one or more of the following: (i) univariate analysis; (ii) multivariate analysis; (iii) principal component analysis (PCA); (iv) linear discriminant analysis (LDA); (v) maximum margin criterion (MMC); (vi) library-based analysis; (vii) soft independent modeling classification (SIMCA); (viii) factor analysis (FA); (ix) recursive partitioning (decision tree); (x) random forest; (xi) independent component analysis (ICA); (xii) partial least squares discriminant analysis (PLSA); (xiii) factor analysis (FA); (ix) decision tree; (xiv) random forest; (xi) independent component analysis (ICA); (xii) partial least squares discriminant analysis (PLSA); (xv) factor analysis (FA); (ix) decision tree; (xv ... recursive partitioning (decision tree); (xv) random forest; (xi) independent component analysis (ICA); (xii) partial least squares discriminant analysis (PLSA); (xii) factor analysis (FA); (ix) factor analysis (FA); (ix) factor analysis (FA); (xv) factor analysis (FA); (ix) factor analysis (FA); (xv) factor analysis (FA); (ix) factor analysis (FA); (ix) factor analysis (FA); (xv) factor analysis (FA); (ix) factor analysis (FA); (ix) factor analysis (FA); (xv) factor analysis (FA); (ix) factor analysis (FA); (ix) factor analysis (FA); (xv) factor analysis (FA); (xv) factor analysis (FA); (ix) factor analysis (FA); (ix) factor analysis (FA); (ix) factor analysis (FA discriminant analysis (PLS-DA); (xiii) orthogonal (partial least squares) projection to latent structure (OPLS); (xiv) OPLS discriminant analysis (OPLS-DA); (xv) support vector machine (SVM); (xvi) (artificial) neural network; (xvii) multilayer perceptron; (xviii) radial basis function (RBF) network; (xix) Bayesian analysis; (xx) cluster analysis; (xxi) kernelization method; and (xxii) subspace discriminant analysis; (xxiii) k-nearest neighbors (KNN); (xxiv) quadratic discriminant analysis (QDA); (xxv) probabilistic principal component analysis (PPCA); (xxvi) non-negative matrix factorization; (xxvii) k-means factorization; (xxviii) fuzzy c-means factorization; and (xxix) discriminant analysis (DA).

[0589] Analyzing one or more sample spectra to classify the samples may include developing a classification model or library using one or more reference sample spectra.

[0590] Analyzing one or more sample spectra to classify the samples may include performing a principal component analysis (PCA) (eg, to perform dimensionality reduction) followed by a linear discriminant analysis (LDA) (eg, to perform classification).

[0591] Analyzing one or more sample spectra to classify the samples may include performing a principal component analysis (PCA) (eg, performing dimensionality reduction) followed by a maximum margin criterion (MMC) process (eg, performing classification).

[0592] Analyzing one or more sample spectra to classify the samples may include defining one or more classes within a classification model or library.

[0593] Analyzing one or more sample spectra to classify the samples may include manually or automatically defining one or more classes within a classification model or library according to one or more classification or clustering criteria.

[0594] One or more classification or clustering criteria for each category can be based on one or more of the following: the distance between one or more pairs of reference points of the reference sample spectrum in the model space; the variance value between each group of reference points of the reference sample spectrum in the model space; and the variance value within a group of reference points of the reference sample spectrum in the model space.

[0595] One or more categories may each be defined by one or more classification definitions.

[0596] One or more classification definitions can include one or more of: a set of one or more reference points within a model space for reference sample spectra, values, boundaries, lines, planes, hyperplanes, variances, volumes, Voronoi cells, and / or positions; and one or more positions within a classification hierarchy.

[0597] Analyzing one or more sample spectra to classify the samples may include classifying one or more unknown sample spectra using a classification model or library.

[0598] Analyzing one or more sample spectra to classify the samples may include manually or automatically classifying the one or more sample spectra according to one or more classification criteria.

[0599] The one or more classification criteria may include one or more of the following:

[0600] The following distances are below the distance threshold or are the lowest of such distances: the distances between one or more projected sample points of one or more sample spectra in the model space and a set of one or more reference points of one or more reference sample spectra, values, boundaries, lines, planes, hyperplanes, volumes, Voronoi cells, or locations in the model space;

[0601] The location of one or more projected sample points of one or more sample spectra in the model space is on one side or the other side of one or more reference sample spectra, values, boundaries, lines, planes, hyperplanes, or one or more reference points located in the model space;

[0602] The locations of one or more projected sample points of the one or more sample spectra in the model space are within one or more volumes or Voronoi cells in the model space; and

[0603] The probability or classification score is above the probability or classification score threshold or is the highest of such probability or classification scores.

[0604] According to an aspect, there is provided a mass analyser and / or an ion mobility analyser comprising a device as described above.

[0605] According to one aspect, a mass spectrometry method and / or an ion mobility spectrometry method is provided, comprising the method as described above.

[0606] The mass spectrometer and / or ion mobility spectrometer may acquire data in negative ion mode only, positive ion mode only, or both positive and negative ion modes. Positive ion mode spectral data and negative ion mode spectral data may be combined or concatenated.

[0607] Different ion mobility drift gases and / or dopants may be used to obtain ion mobility spectrometry data. This data may then be combined or concatenated.

[0608] Contain and relate to use open type ionization ion source by target (its details are provided in this paper elsewhere) to produce each embodiment of smoke, aerosol or steam. Aerosol, smoke or steam can then be mixed with matrix and be drawn into the vacuum chamber of mass spectrometer and / or ion mobility spectrometer. Mixture can be made to impact collision surface, cause aerosol, smoke or steam to be ionized by impact ionization, thereby produce analyte ion. Then can carry out mass analysis and / or ion mobility analysis to obtained analyte ion (or by the fragment or product ion derived from analyte ion), and can make obtained mass spectrum data and / or ion mobility spectrum data experience multivariate analysis or other mathematical treatment to determine one or more characteristics of target in real time.

[0609] According to one embodiment, the first means for generating an aerosol, mist or vapor from a target may include means for utilizing an RF voltage, such as a continuous RF waveform.

[0610] Other embodiments are contemplated in which the first device for generating an aerosol, smoke or vapor from a target may comprise an argon plasma coagulation ("APC") device. An argon plasma coagulation device involves the use of an ionized argon gas (plasma) jet introduced through a probe. The probe may be passed through an endoscope. Because the probe is placed at a distance from the target, argon plasma coagulation is essentially a non-contact process. Argon gas is emitted from the probe and then ionized by a high voltage discharge (e.g., 6 kV). A high frequency current is then conducted through the gas jet, causing coagulation of the target at the other end of the jet. The coagulation depth is typically only a few millimeters.

[0611] The first device, surgical or electrosurgical tool, device or probe or other sampling device or probe disclosed in any aspect or embodiment of the present invention may include a non-contact surgical device, such as a hydrosurgical device, a surgical water jet device, an argon plasma coagulation device, a hybrid argon plasma coagulation device, a water jet device, and a laser device. One or more.

[0612] Non-contact surgical devices can be defined as surgical devices arranged and adapted to dissect, fragment, liquefy, aspirate, fulgurate or otherwise destroy biological tissue without physically contacting the tissue. Examples include laser devices, hydrosurgical devices, argon plasma coagulation devices, and hybrid argon plasma coagulation devices.

[0613] Because non-contact devices may not make physical contact with tissue, the procedure may be considered relatively safe and can be used to treat delicate tissues with low intracellular connections, such as skin or fat.

[0614] According to various embodiments, the mass spectrometer and / or ion mobility spectrometer may acquire data in negative ion mode only, in positive ion mode only, or in both positive and negative ion modes. Positive ion mode spectral data may be merged or concatenated with negative ion mode spectral data. Negative ion mode can provide spectra that are particularly suitable for classifying aerosol, smoke or vapor samples, such as aerosol, smoke or vapor samples from targets containing lipids.

[0615] Ion mobility spectrometry data can be acquired using different ion mobility drift gases, or dopants can be added to the drift gas to induce a change in the drift time of one or more species. This data can then be combined or concatenated.

[0616] Obviously, the requirement to add matrix or reagents directly to the sample may hinder the ability to perform in vivo analysis of tissues and, more generally, the ability to provide rapid and simple analysis of target materials.

[0617] According to other embodiments, the open ionization ion source may include an ultrasonic ablation ion source or a hybrid electrosurgery-ultrasonic ablation source to produce a liquid sample that is then aspirated in the form of an aerosol. The ultrasonic ablation ion source may include focused or unfocused ultrasound.

[0618] Optionally, the first device comprises or forms part of an ion source selected from the group consisting of: (i) a rapid evaporation ionization mass spectrometry ("REIMS") ion source; (ii) a desorption electrospray ionization ("DESI") ion source; (iii) a laser desorption ionization ("LDI") ion source; (iv) a thermal desorption ion source; (v) a laser diode thermal desorption ("LDTD") ion source; (vi) a desorption current dynamic focusing ("DEFFI") ion source; (vii) a dielectric barrier discharge ("DBD") ion source; ”) plasma ion source; (viii) atmospheric pressure solid analysis probe (“ASAP”) ion source; (ix) ultrasound assisted spray ionization ion source; (x) easy open acoustic spray ionization (“EASI”) ion source; (xi) desorption atmospheric pressure photoionization (“DAPPI”) ion source; (xii) paper spray (“PS”) ion source; (xiii) jet desorption ionization (“JeDI”) ion source; (xiv) touch spray (“TS”) ion source; (xv) nano DESI ion source; (xvi ) laser ablation electrospray ("LAESI") ion source; (xvii) direct analysis in real time ("DART") ion source; (xviii) probe electrospray ionization ("PESI") ion source; (xix) solid probe assisted electrospray ionization ("SPA-ESI") ion source; (xx) ultrasonic surgical aspirator ("CUSA") device; (xxi) hybrid CUSA-diathermal device; (xxii) focused or unfocused ultrasound ablation device; (xxiii) hybrid focused or unfocused ultrasound ablation and diathermy device; (xxiv) microwave resonant device; (xxv) pulsed plasma RF dissection device; (xxvi) argon plasma coagulation device; (xxvi) hybrid pulsed plasma RF dissection and argon plasma coagulation device; (xxvii) hybrid pulsed plasma RF dissection and JeDI device; (xxviii) surgical water / saline jet device; (xxix) hybrid electrosurgery and argon plasma coagulation device; and (xxx) hybrid argon plasma coagulation and water / saline jet device. BRIEF DESCRIPTION OF THE DRAWINGS

[0619] Various embodiments will now be described by way of example only and with reference to the accompanying drawings, in which:

[0620] Figure 1 Schematic representation of the various microorganisms present in the human microbiome;

[0621] Figure 2 Schematically shows the various mucosa or mucosal membranes present in the human body;

[0622] Figure 3 A mucosa containing biological tissue and bacteria is schematically shown;

[0623] Figure 4 Schematically illustrates how analytes present in the mucosa can be used to help identify a variety of clinical conditions;

[0624] Figure 5 Schematic illustration of how metabolomic profiling of analytes from the mucosa can help identify clinical conditions such as allergy, inflammation, and preterm birth;

[0625] Figure 6 Various microbial analysis pathways and real-time rapid direct analysis methods using open mass spectrometry according to various embodiments are shown;

[0626] Figure 7 Describing desorption electrospray ionization ("DESI") techniques according to various embodiments;

[0627] FIG8 schematically illustrates a desorption electrospray ionization (“DESI”) mass spectrometry setup for swab analysis according to various embodiments;

[0628] Fig. 9 Schematically illustrates mucosa sampled from a selected human body part (e.g., urogenital tract, oral cavity, or nasal cavity) using a medical cotton swab as a sampling device according to various embodiments, wherein the surface of the medical swab can then be directly analyzed by desorption electrospray ionization ("DESI") mass spectrometry without prior sample preparation procedures;

[0629] Fig. 10A Shown are average negative ion desorption electrospray ionization ("DESI") mass spectra from vaginal, oral, and nasal mucosa recorded using a Xevo G2-S Q-Tof (RTM) mass spectrometer, Fig. 10B PCA and MMC score plots acquired from vaginal mucosa (n=68), oral mucosa (n=15), and nasal mucosa (n=20) using desorption electrospray ionization ("DESI") mass spectrometry are shown;

[0630] Fig.11Desorption electrospray ionization ("DESI") mass spectra of vaginal, oral, and nasal mucosa obtained from medical cotton swabs in negative ion mode, as well as principal component analysis (PCA) and maximum margin criterion analysis, provide separation between different mucosal classes (nasal, oral, vaginal mucosa), and obtain prediction accuracy in the range of 92-100% by leave one out cross validation;

[0631] Fig.12 The desorption electrospray ionization ("DESI") mass spectrum of pregnant vaginal mucosa obtained from a medical cotton swab in negative ion mode is shown, in which the urogenital mucosa was found to produce cholesterol sulfate [MH] with a mass-to-charge ratio of 465.41 - As the most abundant lipid species, as well as different glycerophospholipid species, such as glycerophosphoethanolamine (PE) [PE(40:7)-H] with a mass-to-charge ratio of 788.50 - , glycerophosphoserine (PS) with a mass-to-charge ratio of 760.50 [PS(34:1)-H] - and glycerophosphoinositol (PI) [PI(36:1)-H] with a mass-to-charge ratio of 863.58 - ;

[0632] Fig.13A Shown are average desorption electrospray ionization ("DESI") mass spectra acquired in negative ion mode from the pregnant group (highlighted in blue) and the non-pregnant group (highlighted in red) in the mass range m / z 150-1000. Fig. 13B shows the principal component analysis and discriminant analysis obtained using the recursive maximum margin criterion ("RMMC"), Fig. 13C A leave-one-out cross-validation analysis showed enhanced separation of the groups of classes with highly accurate identification (>80%) based on chemical signatures in the vaginal mucosa. Fig.13D Box plots are shown indicating significant differences in the abundance of selected peaks between non-pregnant and pregnant vaginal mucosa, primarily in the mass to charge ratio ("m / z") range 550-1000, Fig.13E Leave-one-out cross validation is shown;

[0633] Fig.14ADesorption electrospray ionization ("DESI") mass spectrometry analysis of a bacterial sample on a swab is shown according to various embodiments and the bacterial sample can be detected using DESI, Fig. 14B Comparison with rapid evaporative ionization mass spectrometry ("REIMS") analysis, and time-of-flight mass analysis of bacterial samples directly from agar plates are shown;

[0634] Fig.15A The average desorption electrospray ionization ("DESI") mass spectra of the pregnant vaginal mucosa are shown for various microbial species analyzed, including Candida albicans, Pseudomonas monteri, Staphylococcus epidermidis, Moraxella catarrhalis, Klebsiella pneumoniae, and Lactobacillus spp. Fig. 15B PCA plots showing the separation of microbial species within the first two components between vaginal mucosa (pregnant and non-pregnant groups) are shown. Fig. 15C The separation between different bacterial species and fungal species is shown;

[0635] Fig.16 Schematically illustrates desorption electrospray ionization ("DESI") mass spectrometry, rapid evaporation ionization mass spectrometry ("REIMS") mass spectrometry, and culture-based analysis of a sample on a swab according to various embodiments;

[0636] Fig.17A shows desorption electrospray ionization ("DESI") mass spectrometry data, where the swab can be continuously rotated while undergoing desorption electrospray ionization ("DESI") ionization to increase signal intensity, Fig. 17B Rapid Evaporative Ionization Mass Spectrometry ("REIMS") mass spectrometry data is shown, wherein a swab may be dipped, soaked, or otherwise immersed in a fluid (such as water) to increase signal intensity prior to undergoing Rapid Evaporative Ionization Mass Spectrometry ("REIMS") analysis;

[0637] Fig.18 schematically illustrates a rapid evaporative ionization mass spectrometry ("REIMS") technique according to various embodiments;

[0638] Fig.19 Describing various advantages and disadvantages associated with standard cotton swabs and coated or chemically modified swabs according to various embodiments;

[0639] Fig. 20A Illustration of the mass spectra obtained when using a standard cotton swab, Fig. 20B shows how to improve sensitivity (especially lipid signal) using modified swabs, Fig. 20C It shows how to improve sensitivity (especially improve lipid signal) using modified swabs according to various embodiments;

[0640] Fig.21Various solid phase microextraction ("SPME") coating materials for mucosal sampling according to various embodiments are shown;

[0641] Fig. 22 Schematically illustrates a solid phase microextraction ("SPME") swab sample preparation workflow for extracting analytes in a saliva matrix, followed by desorption electrospray ionization ("DESI") mass spectrometry analysis according to various embodiments;

[0642] Fig.23A Shown are saliva mass spectra obtained using a standard medical swab in negative ion mode (left) and positive ion mode (right). Fig. 23B shows the mass spectra of saliva obtained in negative ion mode (left) and positive ion mode (right) using a standard medical swab coated with three layers of C18 (octadecyl) adsorbent, Fig.23C shows the mass spectra of saliva obtained in negative ion mode (left) and positive ion mode (right) using a standard medical swab coated with three layers of C18 (octadecyl) end capped (EC) adsorbent, Fig.23D shows the mass spectra of saliva obtained in negative ion mode (left) and positive ion mode (right) using a standard medical swab coated with three layers of hydrophilic-lipophilic balance (HLB) adsorbent, Fig.23E Shown are saliva mass spectra obtained in negative ion mode (left) and positive ion mode (right) using a standard medical swab coated with three layers of divinylbenzene (DVB) weak anion exchange (WAX) adsorbent;

[0643] Fig.24 shows the spectra observed when a stool sample was analyzed using rapid evaporative ionization mass spectrometry ("REIMS") analysis;

[0644] Fig.25A A desorption electrospray ionization ("DESI") device is shown, Fig.25B shows a graph of intensity versus inlet capillary temperature for the analysis of fatty acids using a Waters Synapt (RTM) mass spectrometer, Fig.25C shows a graph of intensity versus inlet capillary temperature for the analysis of fatty acids using a Waters Xevo (RTM) mass spectrometer, Fig.25D shows a graph of intensity versus inlet capillary temperature for the analysis of phospholipids using a Waters Synapt (RTM) mass spectrometer, Fig.25E shows a graph of intensity versus inlet capillary temperature for the analysis of phospholipids using a Waters Xevo (RTM) mass spectrometer;

[0645] Fig.26 A needle biopsy procedure is shown in which a biopsy core is extracted from a patient using a biopsy needle;

[0646] Fig. 27 A biopsy needle is shown that includes a rapid vaporization ionization mass spectrometry ("REIMS") electrode according to one embodiment;

[0647] Fig.28 An analysis method is shown, which includes constructing a classification model according to various embodiments;

[0648] Fig.29 A set of reference sample spectra obtained from two categories of known reference samples are shown;

[0649] Fig.30 A multivariate space is shown having three dimensions defined by an intensity axis, wherein the multivariate space comprises a plurality of reference points, each reference point corresponding to a set of three peak intensity values ​​derived from a reference sample spectrum;

[0650] Fig.31 The general relationship between the cumulative variance of the PCA model and the number of components is shown;

[0651] Fig.32 shows a PCA space having two dimensions defined by the principal component axes, wherein the PCA space contains a plurality of transformed reference points or scores, each corresponding to Fig.30 reference point;

[0652] Fig.33 shows a PCA-LDA space with a single dimension or axis, where LDA is based on Fig.32 The PCA space is performed, and the PCA-LDA space contains multiple other transformed reference points or category scores, each of which corresponds to Fig.32 The transformation reference point or score;

[0653] Fig.34 A method of analysis is shown, which includes using a classification model according to various embodiments;

[0654] Fig.35 A sample spectrum obtained from an unknown sample is shown;

[0655] Fig.36 Shows Fig.33 The PCA-LDA space is further composed of Fig.35 PCA-LDA projected sample points derived from the peak intensity values ​​of the sample spectrum;

[0656] Fig.37 An analysis method is shown, which includes constructing a classification library according to various embodiments; and

[0657] Fig.38A method of analysis is shown that includes using a classification library according to various embodiments. DETAILED DESCRIPTION

[0658] Various embodiments will now be described in more detail.Some of the embodiments described in more detail relate to analyzing standard medical swabs using a desorption electrospray ionization ("DESI") ion source.

[0659] However, other embodiments are contemplated that may use different open ionization ion sources.

[0660] Open ion source

[0661] Various embodiments as described herein are described in the context of generating a spray of charged droplets using a desorption electrospray ionization ("DESI") ion source. However, other embodiments are also contemplated in which other means of generating analyte ions are used.

[0662] The device or ion source may comprise an open ionization ion source characterized by the ability to produce analyte ions from a native or unmodified target. In contrast, other types of ionization ion sources, such as matrix-assisted laser desorption ionization ("MALDI") ion sources, require the addition of a matrix or reagent to the sample prior to ionization.

[0663] Obviously, the requirement to add matrix or reagents to the sample impedes the ability to perform in vivo analysis of tissues and, more generally, the ability to provide rapid and simple analysis of target materials.

[0664] Thus, in comparison, open ionization techniques are particularly advantageous, firstly because they do not require the addition of matrices or reagents (and are therefore suitable for analysis of in vivo tissues), and secondly because they enable rapid and simple analysis of target materials.

[0665] A variety of different open ionization techniques are known and are contemplated to be within the scope of the present invention. Desorption electrospray ionization ("DESI") was the earliest open ionization technique developed and was disclosed in 2004. Since 2004, a variety of other open ionization techniques have been developed. The difference between these open ionization techniques is their precise ionization methods, but they all have the same general ability to directly generate gas phase ions from natural (i.e., untreated or unmodified) samples. A specific advantage of the various open ionization techniques within the scope of the present invention is that the various open ionization techniques do not require any sample preparation in advance. Therefore, the various open ionization techniques are capable of analyzing in vivo and ex vivo tissue samples without the time and expense of adding matrix or reagents to tissue samples or other target materials.

[0666] A list of open ionization techniques that are contemplated to fall within the scope of the present invention is given in the following table:

[0667]

[0668]

[0669]

[0670] According to one embodiment, the open ionization ion source may comprise a rapid evaporative ionization mass spectrometry ("REIMS") ion source, in which an RF voltage is applied to electrodes to generate an aerosol or plume of surgical smoke by Joule heating.

[0671] However, it should be understood that numerous other open ion sources may also be utilized, including those mentioned above. For example, according to another embodiment, the open ionization ion source may include a laser ionization ion source. According to one embodiment, the laser ionization ion source may include a mid-infrared laser ablation ion source. For example, there are several lasers that emit radiation close to or at 2.94 μm, which corresponds to a peak in the absorption spectrum of water. According to various embodiments, the open ionization ion source may include a laser ablation ion source with a wavelength close to 2.94 μm, that is, based on the high absorption coefficient of water at 2.94 μm. According to one embodiment, the laser ablation ion source may include an Er:YAG laser that emits 2.94 μm radiation.

[0672] Other embodiments are contemplated in which a mid-infrared optical parametric oscillator ("OPO") may be used to generate a laser ablation ion source having a wavelength longer than 2.94 μm. For example, a ZGP-OPO pumped by Er:YAG may be used to generate laser radiation having a wavelength of, for example, 6.1 μm, 6.45 μm, or 6.73 μm. In some cases, it may be advantageous to use a laser ablation ion source having a wavelength shorter or longer than 2.94 μm because only surface layers will be ablated and the thermal damage caused may be less. According to one embodiment, a Co:MgF2 laser may be used as a laser ablation ion source, wherein the laser light may be tuned from 1.75 μm to 2.5 μm. According to another embodiment, a laser ablation ion source having a wavelength between 2.9 and 3.1 μm may be generated using an optical parametric oscillator ("OPO") system pumped by a Nd:YAG laser. According to another embodiment, a CO with a wavelength of 10.6 μm may be used. 2 Lasers produce aerosols, smoke, or vapor.

[0673] According to other embodiments, the open ionization ion source may include an ultrasonic ablation ion source to generate a liquid sample, which is then aspirated in the form of an aerosol. The ultrasonic ablation ion source may include a focused or unfocused source.

[0674] According to one embodiment, the first device for generating an aerosol, smoke or vapor from one or more areas of the target may include an electrosurgical tool utilizing a continuous RF waveform. According to other embodiments, a radio frequency tissue dissection system may be used, which is arranged to supply pulsed plasma RF energy to the tool. The tool may include, for example, a PlasmaBlade (RTM). The operating temperature of the pulsed plasma RF tool is lower than that of conventional electrosurgical tools (e.g., 40-170°C compared to 200-350°C), thereby reducing the depth of thermal damage. By inducing an electrical plasma along the cutting edge of a thin insulating electrode, both the cutting and coagulation operating modes can use pulsed waveforms and duty cycles.

[0675] Real-time rapid analysis of medical swabs using desorption electrospray ionization ("DESI") mass spectrometry

[0676] According to various embodiments, a swab, medical swab, or standard medical swab can be directly analyzed using desorption electrospray ionization ("DESI") mass spectrometry, and in particular, specific microorganisms on the surface of the swab can be identified in a short time by their chemical characteristics.

[0677] Rapid identification of specific microorganisms from the swab surface enables rapid diagnosis of various infections. In addition, biomarkers such as metabolomics markers, inflammatory markers and / or microbial markers can be analyzed, e.g., identified or determined, for example, which enables rapid analysis, e.g., identification, of different diseases, such as cancer, dysbiosis, infection and / or any other disease listed elsewhere herein.

[0678] Various embodiments are described in more detail below that relate to mucosal analysis, such as diagnosis.

[0679] One of the many potential applications of the various techniques disclosed herein is the ability to identify whether a patient has an increased risk of experiencing premature birth by analyzing vaginal mucosal samples.The results according to the various embodiments can optionally be compared to standard microbiological tests.

[0680] A real-time rapid medical swab analysis approach is disclosed that utilizes desorption electrospray ionization ("DESI") mass spectrometry to reveal biomarkers, such as pathogenicity and / or inflammatory metabolomic markers.

[0681] Specifically, various chemically modified swabs for use with desorption electrospray ionization ("DESI") are disclosed. The various chemically modified swabs were found to exhibit improved sensitivity compared to conventional (non-modified) swabs.

[0682] It has also been discovered that significantly increased signal intensity can be obtained by rotating or continuously rotating the swab while analyzing the swab using a desorption electrospray ionization ("DESI") ion source.

[0683] Other embodiments are disclosed below that relate to methods of rapid evaporative ionization mass spectrometry ("REIMS") analysis of swabs (rather than desorption electrospray ionization ("DESI") analysis) in which the swab is dipped, soaked, or otherwise immersed in a fluid (such as water) prior to undergoing rapid evaporative ionization mass spectrometry ("REIMS") analysis.

[0684] Soaking the swab in a fluid such as water prior to rapid evaporation ionization mass spectrometry was found to have the effect of increasing signal intensity.

[0685] According to various other embodiments, for example, the swab may be subjected to desorption electrospray ionization ("DESI") mass spectrometry for poison screening, such as on-site emergency poison screening, drug testing, such as roadside drug testing, doping testing, and the like.

[0686] Desorption Electrospray Ionization ("DESI") Mass Spectrometry of Mucosal Samples

[0687] Various embodiments, which will now be described in greater detail, relate to a non-invasive mucosal analysis approach, such as a diagnostic approach.

[0688] Medical swabs are standard mucosal collection devices that are often used to diagnose pathogen-related diseases. Conventional clinical microbiology techniques for mucosal swab diagnosis are time-consuming, lack sensitivity, and are usually qualitative. Standard medical swabs that have been used to obtain mucosal samples are sent to a microbiology laboratory, where the samples are analyzed by culturing microorganisms. However, this conventional route usually takes 24-48 hours, delaying the patient's diagnosis.

[0689] In contrast, various embodiments, which will now be described in greater detail, enable analysis of mucosal samples immediately or in real time, thereby avoiding the 24-48 hour delay common with conventional techniques.

[0690] Specifically, according to various embodiments, a method is provided that includes providing a biological sample on a swab, directing a spray of charged droplets onto a surface of the swab to generate a plurality of analyte ions, and analyzing the analyte ions.

[0691] Various embodiments relate to rapid and direct analysis methods of medical swabs by desorption electrospray ionization mass spectrometry without the need for extensive extraction protocols. According to various embodiments, ionization of mucosal biomass occurs directly from a medical swab, such as a standard medical rayon swab, which may be rotating, prior to analysis in a mass spectrometer and / or ion mobility spectrometer, for example, prior to online chemical monitoring. According to various embodiments, multivariate modeling of the collected mass spectral fingerprints enables differentiation of different mucosal surfaces; characterization of biochemical changes, such as those induced by pregnancy; and / or rapid identification of intact bacterial and fungal species. According to various embodiments, direct analysis of medical swabs by desorption electrospray ionization mass spectrometry can be used in a variety of clinical applications, including rapid mucosal diagnostics and / or characterization of clinically relevant changes in mucosal biochemistry.

[0692] Various embodiments relate to a non-invasive and culture-independent method for directly analyzing clinical swabs, e.g., in a matter of minutes, using desorption electrospray ionization ("DESI") to achieve a profile of the mucosa, e.g., a metabolomic profile. These swabs can be used to obtain rapid diagnosis of diseases, including, e.g., (i) microbial infections; (ii) dysbiosis; (iii) immune disorders; (iv) cancer; and / or any other disease listed elsewhere herein.

[0693] As further described below, a total of n=85 mucosal models were collected from three cohorts (urogenital tract, nasal cavity, and oral cavity). The mucosal samples were subjected to desorption electrospray ionization ("DESI") mass spectrometry, and the resulting mass spectrometry data were subjected to multivariate statistical analysis. Multivariate statistical analysis was able to separate different mucosal classes and biomarker changes that may be associated with the diverse microbiome within the mucosa.

[0694] The microbiome consists of communities of microorganisms that exist in humans or non-human animals, such as humans. Humans and non-human animals have co-evolved with microorganisms in a symbiotic system. The complex interactions of microbial communities affect health and disease.

[0695] Figure 1 Describe the various microorganisms that may be present in the human microbiome. Figure 1 As shown in , the human microbiome can include various bacteria, fungi, archaea, viruses, yeasts, protozoa, etc., which can be found in, for example, the mouth, pharynx, respiratory system, skin, stomach, intestines and / or urogenital tract, etc.

[0696] "Microbe", also known as microorganism, is an organism that is so small that it cannot be seen by the naked eye, that is, it is microscopic. The microorganism can be selected from bacteria, fungi, archaea, algae, protozoa and viruses. Although the terms bacteria, fungi, archaea, algae, protozoa and viruses theoretically represent the plural form, they are also used to represent the singular form, which is a common usage. Therefore, the terms "bacteria" and "bacterium" are used interchangeably in this article; the terms "fungi" and "fungus" are used interchangeably in this article; the terms "archaea" and "archaeum" are used interchangeably in this article; the terms "protozoa" and "protozoum" are used interchangeably in this article; the terms "viruses" and "virus" are used interchangeably in this article.

[0697] In the case of microorganisms, analysis can optionally be at any taxonomic level, for example, at the Kingdom, Phylum or Division, Class, Order, Family, Genus, Species and / or Strain level.

[0698] "Taxonomy" is the classification of organisms, with each level of classification being called a "taxon" (plural: taxa). Organisms can be divided into the following taxa in increasing order of specificity: kingdom, phylum, class, order, family, genus, species, and strain. Each taxon can be further divided. It is important to understand that within the wider scientific community there is some disagreement about certain taxonomic classifications. There may also be no consensus on the nomenclature of some microorganisms, resulting in more than one name for a particular microorganism or the same name for two different microorganisms.

[0699] For simplicity, the term "type" of a microorganism is used to refer to a microorganism that is distinct from other microorganisms at any taxonomic level.

[0700] In some embodiments, the microorganism can be selected from bacteria, fungi, archaea, algae and protozoa. In some embodiments, it can be selected from bacteria and fungi. In some embodiments, it can be selected from bacteria.

[0701] The microorganism may be unicellular or multicellular. If the microorganism is a fungus, it may optionally be filamentous or unicellular, such as a yeast.

[0702] The fungus may optionally be a yeast. It may optionally be selected from the genus Aspergillus, Arthroascus, Brettanomyces, Candida, Cryptococcus, Debaryomyces, Geotrichum, Pichia, Rhodotorula, Saccharomyces, Trichosporon and Zygotorulaspora.

[0703] It can optionally be selected from the following species: Arthroascus schoenii, Brettanomyces bruxellensis, Candida albicans, C. ascalaphidarum, C. amphixiae, C. antarctica, C. argentea, C. atlantica, C. atmosphaerica, C. blattae, C. bromeliacearum, C. carpophila, C. carvajalis, C. cerambycidarum, C. chauliodes, C. corydali, C. dosseyi, C. dubliniensis, C. ergatensis, C. fructus, C. glabrata, C. fermentati, C. guilliermondii, C. haemulonii, C. insectamens, C. insectorum, C. intermedia, C. jeffresii, C. kefyr, C. keroseneae, C. krusei, C. lusitaniae, C. lyxosophila, C. maltosa, C. marina, C. membranifaciens, C. milleri, C. mogii, C. oleophila, C. oregonensis, C. parapsilosis, C. quercitrusa, C. rugosa, C. sake, C. shehatea, C. temnochilae, C. cellulolyticumtenuis), C. theae, C. tolerans, C. tropicalis, C. tsuchiyae, C. sinolaborantium, C. sojae, C. subhashii, C. viswanathii, C. utilis, C. ubatubensis, C. zemplinina, Cryptococcus neoformans, Cryptococcus uniguttulatus, Debaryomyces carsonii, Geotrichum capitatum, Trichosporon asahii, Trichosporon mucoides, Trichosporon inkin, Saccharomyces cerevisiae, Pichia acaciae, Pichia anomala, Pichia capsulata, Pichia farinosa, Pichia guilliermondii, Pichia spartinae, Pichia ohmeri, Rhodotorula glutinous, Rhodotorula mucilaginosa, Saccharomyces boulardii, Saccharomyces cerevisiae and / or Zygotorulaspora florentinus.

[0704] The protozoa may optionally be selected from the group of amoeba, flagellate, ciliate or sporozoa. It may optionally be selected from the genus Acanthamoeba, Babesia, Balantidium, Cryptosporidium, Dientamoeba, Entamoeba, Giardia, Leishmania, Naegleria, Plasmodium, Paramecium, Trichomonas, Trypanosoma, Typanosoma and Toxoplasma.

[0705] The protozoa may optionally be of the following species: Balantidium coli, Entamoeba histolytica, Giardia lamblia (also known as Giardia intestinalis or Giardia duodenalis), Leishmania donovani, L. tropica, L. brasiliensis, Plasmodium falciparum, falciparum, P.vivax, P.ovale, P.malariae, P.knowlesi, P.reichenowi, P.gaboni, P.mexicanum, P.floridense, Trypanosoma brucei, Typanosoma evansi, Trypanosoma rhodesiense, Trypanosoma cruzi, and Toxoplasma gondii.

[0706] The bacteria may be optionally selected from the group consisting of Aquficae, Thermotogae, Thermodesulfobacteria, Deinococcus-Thermus, Chrysiogenetes, Chloroflexi, Thermomicrobia, Nitrospira, Deferribacteres, Cyanobacteria, Chlorobi, Proteobacteria, Firmicutes. Firmicutes, Actinobacteria, Planctomycetes, Chlamydiae, Spirochaetes, Fibrobacteres, Acidobacteria, Bacteroidetes, Fusobacteria, Verrucomicrobia, Dictyoglomi, Gemmatomonadetes and / or Lentisphaerae.

[0707] The bacteria may optionally be selected from the class Actinobacteria, Alphaproteobacteria, Bacilli, Betaproteobacteria, Clostridia, Deltaproteobacteria, Epsilonproteobacteria, Flavobacteriaceae, Fusobacteria, Gammaproteobacteria, Mikeiasis, Mollicute or Negativicutes.

[0708] The bacteria may optionally belong to the following orders: Aeromonadales, Actinomycetales, Bacillales, Bacteroidales, Bifidobacteriales, Burkholderiales, Campylobacterales, Caulobacterales, Cardiobacteriales, Clostridiales, Enterobacteriales, Flavobacteriales, The invention further comprises the following members: Flavobacteriales, Fusobacteriales, Lactobacillales, Micrococcales, Neisseriales, Pasteurellales, Pseudomonadales, Rhizobiales, Rhodospirillales, Selenomonadales, Vibrionales and / or Xanthomonadales.

[0709] The bacteria may optionally be selected from the following families: Acetobacteraceae, Alcaligenaceae, Bacillaceae, Bacteroidaceae, Burkholderiaceae, Caulobacteraceae, Comamonadaceae, Enterobacteriaceae, Flavobacteriaceae, Fusobacteriaceae, Nocardiaceae, Prevotellaceae, Porphyromonadaceae, Pseudomonadaceae, Rikenellaceae, Rhizobiaceae and / or Sutterellaceae.

[0710] The bacteria may optionally belong to the genus Abiotrophia, Achromobacter, Acidovorax, Acinetobacter, Actinobacillus, Actinomadura, Actinomyces, Aerococcus, Aeromonas, Anaerococcus, Anaplasma, Bacillus, Bacteroides, roides), Bartonella, Bifidobacterium, Bordetella, Borrelia, Brevundimonas, Brucella, Burkholderia, Campylobacter, Capnocytophaga, Chlamydia, Citrobacter, Chlamydophila, Chryseobacterium, Clostridium, Comamonas, Corynebacterium, Coxiella, Cupriavidus, Delftia, Dermabacter, Ehrlichia, Eikenella, Enterobacter, Enterococcus, Escherichia, Erysipelothrix ipelothrix), Facklamia, Finegoldia, Francisella, Fusobacterium, Gemella, Gordonia, Haemophilus, Helicobacter, Klebsiella, Lactobacillus, Legionella, Leptospira, Listeria,Micrococcus, Moraxella, Morganella, Mycobacterium, Mycoplasma, Neisseria, Nocardia, Orientia, Pandoraea, Pasteurella, Peptoniphilus, Peptostreptococcus, Plesiomonas, Porphyromonas, Pseudomonas, Prevotella, Proteus eus, Propionibacterium, Rhodococcus, Ralstonia, Raoultella, Rickettsia, Rothia, Salmonella, Serratia, Shigella, Staphylococcus, Stenotrophomonas, Streptococcus, Tannerella, Treponema, Ureaplasma, Vibrio, and / or Yersinia.

[0711] The bacteria may optionally belong to a species selected from, for example, Abiotrophia defective, Achromobacter xylosoxidans, Acidovoraxavenae, Acidovorax citrulli, Akkermansia muciniphila, Bacillus anthracis, B. cereus, B. subtilis, B. licheniformis, Bacteroides fragilis, Bartonella henselae, Bartonella quintana, Bordetella pertussis, Borrelia burgdorferi, Borrelia garinii, Borrelia afzelii), Borrelia recurrentis, Brucella abortus, Brucella canis, Brucella melitensis, Brucella suis, Burkholderia cepacia, Burkholderia genomovars, Campylobacter jejuni, Chlamydia pneumoniae, Chlamydia trachomatis, Chlamydophila psittaci, Citrobacter koseri, Clostridium botulinum, Clostridium difficile, C. perfringens, C. tetani, Corynebacterium diphtheriae), C. striatum, C. minutissimum, C. imitans, C.Amycolatum, Delftia acidovorans, Enterobacter aerogenes, Enterobacter cloacae, Enterococcus faecalis, Enterococcus faecium, Escherichia coli, Francisella tularensis, Fusobacterium nucleatum, Haemophilus influenzae, Helicobacter pylori, Klebsiella oxytoca, Klebsiella pneumoniae, Legionella pneumophila, Leptospira interrogans, Leptospira santarosai, Leptospira weilii, Leptospira noguchii, Listeria ivanovii, Listeria monocytogenes, Micrococcus luteus, Morganella morganii, Moraxella catarrhalis, Mycobacterium avium, Mycobacterium fortuitum, Mycobacterium leprae, Mycobacterium peregrinum, Mycobacterium tuberculosis, Mycobacterium ulcerans, Mycoplasma pneumoniae, Neisseria gonorrhoeae, Neisseria lactamica, Neisseria meningitidismeningitidis, Nocardia asteroids, Proteus mirabilis, Pseudomonas aeruginosa, Rhodococcus equi, Rhodococcus pyridinivorans, Rickettsia rickettsii, Salmonella typhi, Salmonella typhimurium, Serratia marcescens, Shigella sonnei, Staphylococcus aureus aureus, S. capitis, S. epidermidis, S. haemolyticus, S. hominis, S. saprophyticus, Stenotrophomonas maltophilia, Streptococcus agalactiae, S. pyogenes, S. pneumonia, Treponema pallidum, Ureaplasma urealyticum, Vibrio cholerae, Yersinia pestis, Yersinia enterocolitica, and Yersinia pseudotuberculosis.

[0712] The virus may optionally be a DNA virus and an RNA virus or a retrovirus. It may optionally be a single-stranded (ss) or double-stranded (ds) virus. More specifically, it may optionally be a ssDNA, dsDNA, dsRNA, ssRNA (positive strand), ssRNA (negative strand), ssRNA (reverse transcription) or dsDNA (reverse transcription) virus.

[0713] It may optionally be selected from one or more of the following: Herpesviridae, optionally selected from Simplexvirus, Varicellovirus, Cytomegalovirus, Roseolovirus, Lymphocryptovirus and / or Rhadinovirus; Adenoviridae, optionally selected from Adenovirus and / or mammalian Mastadenovirus; Papillomaviridae, optionally selected from the genus Alphapapillomavirus, the genus Betapapillomavirus, the genus Gammapapillomavirus, the genus Mupapillomavirus and / or the genus Nupapillomavirus; Polyomaviridae, optionally selected from the genus Polyomaviridae Poxviridae, optionally selected from the genus Molluscipoxvirus, Orthopoxvirus and / or Parapoxvirus; Anelloviridae, optionally selected from the genus Alphatorquevirus, Betatorquevirus and / or Gammatorquevirus; Mycodnaviridae, optionally selected from the genus Mycodnavirus. naviridae, optionally selected from the fungal genus Gemycircular-virus; Parvoviridae, optionally selected from the genus Erythrovirus, Dependovirus and / or Bocavirus; Reoviridae, optionally selected from the genus Coltivirus, Rotavirus and / or Seadornavirus;Coronaviridae, optionally selected from the genus Alphacoronavirus, Betacoronavirus and / or Torovirus; Astroviridae, optionally selected from the genus Mamastrovirus; Caliciviridae, optionally selected from the genus Norovirus and / or Sapovirus; Flaviviridae, optionally selected from the genus Flaviviridae rus), Hepacivirus and / or Pegivirus; Picornaviridae, optionally selected from Cardiovirus, Cosavirus, Enterovirus, Hepatovirus, Kobuvirus, Parechovirus, Rosavirus and / or Salivirus; Togaviridae, optionally selected from Alphavirus and / or Rubivirus; Rhabdoviridae, optionally selected from Lyssavirus and / or Vesiculovirus; Filoviridae, optionally selected from Ebolavirus and / or Marburgvirus; Paramyxoviridae, optionally selected from Henipavirus, Heffalumpvirus, Morbillivirus, orbilivirus), Respirovirus, Rubulavirus, Metapneumovirus and / or Pneumovirus; Arenaviridae, optionally selected from Arenavirus; Bunyaviridae, optionally selected from Hantavirus, Nairovirus, Orthobunyavirus and / or Phlebovirus;Orthomyxoviridae, optionally selected from Influenzavirus A, Influenzavirus B, Influenzavirus C and / or Thogotovirus; Retroviridae, optionally selected from Gammaretrovirus, Deltaretrovirus, Lentivirus, Spumavirus; Epadnaviridae, optionally selected from Orthohepadnavirus; Hepevirus; and / or Deltavirus.;

[0714] The microorganism may optionally be pathogenic or non-pathogenic. Pathogenic microorganisms may also be referred to as "pathogens," which may be defined as microorganisms that are capable of causing disease in a host such as a plant or an animal. Pathogens may optionally be obligate pathogens or opportunistic pathogens.

[0715] The ability of a microorganism to cause disease depends on both its intrinsic virulence factors and the host's ability to defeat the microorganism. The distinction between nonpathogens and opportunistic pathogens is therefore not clear-cut, since, for example, a microorganism that cannot infect a host with a healthy immune system can easily infect an immunocompromised host.

[0716] For example, Neisseria gonorrhoeae is an obligate pathogen, Pseudomonas aeruginosa and Candida albicans are often referred to as opportunistic pathogens, and Lactobacillus acidophilus and Bifidobacterium bifidum are generally considered non-pathogens and may be referred to as "commensal."

[0717] Drugs such as antimicrobial and / or anti-inflammatory drugs can also create an environment where microorganisms will grow as opportunistic pathogens. Thus, medication can alter the microbiome. The method can therefore optionally include analyzing the microbiome, such as the mucosal microbiome, to analyze the response to the drug.

[0718] Pathogenic microorganisms can optionally be characterized by the expression of one or more virulence factors, which are factors that can achieve or promote host infection. Virulence factors can optionally be selected from factors that mediate the following: cell adhesion, cell growth, the ability to bypass or overcome host defense mechanisms and / or toxin production. Toxins can be selected from exotoxins and endotoxins. The method can optionally include analyzing one or more virulence factors.

[0719] Symbiotic microorganisms are such that they are part of the natural flora of a person or animal and, in equilibrium, are non-pathogenic.

[0720] The community of microorganisms in a particular environment can be referred to as the "microbiome". The microbiome can be a complex mixture of a large number and variety of different microorganisms. It is estimated that the gastrointestinal (GI) microbiome contains more than 100 trillion microorganisms, representing at least hundreds or even more than a thousand different species. The intestinal microbiome of a healthy person is dominated by Bacteroidetes and Firmicutes, but there is usually also a smaller proportion of Proteobacteria, Verrucomicrobia, Actinobacteria, Fusobacteria, and Cyanobacteria.

[0721] The microbiome in the same person or animal can vary depending on the environment, so a person's gastrointestinal (GI) microbiome may be different from that person's nasal microbiome. The GI microbiome can be further divided into different GI regions, such as the stomach, duodenum, jejunum, ileum and / or colon. The luminal microbiome can also be different from the mucosal microbiome. Each microbiome can also vary from individual to individual. Disturbances in the normal microbiome can be referred to as "dysbiosis." Dysbiosis can cause or be associated with disease, such as any of the diseases mentioned herein. The method can optionally include analyzing the microbiome to analyze dysbiosis. The GI microbiome can also be referred to as "gut flora."

[0722] The microbiome changes during pregnancy, so profiling the female (human or animal) microbiome enables pregnancy analysis. Pregnancy dysbiosis is associated with complications, such as an increased risk of preterm birth.

[0723] Dysbiosis can include the presence of one or more types of microorganisms that are not normally present or not previously present in a particular microbiome. More commonly, however, dysbiosis can include a relative increase in the proportion of one or more specific microorganisms, and / or a relative decrease in the proportion of one or more specific microorganisms.

[0724] As mentioned above, the mucosa comprises a mucus layer. Microorganisms such as bacteria can adhere to the mucus layer and / or partially or completely infiltrate the mucus layer. Microbial adhesion and / or proliferation can be affected by: carbohydrate modifications present on the mucin; antimicrobial agents, such as antimicrobial peptides derived from the host; drugs; diet; and / or toxins, such as toxins produced by (pathogenic) microorganisms.

[0725] In at least about 80% of healthy people, the mucosal (epithelial) surface below the mucus layer is free of microorganisms. The thickness of the mucus layer and its coverage can vary, for example, they can decrease with increasing severity of inflammation. Under certain conditions, such as in disease, microorganisms can infiltrate and / or adhere to the mucus layer, epithelium and / or lamina propria (LP). For example, bacteria are often found in mucus from biopsy specimens of subjects with ulcerative colitis, SLC and / or acute appendicitis. The concentration of microorganisms in the mucus layer can be negatively correlated with the number of white blood cells.

[0726] The term "mucosal microbiome" is used herein to refer to a microbiome associated with the mucosa, including a microbiome that has infiltrated the mucosa and a microbiome that is associated with the mucus layer (eg, by adherence or partial or complete infiltration).

[0727] The methods can optionally include infection analysis, such as diagnosing an infection, analyzing the genotype or phenotype of a microorganism causing an infection, monitoring the progression of an infection, and / or monitoring the response of an infection to treatment.

[0728] The method may optionally include an inoculation analysis. This may include, for example, analyzing targets before and after inoculation. Optionally, the subject may be challenged with the microorganism to which the inoculation is directed after inoculation, and then the appropriate targets may be analyzed to determine whether the microorganism is present or how much of the microorganism is present. The presence or amount of the microorganism may indicate the effectiveness of the inoculation, for example, the absence or low amount of the microorganism may indicate a successful inoculation, while the presence or high amount of the microorganism may indicate that the vaccine is insufficient or ineffective.

[0729] The mucous membrane can be thought of as a protective layer responsible for trapping pathogens within the human body.

[0730] Mucosa lines several passages and cavities of the body, especially those that are open to the external environment, including the oropharyngeal cavity, the gastrointestinal (GI) tract, the respiratory tract, the urogenital tract, and the exocrine glands. Thus, the mucosa may optionally be selected from bronchial mucosa, endometrium (uterine mucosa), esophageal mucosa, gastric mucosa, intestinal mucosa (intestinal mucosa), nasal mucosa, olfactory mucosa, oral mucosa, penile mucosa, and / or vaginal mucosa.

[0731] Broadly speaking, the mucosa comprises a mucus layer (inner mucus layer); an epithelium; a basement membrane; a lamina propria (LP), which is a connective tissue layer; and a muscularis mucosae, which is a thin layer of smooth muscle. Therefore, unless otherwise indicated, the term "mucosal membrane" is used herein to refer to this entire complex, and the term "mucosal membrane" can be used interchangeably with the term "mucosa". The mucosa may also be covered with an additional outer mucus layer, which is usually loosely associated with the mucosa. Any reference to "mucosa" herein may include reference to this additional outer mucus layer. Adjacent to the mucosa is the submucosa.

[0732] The inner slime layer can be degraded by microorganisms. For example, mucin monosaccharides can be used as an energy source by bacteria, such as commensal bacteria. Therefore, it is very important to continuously renew the inner slime layer.

[0733] Epithelium is a single or multiple layer of epithelial cells. Epithelium may include, for example, intra-epithelial lymphocytes (IEL), endocrine cells, goblet cells, enterocytes and / or Paneth cells.

[0734] The basement membrane may comprise various proteins, in particular structural or adhesion proteins, such as laminin; collagens, such as type IV collagen; proteoglycans; and / or calcium-binding proteins, such as fibulin.

[0735] The lamina propria is connective tissue that may contain, for example, plasma cells, eosinophils, histiocytes, mast cells and / or lymphocytes. Neutrophils are not normally present in the lamina propria of a healthy person.

[0736] As discussed below, the mucosa may also include, for example, antigen presenting cells (APCs) and microfold cells (M cells). The mucosa may include one or more different types of regulatory immune cells, including intestinal intraepithelial lymphocytes (IELs), Foxp3(+) regulatory T cells, regulatory B cells, alternatively activated macrophages, dendritic cells, and / or innate lymphocytes.

[0737] Mucous membranes normally secrete mucus, which forms a mucus layer between the mucosal epithelium and the lumen. The mucus layer may have a protective function. The major components of mucus are mucins, which are produced by specialized mucosal cells called goblet cells. Mucins are glycoproteins characterized by a high content of O-linked oligosaccharides. The amount of the protein portion attached to the carbohydrate portion, as well as the exact identity of the carbohydrate portion, can vary widely.

[0738] The mucosa creates a barrier between the sometimes hostile external environment and the internal environment. However, the mucosa is also responsible for nutrient absorption and waste product secretion, which requires a selectively permeable barrier. These functions place the mucosal epithelium at the center of the interaction between the mucosal immune system and the luminal contents, including dietary antigens and microbial products. Therefore, many physiological and immune stimuli trigger responses in the mucosa. Dysfunctional responses can lead to disease.

[0739] The mucosal immune system is a local specific immune tissue. The mucosal immune systems in different organs share similar anatomical organizations and features. The GI mucosal immune system is best understood and discussed below for illustrative purposes. The GI mucosal immune system consists of three major compartments: the epithelial layer; the lamina propria (LP); and mucosal-associated lymphoid tissue (MALT), which may be referred to as gut-associated lymphoid tissue in the gastrointestinal tract, and which contains Peyer's patches and isolated lymphoid follicles.

[0740] Dendritic cells can project dendrites onto the epithelium, thereby taking up antigens and migrating them to the LP, secondary lymphoid tissues, and draining lymph nodes, where the antigens sensitize naive T cells. Microfold cells (M cells) located in the epithelium of Peyer's patches can pass antigens to dendritic cells, macrophages, and other antigen-presenting cells. Naive T cells in secondary lymphoid tissues can be activated after sensitization by antigen-presenting cells and return to the LP (called LPL) or infiltrate into the inflamed epithelium.

[0741] The gastrointestinal (GI) tract can be divided into four concentric layers that surround the lumen in the following order: (i) mucosa; (ii) submucosa; (iii) muscularis; and (iv) adventitia or serosa.

[0742] Therefore, the GI mucosa is the innermost layer of the gastrointestinal tract. This layer is in direct contact with the digested food. In the GI mucosa, the epithelium is responsible for most of the digestive, absorptive, and secretory processes, while the muscularis mucosa aids the passage of materials and enhances the interaction between the epithelial layer and the luminal contents through agitation and peristalsis.

[0743] The GI mucosa is highly specialized in each organ of the gastrointestinal tract to handle different conditions. Most of the changes probably occur in the epithelium.

[0744] Different types of mucosa are different from each other and the inventors have demonstrated that the methods of various embodiments described herein may optionally be used, for example, to distinguish between different types of mucosa, such as vaginal mucosa, nasal mucosa, and oral mucosa.

[0745] Figure 2 Describe the various mucous membranes that exist in the human body.

[0746] Mucosa 200 comprises a layer of epithelial tissue that lines all passages in the human body that lead to the external environment, including the nose and portions of the digestive tract, urogenital tract, and respiratory tract. Mucosa generally acts as a protective barrier for trapping pathogens such as bacteria, viruses, and fungi. For example, the mucosa of the oral cavity, respiratory tract, and urogenital tract are composed of epithelial tissue and an underlying thin layer that is directly exposed to the external environment, making it a primary site for innate and acquired protection against host infection.

[0747] like Figure 2 As shown in , mucosa are present in the mouth, pharynx and respiratory system 201 as well as the gastrointestinal tract 202 and urogenital tract 203, and include the endometrium, intestinal membranes, gastric membranes, oral membranes, vaginal membranes, esophageal membranes, gingival membranes, nasal membranes, buccal membranes and bronchial membranes.

[0748] Studies as part of the Human Microbiome Project have revealed that the colonization of different microbial species within the mucosa has a huge impact on human health and disease. As discussed elsewhere in this article, many diseases (e.g., cancer, infection, etc.) are associated with the mucosa. The host-microbiome interaction at the mucosal surface has an important impact not only on pathology and disease, but also on health status. For example, the symbiotic vaginal microbiota excretes antimicrobial compounds and metabolites into the cervicovaginal mucosa, which regulates its physiological and immune properties. We believe that this mechanism provides protection against pathobiont colonization of the reproductive tract during pregnancy, which is the main cause of premature birth. The nasal mucosal surface is a key regulator of allergic inflammation and airway obstruction symptoms (such as asthma).

[0749] Therefore, mucosa is an easily accessible and clinically highly relevant sample for analyzing diseases, e.g. for diagnosing diseases, such as microbial and / or cancer-related diseases, etc., and mucosal diagnostics represents an important field with broad clinical applications.

[0750] like Figure 3As shown in FIG. 3 , a typical mucosa may be present in a lumen 300 and may include mucus 301, bacteria 302, lymphatic vessels 303, blood vessels 304, mucosal glands 305, and a submucosa 306. Figure 3 As illustrated, the biological tissue of the mucosa itself (e.g., mucus 301) and / or bacteria present in or associated with the mucosa 302 represent potential analytes / biomarkers. For example, membrane lipids and / or inflammatory markers of the mucosa, and / or complex lipids and / or signaling molecules of intact bacterial cells represent potential analytes / biomarkers.

[0751] Methods according to various embodiments may include analyzing a mucosal target, such as on a swab or biopsy. Optionally, the method may include analyzing a mucosal target to analyze the cellular composition of the mucosa; to analyze a disease; to analyze a response to a drug; to analyze a response to a specific food, diet, and / or dietary change; to analyze mucosal microbes; to analyze the interaction of microbes with the mucosa; and / or to analyze a mucosal microbiome.

[0752] Analyzing the cellular composition of the mucosa can, for example, analyze the presence or absence and / or ratio of one or more cell types, which can be optionally selected from any cell type listed herein. Optionally, the method can include analyzing MALT and / or Peyer's spots. Optionally, the method can include analyzing the phenotype and / or genotype of one or more cell types, which can be optionally selected from any cell type listed herein.

[0753] Optionally, the method may include analyzing mucosal changes, which may optionally be, for example, changes in mucosal cell composition, interactions of microorganisms with the mucosa, and / or changes in the mucosal microbiome. "Changes" in the mucosa mean that the mucosa is different from what it normally looks like in a healthy subject; it is different in one location from another location within the same subject; and / or it is different from what it looked like when it was analyzed at an earlier time point. Mucosal changes may optionally be caused by or associated with, for example, disease, a reaction to a substance such as a drug, and / or a reaction to food, diet, and / or a change in diet.

[0754] The disease may optionally be selected from an autoimmune disorder, an inflammatory disease, tropical sprue, a food intolerance, an infection, cancer and / or any of the disorders mentioned herein.

[0755] More specifically, the disease can be optionally selected from, for example, asthma, coeliac disease, gastritis, peptic duodenitis, gluten-sensitive enteropathy; allergies and / or intolerances to allergens, such as milk, soy, tree nuts, eggs, wheat, meat, fish, shellfish, peanuts, seeds (such as sesame, sunflower seeds and / or poppy seeds), garlic, mustard, coriander and / or onions; Hashimoto's thyroiditis; irritable bowel syndrome; Graves' disease; reactive arthritis; psoriasis; multiple sclerosis; systemic lupus erythematosus (SLE or lupus); ankylosing spondylitis; progressive systemic sclerosis (PSS); glomerulonephritis; autoimmune enteropathy; IgA deficiency; common variable immunodeficiency disease; Crohn's disease; disease); colitis, such as lymphocytic colitis, collagenous colitis and / or ulcerative colitis; diffuse lymphocytic gastroenteritis; ulcers; intestinal T-cell lymphoma; infections, such as pharyngitis, bronchitis and / or infection with microorganisms selected from, for example, Giardia, Cryptosporidium, Helicobacter and / or any other microorganisms mentioned herein; and / or cancer, details of which are discussed elsewhere herein.

[0756] The method may, for example, optionally include analyzing the interaction of mucous membranes with microorganisms, or mucous membrane changes caused by such interactions or related to such interactions. Optionally, the interaction may be, for example, microbial localization transfer to the mucous membrane, such as the localization transfer of symbiotic bacteria. The method may, for example, optionally include analyzing the mucous membrane microbiome, or mucous membrane changes caused by the mucous membrane microbiome or related to the mucous membrane microbiome. The method may, for example, optionally include analyzing infection, or mucous membrane changes caused by infection or related to infection. The analysis of microorganisms, microbial interactions, infection and / or microbiome is also discussed elsewhere herein.

[0757] As mentioned above, IEL is a normal component of the small intestinal mucosa. They play an important role in immune surveillance and immune activation. In healthy people, the vast majority of IELs belong to T cell types and express the α / β T cell receptors on their surfaces. It is generally believed that in the intestinal mucosa of healthy people, every 100 epithelial cells have no more than about 20 lymphocytes.

[0758] An increased number of lymphocytes in a mucosal specimen may optionally indicate an alteration, such as a disease, a response to a drug, and / or a microbial alteration. Thus, the term "elevated" or "increased" IEL content is used to refer to more than 20 IELs per 100 epithelial cells in the intestinal mucosa, optionally at least 22, 24, 25, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 65, 70, 75, or 80 IELs per 100 epithelial cells in the intestinal mucosa.

[0759] Under normal conditions, more than 2-3% of T lymphocytes do not express the T lymphocyte gamma-delta receptor. Thus, an increased percentage of T lymphocytes expressing this receptor may indicate an alteration, such as disease, response to a drug, and / or microbial alteration. Thus, a method may include determining the presence or percentage of T lymphocyte gamma-delta receptor expression. For example, in celiac disease, 20-30% of mucosal T lymphocytes may express this receptor.

[0760] Thus, the method may optionally include analyzing lymphocytes in the target, which lymphocytes may optionally be T lymphocytes, such as gamma-delta receptor positive T lymphocytes. Optionally, the number of lymphocytes in the target may be analyzed to determine whether it is increased or decreased. Optionally, the phenotype and / or genotype of the lymphocytes may be analyzed.

[0761] Polymorphonuclear leukocytes (PMNs), also known as neutrophils, are the most abundant white blood cell population in the blood, accounting for 50-60% of circulating white blood cells (25×10 9 PMNs are key components of the innate immune response, which is necessary to protect the host, for example, from microbial pathogens while also minimizing harmful effects mediated by dying or injured cells.

[0762] PMNs can perform various antimicrobial functions, such as degranulation and phagocytosis. They are the only ones that can form large amounts of reactive oxygen species and other toxic molecules that can weaken and / or destroy pathogens. Once PMNs come into contact with invading microorganisms, they can generate reactive oxygen species through an oxidative burst of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase.

[0763] PMNs may also have different pools of intracellular granules containing antimicrobial peptides, such as α-defensins and / or cathelicidins; myeloperoxidase; hydrolases, such as lysozymes, sialidases, and / or collagenases; proteases, such as cathepsin G; azurocidin and / or elastase; cationic phospholipases; and / or metal chelators, such as lactoferrin. These granules may be released upon contact with microorganisms.

[0764] PMNs can also characterize tissues as neutrophil extracellular traps (NETs). NETs can consist of nuclear contents (DNA and chromatin) mixed with toxic molecules from intracellular granules and the cytosol. Invading microorganisms can be trapped in these NETs and effectively destroyed.

[0765] During intestinal inflammation, resident monocytes help recruit neutrophils by producing macrophage-derived chemokines. Neutrophils present in the blood sense the chemoattractant gradient and cross the vascular endothelium to the intestinal lamina propria. In this way, neutrophils are recruited to the site of infection or inflammatory stimulation within a few minutes. The response usually reaches its peak at 24-48 hours. Under certain physiological or pathological conditions, neutrophils can cross the epithelium into the intestinal lumen.

[0766] At sites of inflammation, neutrophils can selectively release monocyte chemoattractants such as CAP18, cathepsin G, and / or azurocidin. Thus, soon after PMNs arrive at the mucosa, macrophages are recruited for a second wave of inflammation that occurs over the next few days.

[0767] Thus, the method can optionally include analyzing the subject for neutrophils. Optionally, the subject can be analyzed for the presence of reactive oxygen species and / or neutrophils that produce reactive oxygen species. Optionally, the subject can be analyzed for the presence of NETs and / or neutrophils that produce NETs. Optionally, the subject can be analyzed for the presence of monocyte chemoattractants and / or neutrophils that produce monocyte chemoattractants.

[0768] According to various embodiments, microbial (e.g., bacterial) and / or animal (e.g., human) mucosal analytes can be characterized, for example, using open-type mass spectrometry-based techniques such as desorption electrospray ionization (“DESI”) techniques and rapid evaporative ionization mass spectrometry (“REIMS”) techniques.

[0769] If through Figure 4As demonstrated, these analytes (eg, membrane lipids and inflammatory markers of mucosal membranes, and complex lipids and signaling molecules of intact bacterial cells) can be used to identify a variety of clinical conditions.

[0770] Therefore, various embodiments are directed to the development of real-time point of care (POC) diagnostic methods for studying various clinical conditions. Specifically, various embodiments are directed to real-time point of care (POC) technology based on mass spectrometry (MS).

[0771] For example, an infection such as pharyngitis, bronchitis, and / or infection with any of the microorganisms mentioned herein, etc. can be identified by, for example, analyzing the microorganism, such as identifying the microorganism.

[0772] Changes in the microbiome can also be analyzed, e.g., detected, such as by identifying microorganisms, and for example, determining changes in the microbiome of a pregnant patient can be used to identify those patients who have an increased risk of premature birth during pregnancy.

[0773] Furthermore, profiling of various analytes, such as biomarkers, obtained from the mucosa can be used to identify various immune disorders (eg, asthma, allergies) as well as to identify cancer and precancerous conditions.

[0774] If through Figure 5 Further illustrate, the metabolomics profile analysis of the analyte obtained from various mucosa using swab can be used to identify multiple clinical conditions.For example, allergies can be identified, such as prostaglandins (PGD2), leukotrienes, histamine, etc., by identifying inflammatory mediators (eicosanoids).Inflammation (such as pharyngitis, pharyngalgia, etc.) can be identified, such as by identifying microorganisms, such as secondary metabolites of bacteria, lipids from bacteria, etc., bacteria such as Streptococcus, Staphylococcus, Haemophilus, etc. It is also possible to, for example, identify healthy mucosa (for example, comprising a stable lactobacillus environment, including such as Lactobacillus crispatus (L.crispatus) leading, Lactobacillus iners (L.iners) leading and / or Lactobacillus gasseri (L.gasseri) mixing, etc.) or unhealthy mucosa (for example, comprising the overgrowth of pathogens, the pathogens include such as Escherichia coli, Atopobium vaginae (Atopobium vaginae), Peptostreptococcus and / or Bacteroides, etc.) to identify premature birth.

[0775] According to various embodiments, mucosal diagnostics enable non-invasive direct mucosal sampling of patients at the clinical point of care.

[0776] According to various embodiments, analytes can be obtained from the mucosa using, for example, a "standard medical swab", i.e., a standard mucosal collection or sampling device that can be used in clinical microbiology (e.g., for obtaining microbial cultures), genetic and drug testing, etc. A "standard medical swab" can include, for example, a cotton pad, i.e., a standard mucosal sample collection device. As will be discussed in more detail below, the medical swab can be moistened or otherwise functionalized.

[0777] For clinical analysis, swabs can be wiped in or in the infected area, for example, for taking body fluid samples rich in microorganisms, such as pus and / or mucosa itself. For conventional microbiological analysis, swabs can then be placed in sterile tubes containing buffer solutions or transport media for storage, and then the tubes are sent to the laboratory for analysis, such as microscopic examination of microbial contents and / or characterization based on culture. For example, the laboratory receiving the tube can wipe the smear contents onto the culture medium, such as on an agar plate. The culture medium can then be incubated to grow the organisms present. Microbiological identification can then be performed under a microscope. Any organisms present in the sample can also be identified, for example, by sequence analysis, such as 16S gene sequencing of bacteria, and / or by using matrix-assisted laser desorption ionization ("MALDI") mass spectrometry, and then comparing mass spectrometry with commercial databases.

[0778] Figure 6 The microbial identification workflow is illustrated and shows the use of a swab to sample the analyte 601 and then transport the swab 602 to a specialized laboratory for microbial culture 603 and further analysis. Figure 6 As shown in , such culture-based analysis can include the use of microscopy imaging 604 and / or matrix-assisted laser desorption ionization ("MALDI") mass spectrometry ("MS") 605, followed by statistical analysis 606, etc. 16s rRNA sequencing 607 is a culture-independent analysis method.

[0779] Although easy to perform, current medical swab analysis for diagnostic purposes is culture-dependent and involves a relatively time-consuming and relatively costly workflow. As a result, the diagnosis and appropriate treatment of pathogen-related diseases are greatly delayed. In addition, approximately 95% of bacteria cannot be cultured for analysis. Conventional methods are therefore limited by their qualitative nature, the time required to obtain results, and the inability to assess the host's response to the presence or absence of specific microbial groups.

[0780] Bacterial 16S rRNA gene sequencing or analyzing samples by matrix-assisted laser desorption ionization (MALDI) mass spectrometry (MS) is also time-consuming and costly.

[0781] The various embodiments described in more detail below provide a rapid and direct way to study clinical samples from mucosa, for example by identifying microorganisms and / or biomarkers characteristic of specific clinical conditions in mucosal samples, thereby allowing for faster diagnosis and treatment of patients.

[0782] Various embodiments are directed to the rapid and direct analysis of analytes present on a swab in real time using open mass spectrometry. The sample surface can be analyzed directly using open ionization mass spectrometry-based techniques. The sample can be analyzed in its native state with little or no prior sample preparation.

[0783] Various embodiments allow for the rapid acquisition of detailed mass spectrometric metabolic fingerprints from a variety of biological materials, including intact bacteria, without the need for extraction or extensive sample preparation protocols.

[0784] In particular, desorption electrospray ionization ("DESI") has been found to be a particularly useful and convenient method for real-time rapid and direct analysis of analytes present on a swab. Desorption electrospray ionization ("DESI") allows for direct and rapid analysis of surfaces without prior sample preparation. Reference will now be made to Figure 7 The technique is described in more detail.

[0785] like Figure 7 As shown in , the desorption electrospray ionization ("DESI") technique is an open ionization method that includes directing a (primary) charged droplet spray 701 onto a surface 702 and / or directly onto a sample surface 704, where an analyte 703 is present on the surface 702. The electrospray mist is pneumatically directed to the sample by a nebulizer 700, where the subsequent splashing (secondary) droplets 705 carry the desorption-ionized analytes (e.g., desorbed lipid ions). The nebulizer 700 can be equipped with a solvent 706, a gas 707 (such as nitrogen), and a voltage from a high voltage ("HV") source 708. After ionization, the ions advance through air, for example, through a transfer capillary 710 into an atmospheric pressure interface 709 of a mass spectrometer and / or ion mobility spectrometer or mass analyzer (not shown).

[0786] Desorption electrospray ionization ("DESI") technology allows for the open ionization of trace samples at atmospheric pressure with minimal sample preparation. Desorption electrospray ionization ("DESI") technology allows, for example, the direct analysis of biological compounds such as lipids, metabolites, and peptides in their native state without any prior sample preparation.

[0787] Embodiments described herein relate to direct analysis of medical swabs using desorption electrospray ionization ("DESI") mass spectrometry. According to various embodiments, chemical signature identification of specific microorganisms, such as bacteria and / or biomarkers, on the swab surface is possible in a relatively short time.

[0788] Various specific embodiments are directed to the rapid diagnosis of infection and / or dysbiosis associated with, for example, premature birth (and these results can optionally be compared with standard microbiological tests).

[0789] Other embodiments relate to real-time rapid analysis of medical swabs using desorption electrospray ionization ("DESI") mass spectrometry to reveal pathogenic and / or inflammatory metabolomic markers.

[0790] In addition, various chemically modified swabs for use with desorption electrospray ionization ("DESI") mass spectrometry are disclosed. These swabs were found to exhibit improved sensitivity compared to conventional (unmodified) swabs.

[0791] It has also been discovered that significantly increased signal intensity can be obtained by rotating or continuously rotating the swab while analyzing the swab using the desorption electrospray ionization ("DESI") technique.

[0792] Desorption electrospray ionization ("DESI") mass spectrometry of medical swabs is relatively simple, for example compared to liquid extraction-based mass spectrometry techniques, including high performance liquid chromatography ("HPLC") mass spectrometry, because no sample preparation steps are required prior to analysis and ionization occurs directly from the (rotatable) medical swab. Desorption electrospray ionization ("DESI") mass spectrometry of medical swabs is also an improvement over so-called "touch spray" ("TS") mass spectrometry, which is limited in reproducibility and control over spray formation and spray stability due to the intense evaporation and drying of the swab.

[0793] Fig. 8A -C illustrates a desorption electrospray ionization ("DESI") mass spectrometry setup for swab analysis according to various embodiments. Fig. 8A As shown in FIG. 8 , a desorption electrospray ionization (“DESI”) nebulizer 800 and a mass spectrometer inlet capillary 801 can be positioned adjacent to a medical swab 802. The nebulizer 800 can be provided with a gas supply 803, a power / solvent supply 804, and can be disposed on a movable nebulizer station 805. The swab 802 can be disposed on a movable swab station 806.

[0794] like Figure 8B As shown in FIG. 8 , the swab 802 can be rotated to obtain different portions of the analyte on the swab 802 . Figure 8B The arrow in shows the movement direction of the swab according to one embodiment.

[0795] Initial experiments optimized the swab-inlet geometry, tip-sample angle and distance, and rotation speed, and provided high reproducibility of desorption electrospray ionization ("DESI") mass spectrometry analysis. The optimal parameters for this setup were found to include a swab-capillary distance of about 1-2 mm, a nebulizer-swab distance of about 1-2 mm, a nebulizer voltage of about 4.3 kV, a solvent flow rate of about 10 μL / min, and a nebulizer gas pressure of about 7 bar.

[0796] Figure 8C Various swab positions and geometries are described according to various embodiments.

[0797] Various embodiments relate to applying desorption electrospray ionization ("DESI") mass spectrometry to directly analyze standard medical swabs, allowing rapid assessment of perturbations in mucosal surface chemistry. Thus, various embodiments relate to the development of non-invasive point-of-care diagnostic technologies, such as for disease detection, such as infection, dysbiosis, cancer and / or inflammatory disease and / or any other disease mentioned elsewhere herein.

[0798] Medical swabs were analyzed by desorption electrospray ionization ("DESI") mass spectrometry in a non-invasive procedure with the goal of extracting chemical information relevant to patient care. In this case, desorption electrospray ionization ("DESI") mass spectrometry represents a rapid and straightforward method for metabolomic profiling of different mucosal models or membranes (e.g., nasal, vaginal, oral) by desorbing and analyzing molecules from the surface of standard medical cotton swabs.

[0799] Because the swab design may be different for each clinical application and the appropriate shape and material must be selected for each type of application, commercially available swabs of different shapes were tested.

[0800] A study was conducted in which samples of vaginal mucosa (n=25 pregnant, n=25 non-pregnant), nasal mucosa (n=20) and oral mucosa (n=15) were taken from patients using medical rayon swabs. Mucosal samples were taken using medical cotton swabs sold as Transwab (RTM) Amies (MWE medical wire, Wiltshire, UK), and then the samples were transferred to sterile tubes without buffer or storage medium solution and stored in a freezer at -80°C.

[0801] Fig. 9 Sampling points for analysis of mucosal tissue collected from the urogenital tract, oral cavity, and nasal cavity using medical cotton swabs 900 are highlighted. Fig. 9As described, the surface of the medical swab 900 is directly analyzed by desorption electrospray ionization ("DESI") mass spectrometry without requiring prior sample preparation procedures.

[0802] Desorption electrospray ionization ("DESI") mass spectrometry experiments were performed using a Xevo G2-S Q-TOF (RTM) mass spectrometer (Waters (RTM), Manchester, UK). The desorption electrospray ionization ("DESI") source comprises an electrospray emitter 901 connected to a gas 902, a solvent 903, and a power supply 904, and an automatic rotatable swab holder 905 with adjustable rotation speed.

[0803] For desorption electrospray ionization ("DESI") mass spectrometry, a medical swab 900 is positioned orthogonally in front of an inlet capillary 906, which is connected to a mass spectrometer and / or ion mobility spectrometer atmospheric pressure interface 907. A mixed methanol:water solution (95:5) spray solvent with a flow rate of about 10 μL / min is used to desorb the sample material. The sprayer 900 is also supplied with nitrogen at about 7 bar and a voltage of about 3.4 kV.

[0804] The mucosa is absorbed from the surface of a rotating swab by gently desorbing the molecules with charged droplets of an organic solvent, followed by transfer of the desorbed ions (eg, lipids) to a mass spectrometer and / or an ion mobility spectrometer.

[0805] Full scan mass spectra (m / z 150-1000) were recorded in negative ion mode. The mass spectral data were then imported into the statistical analysis toolbox and processed. For data analysis and extraction of specific molecular ion patterns, unsupervised principal component analysis ("PCA") and recursive maximum margin criterion ("RMMC") methods were applied to improve supervised feature extraction and class information, and leave-one-out cross validation ("CV") was used to determine the classification accuracy within the data set.

[0806] Fig. 10A and Fig. 10B Shown are the results of desorption electrospray ionization ("DESI") mass spectrometry analysis of the swabs and multivariate statistical analysis including principal component analysis (PCA) and recursive maximum margin criterion (RMMC), which were used to identify lipid pattern characteristics of different mucosal models.

[0807] Fig. 10A Shown are average negative ion mode desorption electrospray ionization ("DESI") mass spectra recorded from vaginal, oral, and nasal mucosa using a Xevo G2-S Q-Tof (RTM) mass spectrometer.

[0808] Fig. 10BShown are principal component analysis ("PCA") and maximum margin criterion ("MMC") score plots acquired from vaginal mucosa (n=68), oral mucosa (n=15), and nasal mucosa (n=20) using desorption electrospray ionization ("DESI") mass spectrometry.

[0809] like Fig. 10A As shown in Figure 2, unique lipid patterns were observed between the different mucosal models. The spectral features of the vaginal and oral mucosa were mainly glycerophospholipids, such as [PS(34:1)-H] with a mass-to-charge ratio ("m / z") of 760.4. - , [PS(36:2)-H] with m / z 788.5 - and [PI(36:1)-H] at m / z 863.4 - .

[0810] like Fig. 10A As shown in Figure 2, the nasal mucosa is characterized by the presence of chlorinated adducts [PC(36:2)+Cl] - m / z 820.5, [PC(34:1)+Cl] - m / z 794.5 and [PI(36:2)-H] - m / z 826.4, in the range of m / z 700-900.

[0811] A characteristic feature of the vaginal mucosa is deprotonated cholesterol sulfate at m / z 465.3, which is consistently observed as the most dominant peak in the spectrum. The chemical assignment of this peak was confirmed by tandem mass spectrometry experiments. This compound is an important component of cell membranes with regulatory functions including stabilization, such as protection of erythrocytes from osmotic lysis and regulation of sperm capacitation.

[0812] Leave-one-patient-out cross validation of a multivariate model containing profiles obtained by analysis of the three mucosal models yielded high classification accuracy. This demonstrates that MS-based profiling of different mucosa allows stratification of patients on the basis of bacterial diversity.

[0813] Similarly, Fig.11 Shown are Fourier transform mass spectrometry (FTMS) mass spectrometry data obtained from vaginal, oral, and nasal mucosa on medical cotton swabs in the negative ion mode over the mass range of m / z 150-1000. Also different metabolic signatures were observed in each mucosal model.

[0814] A total of 300 to 1000 isotopically neutral and adducted spectral features found in the mucosa were attempted to be identified by accurate mass, isotopic cluster distribution, and tandem mass spectrometry experiments; adducts included small human primary metabolites such as cholesterol sulfate; bacterial secondary metabolites including lactate and glycerophospholipids.

[0815] Fig.12 A more detailed representation of a desorption electrospray ionization ("DESI") mass spectrum associated with pregnant vaginal mucosa, obtained using a medical cotton swab in negative ion mode, is shown. The urogenital mucosa was found to produce cholesterol sulfate [MH] at m / z 465.41. - As the most abundant lipid species as well as different glycerophospholipid species such as glycerophosphoethanolamine (PE) [PE(40:7)-H] at m / z 788.50 - , glycerophosphoserine (PS) with m / z 760.50 [PS(34:1)-H] - and phosphoinositide glycerol (PI) [PI(36:1)-H] at m / z 863.58 - .like Fig.12 As shown in , the chemical assignment of the cholesterol sulfate peak was confirmed by tandem mass spectrometry experiments.

[0816] Fig.11 The mass spectrometry data of further use median standardization, background subtraction, Savitzky-Golay peak detection, peak alignment and logarithmic transformation processing.After data processing, multivariate statistical analysis is applied to the data set to characterize a unique mucosal model based on its metabolic profile.Multivariate statistical analysis tools are used to analyze the data set, including principal component analysis (PCA) and maximum margin criterion (MMC).

[0817] like Fig.11 As shown in , the PCA score plot as well as the MMC score plot revealed the separation of different mucosal types within the first two components, and a prediction accuracy between 92-100% was obtained by leave-one-out cross-validation.

[0818] It will be appreciated that the analysis according to the various embodiments yields a characteristic profile of each sample type that is clearly distinguishable, for example, by using PCA, MMC and / or leave-one-out cross-validation analysis. These results demonstrate the use of desorption electrospray ionization ("DESI") mass spectrometry, for example, compared to 16S rRNA sequencing, as a rapid bacterial identification method to characterize the human mucosal model, for example, based on its metabolic signature excreted by characteristic bacteria.

[0819] Other embodiments are contemplated in which chemical biomarkers can be measured in human mucosa that are reliable predictors in conditions such as dysbiotic disease, inflammation, cancer, and / or infectious disease.

[0820] Pregnancy includes major changes in circulating hormone (e.g., estrogen and progesterone) levels and their secondary metabolites. In addition, pregnancy is associated with decreased vaginal microbial diversity and increased stability. As described below, the differences in the chemical characteristics of the vaginal mucosa between normal pregnancy and non-pregnancy states can be easily determined using desorption electrospray ionization ("DESI") mass spectrometry according to various embodiments.

[0821] A clinical cohort of pregnant (n=22, gestational age between 26 and 40 weeks) and non-pregnant mucosa (n=22) was evaluated in more detail to reveal differences in metabolic profiles resulting from alterations in the vaginal microbiome during pregnancy. Desorption electrospray ionization ("DESI") mass spectra were acquired from both groups in the negative ion mode over the mass range m / z 150-1000. A variety of different metabolites were detected in the vaginal mucosa.

[0822] Fig.13A Shown are average desorption electrospray ionization ("DESI") mass spectra acquired in negative ion mode over the mass range m / z 150-1000 from the pregnant and non-pregnant groups. Fig.13A Comparison of the average spectra shown in shows spectral differences between the metabolic profiles of non-pregnant and pregnant mucosa, especially in the mass of lipids in the m / z 550-900 range.

[0823] Further data analysis including unsupervised PCA and RMMC analysis revealed a clear separation between the two groups, and a high (>80%) classification accuracy was determined using leave-one-out cross-validation.

[0824] Fig. 13B and Fig. 13C Shown are the results of multivariate statistical analysis of pregnant (n=22) and non-pregnant (n=22) vaginal mucosa using desorption electrospray ionization ("DESI") mass spectrometry.

[0825] Fig. 13B The principal component analysis and discriminant analysis using RMMC are shown. Fig. 13C A leave-one-out cross-validation analysis is shown.

[0826] Fig.13D Shown are box plots indicating significant differences in the abundance of selected lipid peaks between non-pregnant and pregnant vaginal mucosa, primarily in the range of m / z 550-1000, obtained by Kruskal-Wallis ANOVA, p<0.005.

[0827] like Fig.13E As shown in , using RMMC, the two groups were well separated in terms of RMMC space, and the classification accuracy was high (>80%) based on the unique metabolic signatures obtained by leave-one-patient-out cross-validation.

[0828] Clinical studies have shown that the diversity of vaginal microbes, such as bacteria, is associated with specific vaginal mucosal metabolites. For example, during healthy pregnancy, the vaginal mucosa is primarily colonized by Lactobacillus species. Importantly, however, a shift toward vaginal dysbiosis during pregnancy may trigger premature birth.

[0829] Using the desorption electrospray ionization ("DESI") mass spectrometry-based techniques disclosed herein, females, such as women, with spontaneous preterm birth can be evaluated and compared to controls to identify biomarkers that can be used to predict preterm birth. In addition, the vaginal mucosa of pregnant females can be analyzed using the desorption electrospray ionization ("DESI") mass spectrometry-based techniques disclosed herein to analyze, such as diagnose or predict, the risk of (spontaneous) preterm birth.

[0830] Mass spectrometry profiling of vaginal mucosa enables early identification of females, such as women, at risk of infection during pregnancy based on the diversity of microorganisms (such as bacteria) in the vaginal mucosa. In addition, this enables targeted therapeutic response strategies.

[0831] Various embodiments are contemplated, including: (i) identifying vaginal mucosal metabolite biomarkers associated with specific microbial (e.g., bacterial) communities, optionally as determined using sequencing microbiome analysis; (ii) profiling of the vaginal mucosa during healthy pregnancy, wherein microbial (e.g., bacterial)-specific metabolites and signatures excreted during healthy pregnancy can be characterized in detail; and (iii) identifying diagnostic and prognostic metabolic signatures from the vaginal mucosa for adverse pregnancy outcomes (e.g., preterm birth).

[0832] Fig.14A Desorption electrospray ionization ("DESI") mass spectrometry analysis of a bacterial (Klebsiella pneumoniae) sample on a swab is shown according to one embodiment. Fig.14A The data shown in show that, according to various embodiments, bacterial samples on swabs can be detected using desorption electrospray ionization ("DESI") mass spectrometry. Fig. 14B A comparison of rapid evaporation ionization mass spectrometry ("REIMS") time of flight ("TOF") mass spectrometry data of corresponding bacterial samples measured directly from agar plates is shown. Peaks highlighted with asterisks were detected by both ionization techniques.

[0833] The cultured six species were further tested for microbiological detection by desorption electrospray ionization ("DESI") swab analysis, including Candida albicans, Pseudomonas monteri, Staphylococcus epidermidis, Moraxella catarrhalis, Klebsiella pneumoniae, and Lactobacillus spp. These species are all important bacterial and fungal species that have been isolated from the vaginal mucosa of pregnant patients and identified by sequence analysis, such as 16S rRNA gene sequencing.

[0834] The swabs were quickly dipped into a solution of diluted biomass from each species in 10 μL of methanol, followed by desorption electrospray ionization ("DESI") mass spectrometry analysis of the swab surface.

[0835] Fig.15A -C shows microbiological analysis of the swab using desorption electrospray ionization ("DESI") mass spectrometry.

[0836] Fig.15A Shown are average desorption electrospray ionization ("DESI") mass spectra of the different microbial species analyzed, including Candida albicans, Pseudomonas monteri, Staphylococcus epidermidis, Moraxella catarrhalis, Klebsiella pneumoniae, and Lactobacillus spp.

[0837] Fig. 15B and Fig. 15C PCA plots are shown showing the separation between vaginal mucosa (pregnant and non-pregnant groups) and between microbial species within the first two components. In addition, separation between different bacterial and fungal species can be observed.

[0838] In such Fig.15A Unique spectral features were observed in the mass spectra shown in Figure 2, yielding the ability to separate between different microbial classes, as well as the PCA score plot ( Fig. 15B and Fig. 15C ) in the ability to separate the vaginal mucosa within the first two components.

[0839] Such results demonstrate the potential of using desorption electrospray ionization ("DESI") mass spectrometry to characterize microbial (e.g., bacterial)-specific and host response metabolite biomarkers and signatures of specific microbial (e.g., bacterial) communities from animal (e.g., human) mucosa on medical swabs.

[0840] It will be appreciated that various embodiments provide a novel desorption electrospray ionization ("DESI") mass spectrometry setup for non-invasive and rapid analysis of mucosal metabolome profiles from the surface of medical swabs. This arrangement has been successfully shown to be able to differentiate between animal (e.g., human) mucosal models and enable microbial identification. The method can readily distinguish between different mucosal sites, biochemical changes induced by physiological events such as pregnancy, and allows for rapid identification of complete bacterial and fungal species.

[0841] Because desorption electrospray ionization ("DESI") mass spectrometry analysis causes minimal sample destruction to a large portion of the sample surface material, according to various embodiments, medical swabs can be optionally sent directly to, for example, a microbiology laboratory for further evaluation, such as culture, microbial identification / confirmation, and / or next generation sequencing analysis after desorption electrospray ionization ("DESI") analysis. Because the desorption electrospray ionization mass spectrometry profiles obtained according to various embodiments contain information describing mucosal biochemistry and microbe-host interactions, all methods according to various embodiments are suitable for a wide range of clinical applications.

[0842] Various embodiments provide a novel point-of-care mucosal screening diagnostic method that uses a standard cotton medical swab as a sampling probe for mucosal uptake and an ionization probe for desorption electrospray ionization ("DESI") mass spectrometry analysis. After data acquisition, the obtained spectra can be compared with spectra collected in a database to provide a rapid diagnosis for the patient, such as within seconds.

[0843] Various embodiments relate to direct metabolomic profiling of specific mucus models (nasal, vaginal, pharyngeal, bronchial, esophageal) from standard medical swab surfaces using desorption electrospray ionization ("DESI") technology. Various embodiments relate to rapid point-of-care diagnostic methods for diseases, which are optionally selected from any of the diseases mentioned herein, such as inflammatory and pathogen-related diseases, such as immune disorders; microbial flora dysbiosis (which can, for example, indicate the risk of premature birth during pregnancy); microbial infection, such as bacterial infection; or detection of cancer or precancerous conditions. Sequential metabolomic profiling of animal (e.g., human) mucosa and detailed statistical analysis can identify disease-specific metabolic profiles and / or taxon-specific microorganisms, such as bacterial markers, in a rapid and robust manner that is beneficial to point-of-care diagnostic methods.

[0844] like Fig.16 As shown in , according to various embodiments, desorption electrospray ionization ("DESI") mass spectrometry 160 of a sample taken 161 onto a swab may be subjected to statistical analysis 162 in order to provide a diagnosis 163 (or prognosis).

[0845] The sample may additionally or alternatively be analyzed 164 by rapid vapor ionization mass spectrometry ("REIMS") mass spectrometry.

[0846] Embodiments are contemplated in which multiple different analytical techniques may be applied to the same swab (or another swab) to additionally perform culture-dependent 165 analysis, such as DNA extraction and PCR analysis, for example to generate complementary 16S rRNA microbiome data.

[0847] like Fig.16 As shown in , any one or more or all of the additional analyses may be used to validate the desorption electrospray ionization (“DESI”) based diagnosis 163 .

[0848] Fig.17A It is demonstrated how continuously rotating a swab while it undergoes desorption electrospray ionization ("DESI") analysis can result in enhanced signal intensity.

[0849] Rapid Evaporative Ionization Mass Spectrometry ("REIMS") Analysis of Swabs

[0850] Various embodiments described herein also relate to a rapid evaporation ionization mass spectrometry ("REIMS") analysis method for swabs, wherein a sample on a swab undergoes rapid evaporation ionization mass spectrometry ("REIMS") analysis. However, this approach is destructive to the swab, and in bipolar mode, the contact closure of the electrodes is limited.

[0851] When the swab is analyzed by rapid evaporation ionization mass spectrometry, the swab may be dipped, soaked, or otherwise immersed in a fluid (eg, water) prior to undergoing rapid evaporation ionization mass spectrometry ("REIMS") analysis.

[0852] like Fig. 17B It is also described in that soaking the swab in fluid prior to rapid evaporation ionization mass spectrometry has also been shown to enhance signal intensity.

[0853] Now we will look at Fig.18 The rapid evaporative ionization mass spectrometry ("REIMS") technique is described in more detail.

[0854] Fig.18 A rapid evaporative ionization mass spectrometry ("REIMS") method is described in which a bipolar forceps 1 is brought into contact with an in vivo tissue 2 of a patient 3. Fig.18 In the example shown in FIG. 1 , a bipolar forceps 1 may be brought into contact with brain tissue 2 of a patient 3 during a surgical procedure on the patient's brain. However, according to various embodiments, and as shown in FIG. Fig. 17B As shown in FIG. 1 , a bipolar forceps 1 can be brought into contact with a sample provided on a medical swab.

[0855] An RF voltage can be applied from an RF voltage generator 4 to the bipolar forceps 1 to locally Joule or diathermically heat tissue 2 or a sample. As a result, an aerosol or surgical plume 5 is generated. The aerosol or surgical plume 5 can then be captured or otherwise aspirated through the irrigation port of the bipolar forceps 1. Thus, the irrigation port of the bipolar forceps 1 is again used as an aspiration port. The aerosol or surgical plume 5 can then be conducted from the irrigation port (aspiration port) of the bipolar forceps 1 to a tube 6 (e.g., a 1 / 8" or 3.2 mm diameter Teflon (RTM) tube). The tube 6 is arranged to transfer the aerosol or surgical plume 5 to the atmospheric pressure interface 7 of a mass spectrometer 8 and / or an ion mobility analyzer.

[0856] According to various embodiments, a matrix comprising an organic solvent (such as isopropyl alcohol) can be added to the aerosol or surgical plume 5 at the atmospheric pressure interface 7. The mixture of the aerosol 3 and the organic solvent can then be arranged to impact a collision surface within the vacuum chamber of the mass spectrometer and / or ion mobility spectrometer 8. According to one embodiment, the collision surface can be heated. Ionization is caused when the aerosol impacts the collision surface, thereby generating analyte ions. The ionization efficiency for generating analyte ions can be enhanced by adding the organic solvent. However, the addition of the organic solvent is not essential.

[0857] The analyte ions generated by causing the aerosol, smoke, or vapor 5 to impact the collision surface then pass through subsequent stages of the mass spectrometer (and / or ion mobility analyzer) and undergo analysis in a mass analyzer or filter and / or ion mobility analyzer, such as mass analysis and / or ion mobility analysis. The mass analyzer or filter can comprise, for example, a quadrupole mass analyzer or a time-of-flight mass analyzer.

[0858] Modified swab

[0859] Various other embodiments are directed to a modified, chemically functionalized, and / or solid-phase microextraction ("SPME") swab approach.

[0860] As discussed above, it has been found that various chemically modified swabs used in conjunction with desorption electrospray ionization ("DESI") mass spectrometry according to various embodiments exhibit improved sensitivity and / or reduced background compared to conventional (unmodified) swabs. The modified swabs described in more detail below exhibit an improved signal-to-noise ratio in a specific mass-to-charge ratio range compared to conventional (unmodified) swabs.

[0861] As Fig.19 shown, standard cotton swabs are commercially available and are relatively non-invasive. However, the swabs contain a fibrous material with absorbent properties, release molecules relatively poorly, provide non-selective extraction, and can result in a relatively high background signal being observed.

[0862] In contrast, coated or chemically modified swabs according to various embodiments can advantageously provide a solid surface, enable selective extraction and exhibit improved sensitivity.

[0863] According to various embodiments, a (standard) medical swab may be wetted or otherwise functionalized with one or more adsorbents in order to: (i) increase the intensity or signal of analyte ions observed within one or more specific mass-to-charge ratio ranges; and / or (ii) reduce the intensity or signal due to undesirable background ions within one or more specific mass-to-charge ratio ranges.

[0864] According to various embodiments, a standard cotton medical swab may be coated with ODS / C18 (octadecyl), polydimethylsiloxane ("PDMS"), Oasis (RTM) MAX (mixed mode cation exchange), Oasis (RTM) hydrophilic-lipophilic balanced (HLB), and / or Oasis (RTM) MCX (mixed mode cation exchange). For example, according to one embodiment, a swab may be provided with a single or multiple layers of adsorbent materials such as C18 (octadecyl), C18 (octadecyl) end-capped (EC), hydrophilic-lipophilic balanced (HLB) particles, and / or divinylbenzene ("DVB"). These various adsorbent materials may be used to enhance the efficiency of extracting certain compounds from the mucosal matrix.

[0865] FIG. 20 illustrates the improved sensitivity provided by using a modified swab, according to various embodiments. Fig. 20A ), swab modified with ODS / C18 ( Fig. 20B ) and swabs modified with Oasis(RTM)HLB ( Fig. 20C ) was used to perform desorption electrospray ionization ("DESI") mass spectrometry analysis of the nasal fluid on the swab. Specifically, a modified lipid signal was observed in the mass-to-charge ratio range of about m / z 730-890.

[0866] Fig.21 A range of differently coated materials are shown, including different forms, shapes and adsorbent materials.

[0867] Biocompatible and functionalized polybutylene terephthalate ("PBT") plastic swabs and fibers can be prepared by, for example, dip-coating the material surface using peroxyacetyl nitrate ("PAN") as a binder for the adsorbent material. Using the dip-coating technique, a single layer coating with a coating thickness of about 5-10 μm can be achieved.

[0868] Saliva desorption electrospray ionization ("DESI") mass spectrometry metabolic profiling of swabs functionalized with C18 (octadecyl), C18 (octadecyl) endcapped (EC), divinylbenzene (DVB) weak anion exchange (WAX), and hydrophilic-lipophilic balanced (HLB) coatings was tested and compared to a standard medical cotton desorption electrospray ionization ("DESI") mass spectrometry approach.

[0869] like Fig. 22 As shown in , after functionalization, the swab may need to be washed 210 and / or conditioned 211 before use. The washing 210 and / or conditioning 211 steps remove any contaminants left over from the manufacturing process that may interfere with desorption electrospray ionization ("DESI") mass spectrometry analysis. For example, washing 210 may include soaking in a first solvent to remove any contaminants left over from the manufacturing process, and conditioning 211 may include soaking in a second solvent to remove any remaining contaminants, including any undesirable first solvent residues. Suitable solvents for these steps include methanol, acetonitrile (ACN), isopropanol, water, and mixtures thereof.

[0870] After the swab contacts the sample or body surface 212, the swab can be washed 214 to remove unbound material that may interfere with the analysis, leaving the analyte of interest bound to the swab. For example, if the analyte of interest is a lipid, unbound salts or polar molecules can be removed by washing 214. The swab is then analyzed 215 by desorption electrospray ionization ("DESI") mass spectrometry or a variation thereof, such as using a desorption electrodynamic focusing ionization ("DEFFI") ion source.

[0871] An optimized workflow was designed in which the swab was heated in MeOH / ACN / (CH 3 ) 2 Wash 210 in CHOH (50:25:25) for about 1 hour and then in MeOH:H 2 O (50:50) for about 1 minute, immersed in an analyte (e.g., saliva) solution for about 2 minutes and 30 minutes to take a sample of the analyte 212, dried 213 for about 5 minutes, and rinsed 214 by immersion in water for about 1 second, and then washed with MeOH:H 2 O(95:5) was subjected to desorption electrospray ionization ("DESI") mass spectrometry 215. This workflow was shown to enhance extraction efficiency and sample cleanup during mucosal analysis.

[0872] Fig.23A -E shows the use of a standard medical swab ( Fig.23A ) and a three-layer swab ( Fig. 23B-E) Comparison of saliva spectra obtained in negative (left) and positive (right) ion modes for a triple-coated swab coated with four different adsorbents (C18 (octadecyl), C18 (octadecyl) end-capped (EC), hydrophilic-lipophilic balanced (HLB), and divinylbenzene (DVB) weak anion exchange (WAX)).

[0873] Fig.23A Saliva spectra obtained using a standard medical swab in negative ion mode (left) and positive ion mode (right) are shown. Fig. 23B shows saliva spectra obtained in negative ion mode (left) and positive ion mode (right) using a standard medical swab coated with three layers of C18 (octadecyl) adsorbent, Fig.23C shows saliva spectra obtained in negative ion mode (left) and positive ion mode (right) using a standard medical swab coated with three layers of C18 (octadecyl) endcapped (EC) adsorbent, Fig.23D shows saliva spectra obtained in negative ion mode (left) and positive ion mode (right) using a standard medical swab coated with three layers of hydrophilic-lipophilic balance (HLB) adsorbent, Fig.23E Shown are saliva spectra obtained in negative ion mode (left) and positive ion mode (right) using a standard medical swab coated with three layers of divinylbenzene (DVB) weak anion exchange (WAX) sorbent.

[0874] The coated swabs produced cleaner background spectra and enhanced lipid sensitivity from the saliva matrix (green highlights) using C18 (octadecyl) swabs in negative ion mode (m / z 700-900) and using hydrophilic-lipophilic balance (HLB) and divinylbenzene (DVB) weak anion exchange (WAX) swabs in positive ion mode (m / z 600-720); however, the medical cotton swabs showed high background peaks, especially in positive ion mode (highlighted peaks).

[0875] Overall, the functionalized swabs were found to improve sensitivity for hydrophobic analytes due to improved selective extraction efficiency of non-polar analytes in the saliva matrix compared to standard cotton swabs.

[0876] Various embodiments described herein provide an optimized desorption electrospray ionization ("DESI") mass spectrometry method for metabolic profiling of mucosal samples from medical swabs.

[0877] Various embodiments facilitate differentiation of different mucosal models. Various embodiments allow bacterial metabolic profiles to be obtained from swabs.

[0878] Furthermore, the functionalized swab according to various embodiments has improved sensitivity for, for example, hydrophobic analytes.

[0879] According to various embodiments, mucosal profiling may be performed using rapid vapor ionization mass spectrometry ("REIMS") as a complementary analytical technique.

[0880] It should be understood that next generation sequencing technology, such as 16S RNA sequencing, can identify and characterize bacteria colonized in human mucosa. However, the clinical implementation of bacterial identification is limited because of the cost and time constraints of this approach. However, the desorption electrospray ionization ("DESI") mass spectrometry of mucosal swabs meets all the criteria set for conventional diagnostic procedures. Using desorption electrospray ionization ("DESI") mass spectrometry to quickly and directly identify metabolite features excreted into animal (e.g., human) mucosa by specific microorganisms (e.g., bacteria) can produce objective biochemical information, which can identify microorganisms, such as bacteria, and enhance the current clinical decision-making (e.g., target antibiotic treatment) in the case of disease analysis (e.g., diagnosis), diseases such as any disease mentioned elsewhere herein, such as infection, dysbiosis, cancer and / or inflammatory diseases.

[0881] Modified swab surface chemistry

[0882] Various embodiments provide a medical swab for use in the methods of various embodiments, wherein the swab has been chemically modified to enhance selectivity for an analyte.

[0883] Medical swabs normally include a head and a shaft, the head is usually called a bud, and the shaft can be connected to the head or be integral with the head. The shaft can be formed of plastic, wood, rolled paper or wire. The head is usually hydrophilic. The head can be formed of cotton, rayon, plastic fibers or foam. Suitable plastics for the shaft and head include polyurethanes and polyesters, such as polyethylene terephthalate (PET) and polybutylene terephthalate (PBT).

[0884] References to cotton, rayon, plastic fiber, or foam swabs in this article refer to the material used to make the swab head.

[0885] In principle, any known medical swab or medical sampling device can be chemically modified (functionalized) to improve its selectivity for a particular analyte of interest prior to analysis using desorption electrospray ionization ("DESI") mass spectrometry or variations thereof. The swab can be disposable (i.e., intended to be used only once). Swabs useful for chemical modification / functionalization include cotton, rayon, polyester, and foam swabs, especially cotton or polyester swabs.

[0886] The chemical modification of the swab includes introducing a suitable functionalized chemical agent on the swab surface. The functionalized chemical agent can be attached to the swab by chemical means, such as by covalent bonds, or by physical means, such as by physical entrapment or by using a suitable adhesive material. In various embodiments, the chemical modification includes forming a coating on the swab surface.

[0887] The nature of the surface functionalization will depend on the type of analyte of interest. After functionalization, the swab surface can be hydrophilic or hydrophobic. It can also contain ionic groups. For example, a hydrophobic or lipophilic surface is good for analyzing lipids, while a charged or hydrophilic surface would be good for analyzing proteins or certain drug metabolites.

[0888] The chemical modification should preferentially bind the analyte of interest, but should not bind the analyte so tightly that it cannot be subsequently removed for analysis by mass spectrometry.

[0889] After the sample is removed from the patient, the functionalized swab can be used to analyze the sample, such as urine. The surface functionalization used in the swab does not have to be biocompatible for such in vitro use. Alternatively, the functionalized swab can be used directly to collect the sample from the patient, for example by wiping the mucous membrane. Swabs intended for in vivo use or direct contact with patients should be biocompatible.

[0890] Surface functionalization can be introduced into the swab by adsorption or absorption and by methods including, but not limited to, solution phase methods, vapor phase methods, chemical vapor deposition, molecular vapor deposition, atomic layer deposition, dip coating, electrochemical coating, or spray coating.

[0891] The functionalized molecule can be chemically attached directly to the swab by reacting the functional groups in the functionalized molecule with the functional groups in the swab material to form a covalent bond. The covalent attachment of the functionalized molecule to the swab can include, but is not limited to, the reaction of swab functional groups such as alcohols, aldehydes, amines, carboxylic acids, or olefin groups to form silyl ethers, ethers, thioethers, carbamates, carbonates, carbon-carbon bonds, carbon-nitrogen bonds, ureas, or esters. For example, a cellulosic swab such as a cotton or rayon swab will contain a plurality of hydroxyl groups that can react with functional groups such as carboxylic acids in the functionalized molecule to form a covalent attachment of the functionalized agent to the swab surface via an ester bond.

[0892] Methods of functionalizing cellulose surfaces are known in the art and include those disclosed in US 2009 / 0126891, the contents of which are incorporated herein by reference.

[0893] Alternatively, chemical modification of the swab may include physical embedding of the swab with a suitable functionalizing agent. This approach may be applicable when the functionalizing agent is a solid particle such as functionalized silica particles or ion exchange resin particles.

[0894] Another alternative is to connect the functionalized particles to the swab surface with a thin layer of adhesive or other binder material. Any known medical grade adhesive can potentially be used, including cyanoacrylate, epoxy adhesive and acrylate adhesive. Suitable such adhesives include Loctite (RTM) medical grade adhesives available from Henkel Corporation, Connecticut, USA. Any inert biocompatible particles can potentially be used, including silica particles; mixed silica particles-organic particles; or carbon particles. Suitable particle diameters are typically about 1 to about 60 microns, and the diameter can be about 2 to about 10 microns, and the diameter can be about 2 to about 5 microns.

[0895] Advantageous swab surface functionalization includes, for example, attaching a solid phase extraction material to the swab surface by physical embedding or by using a suitable adhesive or binder material. As used herein, the term "solid phase extraction material" refers to a solid material suitable for use as a stationary phase in gas or liquid chromatography. Such materials are typically in particulate form and are typically based on silica or polymer resins. The solid phase extraction material used for lipid analysis can be a reverse phase stationary phase.

[0896] It is well known that functionalized silica and mixed silica particles / organic particles can be used as stationary phases in liquid chromatography. Methods for functionalizing silica surfaces to make them more selective for analytes of interest are known in the art. Suitable modifications include those disclosed in US 2012 / 0141789 and US 2008 / 0073512, the disclosures of which are incorporated herein by reference.

[0897] Surface modifiers used for chromatographic stationary phases typically include organic functional groups that impart certain chromatographic functional groups to the stationary phase. Functional groups on the surface of the stationary phase particles can be derivatized by reaction with suitable modifiers. Silica particles have silanol groups, while silica / organic hybrid particles can have both organic groups and silanol groups, which can be derivatized.

[0898] Suitable surface modifiers for chromatographic stationary phases include those having the formula Z a (R') b Si-R 2 Those where Z=Cl, Br, I, C 1 -C 5an alkoxy group, a dialkylamino group or a trifluoromethanesulfonate group; a and b are each an integer from 0 to 3, provided that a+b=3; R 1 It is C 1 -C 6 A linear alkyl group, a cycloalkyl group or a branched alkyl group, and R 2 It is a functional group.

[0899] R' may be selected from the group consisting of methyl, ethyl, propyl, isopropyl, butyl, tert-butyl, sec-butyl, pentyl, isopentyl, hexyl and cyclohexyl.

[0900] Functional group R 2 It may include alkyl, alkenyl, alkynyl, aryl, cyano, amino, diol, nitro, ester, cation or anion exchange groups, alkyl or aryl groups containing embedded polar functional groups or chiral moieties. Suitable R 2 Examples of functional groups include chiral moieties; 1 -C 30 Alkyl groups, including C 1 -C 20 , such as octyl (C 8 ), octadecyl (C 18 ) and triacontyl (C 30 ); alkylaryl, such as C 1 -C 4 -phenyl; cyanoalkyl, such as cyanopropyl; diol, such as propyldiol; amino, such as aminopropyl; and alkyl or aryl groups containing embedded polar functional groups and chiral moieties, embedded polar functional groups such as carbamate functional groups, as disclosed in U.S. Pat. No. 5,374,755. Such groups include those having the following general formula:

[0901]

[0902] wherein l, m, o, r and s are 0 or 1, n is 0, 1, 2 or 3, p is 0, 1, 2, 3 or 4, and q is an integer from 0 to 19; R 3 is selected from the group consisting of hydrogen, alkyl, cyano and phenyl; and Z, R', a and b are as defined above. The carbamate functional group may have the general structure shown below:

[0903]

[0904] Where R 5 It may be, for example, cyanoalkyl, tert-butyl, butyl, octyl, dodecyl, tetradecyl, octadecyl or benzyl. Advantageously, R 5 It is octyl, dodecyl or octadecyl.

[0905] R 2 Can be C1 -C 30 alkyl, and may be C 1 -C 20 alkyl.

[0906] Particularly advantageous surface modifiers are selected from the group consisting of octyltrichlorosilane, octadecyltrichlorosilane, octyldimethylchlorosilane and octadecyldimethylchlorosilane, and may especially be octyltrichlorosilane or octadecyltrichlorosilane.

[0907] Particulate solid phase extraction materials are commercially available from Waters Corporation, USA. Particularly suitable materials of this type are silica particles functionalized with octadecyl (C18) or polydimethylsiloxane (PDMS) groups.

[0908] Alternatively, the swab may be functionalized by attaching a particulate polymer resin to the swab surface, for example by physical embedding or by using a suitable adhesive or binding agent. Suitable such resins are known in the art as stationary phases for use in chromatography. These stationary phases include reversed phase stationary phases, ion exchange stationary phases, and mixed mode stationary phases, including ion exchange-reversed phase stationary phases.

[0909] Reversed phase chromatography stationary phases may be particularly suitable for preparing swabs for lipid analysis. Suitable reverse phase stationary phases include polydivinylbenzene (DVB) and copolymers of N-vinylpyrrolidone and divinylbenzene, such as Oasis (RTM) HLB available from Waters Corporation.

[0910] Mixed-mode ion exchange / reverse phase adsorbents based on modified N-vinylpyrrolidone / divinylbenzene copolymers can also be used. Some of these copolymers have sulfonated or carboxylated benzene rings that can provide cation exchange functionality. 2 -piperazine or quaternary ammonium (such as -CH 2 N + (CH 3 ) 2 (C 4 H 9 )) Such copolymers modified by linking some benzene rings can provide anion exchange functionality. Suitable such adsorbents are available from Waters Corporation under the trade names Oasis (RTM) MCX, Oasis (RTM) WCX, Oasis (RTM) MAX and Oasis (RTM) WAX.

[0911] The solid particles for attachment to the swab may also be functionalized with a polymer coating. Polymer coatings are known in the literature and can generally be provided by polymerizing or polycondensing physically adsorbed monomers onto the particle surface without chemically bonding the polymer layer to the support (Type I); polymerizing or polycondensing physically adsorbed monomers onto the surface and chemically bonding the polymer layer to the support (Type II); immobilizing physically adsorbed prepolymers onto the support (Type III); and chemically adsorbing presynthesized polymers onto the support surface (Type IV): see, e.g., Hanson et al., J. Chromat. A656 (1993) 369-380, which is incorporated herein by reference.

[0912] Any polymer used to make solid phase extraction materials can potentially be used to form the polymer coating on the swab surface. Suitable coating polymers include polydivinylbenzene (DVB), copolymers of N-vinylpyrrolidone and divinylbenzene, and polydimethylsiloxane.

[0913] Coating the particles with a polymer can be used in conjunction with other surface modifications as described above. Specifically, the SPEM particles can be embedded in a polymer coating such that the coating acts as a binding agent to entrap the particles on or near the swab surface.

[0914] Functionalized swabs can also be prepared by forming a solution or dispersion of the chemical modifier and dipping the swab into the solution or dispersion. The swab is then dried and the dipping step is repeated as needed. Such dipping can achieve a monolayer of the chemical modifier on the swab surface. The thickness of the layer formed by this method can be in the range of 5-10 microns.

[0915] Fibers used as solid phase microextraction (SPME) media are known in the art. Such fibers normally comprise fused silica fibers or metal wires with a thin polymer coating. For example, fused silica fibers coated with C18 have been proposed to sample drugs in urine (Kennedy et al., Analytical Chemistry, Vol. 82, No. 17, Sept. 1, 2010). The swab used in various embodiments may further comprise such fibers.

[0916] In various embodiments, the swab does not include any fused silica based fibers or any metallic fibers / wires.

[0917] Swab sterilization

[0918] If the functionalized swab is used to analyze a sample taken from a patient, the swab does not necessarily need to be sterilized before use. For example, a swab used to analyze a urine sample may not need to be sterilized before use. However, any swab intended to come into contact with a patient will need to be sterilized before use. The functionalized swabs of various embodiments can potentially be sterilized using any standard technique known in the art for sterilizing medical swabs. Suitable sterilization methods include, but are not limited to, autoclaving, heating, gamma radiation, and ethylene oxide sterilization.

[0919] Multiple analyses of the same swab

[0920] As discussed above, a particular benefit of using desorption electrospray ionization ("DESI") mass spectrometry to analyze a sample provided on a medical swab is that multiple different analyses can be performed on the same sample, ie, the same swab.

[0921] Performing multiple different analyses of or on the same sample can provide multiple different sets of information about the same sample in a particularly convenient and efficient manner. This is particularly possible because desorption electrospray ionization ("DESI") mass spectrometry is a relatively non-destructive analytical technique, and also because various commercial analytical techniques have been optimized to use samples provided on medical swabs, such as culture techniques and nucleic acid sequencing techniques, such as 16S rRNA sequencing techniques.

[0922] Thus, after collecting a single sample onto a swab, the sample on the swab may be analyzed multiple times using a plurality of different analytical techniques, wherein at least one of the techniques (eg, the first technique used) comprises desorption electrospray ionization ("DESI") mass spectrometry.

[0923] According to various other embodiments, data directed analysis can be performed on a sample on a swab (the same sample on the same swab). For example, depending on the results of a first (e.g., desorption electrospray ionization ("DESI") mass spectrometry) analysis and / or in the case of detecting ions of interest using a first (e.g., desorption electrospray ionization ("DESI") mass spectrometry) analysis, further analysis can be selected, altered, and / or optimized and performed.

[0924] In these embodiments, one or more of the analytical techniques may comprise a culture analytical method, e.g., in which a swab is contacted with a solid or liquid culture medium, e.g., swabbed onto or dipped into the culture medium, the culture medium is incubated, and then the culture medium is examined, e.g., under a microscope, to identify any microorganisms present.

[0925] One or more of the analytical techniques may include gene sequencing methods to identify the microorganism, such as 16S rRNA sequencing methods.

[0926] One or more of the analytical techniques may include matrix-assisted laser desorption ionization ("MALDI") methods to identify microorganisms.

[0927] One or more of the analytical techniques may include rapid vapor ionization mass spectrometry ("REIMS") methods to identify microorganisms.

[0928] As mentioned above about Fig.16 As shown and described, any one or more or all of the additional analyses may be used to confirm and / or supplement the desorption electrospray ionization ("DESI") based identification or diagnosis.

[0929] Analyze swabs containing stool or body fluid specimens using open ionization mass spectrometry, such as DESI and / or REIMS techniques. son

[0930] Analysis of stool or body fluid specimens according to various embodiments can provide information about disease and / or microbiome, optionally the mucosal microbiome and / or the microbiome of the GI cavity. Thus, optionally, the method can include analyzing a swab containing a stool and / or body fluid specimen. For example, a stool and / or body fluid specimen can be analyzed for the presence of cells, compounds, and / or microorganisms.

[0931] The method can optionally analyze metabolic differences between various conditions, which can optionally be selected from any of the conditions listed elsewhere herein, such as irritable bowel syndrome, colorectal cancer, and / or inflammatory bowel disease. The method can optionally analyze, for example, diagnose microbial infections and / or mixed microbial communities by identifying taxonomically specific biomarkers.

[0932] The cell can be, for example, a mammalian cell, a white blood cell, a red blood cell, a fetal cell, and / or a cancer cell.

[0933] Optionally, stool and / or body fluid specimens may be analyzed for the presence of microorganisms and / or for the microbiome. Details of analysis of microorganisms and / or microbiome are provided elsewhere herein.

[0934] Optionally, the presence of compounds in feces and / or body fluid specimens can be analyzed. The compound can, for example, comprise or consist of a biomolecule, an organic compound, and / or an inorganic compound. It can optionally be selected from any of the compounds listed elsewhere herein. Optionally, it can be bile, hemoglobin, or a derivative of any of them.

[0935] Optionally, stool and / or body fluid samples other than blood may be analyzed for the presence of blood. For example, the presence of blood in urine may indicate infection or other disease. For example, the presence of blood in a stool sample may optionally be used to analyze bleeding in the gastrointestinal tract and / or anus. Optionally, bleeding may indicate a disease, such as selected from the group consisting of anal fissures, diverticular disease, polyps, inflammatory diseases, angiodysplasia, and / or any of the diseases mentioned elsewhere herein.

[0936] Optionally, stool and / or body fluid samples may be analyzed for the presence of bile or its derivatives, for example for liver disease and / or kidney disease and / or any of the diseases mentioned elsewhere herein.

[0937] Analysis of the stool specimen may optionally include the use of clamp-based rapid evaporative ionization mass spectrometry ("REIMS"), in which a sample of the stool specimen may be taken between clamps and then the probes may be pulled together.

[0938] Fig.24 Shown is the spectrum observed when analyzing a stool sample using the rapid evaporative ionization mass spectrometry ("REIMS") technique.

[0939] Desorption electrospray ionization ("DESI") nebulizer with heated transfer capillary

[0940] Fig.25A One embodiment is shown and includes a desorption electrospray ionization (DESI) nebulizer 300 in which a solvent capillary 302 may be arranged to direct charged droplets 304 of solvent to a swab surface 310. A sample 311 may be located on the swab surface 310, which may contain analyte particles. Charging of the solvent droplets may be achieved using a high voltage power supply 306 in contact with the capillary 302. The high voltage power supply 306 may include an electrode 307, which may contact any portion of the capillary 302, such that it is operable to charge the solvent droplets as they exit the outlet end 303 of the capillary 302. The outlet end 303 of the capillary may face the swab surface 310.

[0941] A sheath gas 308 (e.g., nitrogen) can be arranged to surround the capillary 302 so as to atomize the solvent as it emerges from the capillary 302 and direct the charged solvent droplets 304 toward the swab surface 310. The sheath gas can be directed through a tube 312, which can be coaxial with the solvent capillary 302, having an inlet 314 at an end distal from the swab surface 310 and an outlet 316 at an end facing the swab surface 310.

[0942] The outlet 316 of the sheath gas tube 312 can be concentric with the outlet end 303 of the capillary tube, which can facilitate atomization of the solvent as it emerges from the capillary tube 302. The solvent emerging from the outlet end 303 of the solvent capillary tube 302 can be atomized by the sheath gas 308. The connector 318 can connect the tube 312 to a gas source suitable for use as a sheath gas. The sheath gas 308 can include nitrogen or standard medical air, and the sheath gas source can be a nitrogen source or a standard medical air source.

[0943] When the solvent droplet 304 contacts the swab, the analyte particles on the swab can be desorbed and the charged droplet and analyte mixture 320 can be transferred to a transfer capillary or transfer device 330, which can lead to an ion analyzer, a mass analyzer or filter, and / or an ion mobility analyzer, and / or a mass spectrometer 340. The charged droplet and analyte mixture can be transferred through an inlet 332 of the transfer capillary or transfer device 330. This can be achieved by placing the opposite end 333 of the transfer capillary or transfer device 330 into a low pressure region 352, such as a vacuum stage of the mass analyzer or filter and / or ion mobility analyzer and / or mass spectrometer 340.

[0944] The charged droplets and analyte mixture (including, for example, analyte ions) may be transferred through ion optics 352 to an analysis region of an ion analyzer and / or ion mobility analyzer and / or mass spectrometer 340. Ion optics 352 may include an ion guide, such as a StepWave (RTM) ion guide.

[0945] The analyte ions may be directed into the analysis region by applying a voltage to the ion optics 352. The analyte ions may then be analyzed by a mass analyzer or filter and / or an ion mobility analyzer and / or a mass spectrometer 340.

[0946] According to one embodiment, the ion analyzer and / or mass analyzer or filter and / or ion mobility analyzer and / or mass spectrometer 340 may include an ion mobility spectrometer. According to another embodiment, the ion analyzer and / or mass analyzer or filter and / or ion mobility analyzer and / or mass spectrometer 340 may include a combination of an ion mobility spectrometer and a mass spectrometer.

[0947] As a result of the analysis, chemical information about the sample 311 can be obtained.

[0948] One or more heaters may be provided to heat Fig.25A For example, a heater may be provided to heat one or more of the solvent capillary 302, the sheath gas tube 312, the swab surface 310, and the transfer or inlet capillary 330.

[0949] The one or more heaters may include a wire heater (e.g., a tungsten coil) and / or may be configured to heat the various components to at least 50° C., 100° C., 200° C., 300° C., 400° C., 500° C., 600° C., 700° C., or 800° C. However, any type of heater having the function of heating the various components may be used, such as a blower or an induction heater.

[0950] Fig.25A A first heater 342 is shown which may be arranged and adapted to heat the transfer or inlet capillary 330 so that the solvent and analyte mixture 320 may be heated prior to forwarding to, for example, a mass analyzer or filter and / or an ion mobility analyzer and / or a mass spectrometer 340 .

[0951] The first heater 342 can be located anywhere along the solvent capillary 330, such as adjacent to or at the inlet 341 of an ion analyzer, a mass analyzer or filter and / or an ion mobility analyzer and / or a mass spectrometer. Alternatively, the first heater 342 can be located adjacent to or at the inlet 332 of the solvent capillary or transfer device 330. The first heater 342 can include a wire heater (e.g., a tungsten coil) and / or can be configured to heat the inlet capillary to at least 50° C., 100° C., 200° C., 300° C., 400° C., 500° C., 600° C., 700° C., or 800° C.

[0952] The second heater 344 may be arranged and adapted to heat the sheath gas tube 312 so that the solvent and / or the sheath gas may be heated.

[0953] The second heater 344 can be located at the end of the tube 312 closest to the swab surface 310 so that the solvent and / or sheath gas can be heated before being directed to the swab surface 310. The second heater 344 can include a wire heater (e.g., a tungsten coil) and / or can be configured to heat the tube 312 and / or the solvent and / or sheath gas to at least 50°C, 100°C, 200°C, 300°C, 400°C, 500°C, 600°C, 700°C, or 800°C.

[0954] The third heater 346 may be arranged and adapted to heat the solvent capillary 302 so that the solvent may be heated.

[0955] The third heater 346 can be located anywhere along the solvent capillary 302, such as proximate the end 305 that is located away from the swab surface 310, so that the solvent can be heated before it is surrounded by the sheath gas tube 312. The third heater 346 can include a wire heater (e.g., a tungsten coil) and / or can be configured to heat the solvent capillary 302 and / or the solvent to at least 50°C, 100°C, 200°C, 300°C, 400°C, 500°C, 600°C, 700°C, or 800°C.

[0956] The fourth heater 348 may be arranged and adapted to heat the swab surface 310 so that the sample 311 and / or the swab surface 310 may be heated. The fourth heater 348 may be located below a portion of the swab surface 310 that is arranged and adapted to hold or accommodate the sample 311. The fourth heater 348 may include a wire heater (e.g., a tungsten coil) and / or may be configured to heat the sample 311 and / or the swab surface 310 and / or the solvent to at least 50°C, 100°C, 200°C, 300°C, 400°C, 500°C, 600°C, 700°C, or 800°C.

[0957] The swab itself may be heated to heat the sample 311 located on the swab. For example, the fourth heater 348 may be a wire heater located within the swab and may be arranged and adapted to heat the end of the swab configured to fix and / or retain the biological sample for analysis.

[0958] Heated ion inlet transfer capillaries (e.g., Fig.25A The effect of the transfer capillary or transfer device 330) shown in FIG.

[0959] The ion transfer capillary or transfer device 330 was heated in the range of 100 to 490° C. using a nickel wire heater. Pork liver slices were used and the intensities of selected fatty acids and phospholipids were compared. It was found that the inlet capillary heating had some effect on the intensity of fatty acids when using the Xevo (RTM) mass spectrometer, but had no effect when using the Synapt (RTM) mass spectrometer. However, the intensity of the monitored phospholipids increased by almost two orders of magnitude.

[0960] Fig.25B -E shows the effect of inlet capillary heating on the absolute intensity. Fig.25B and Fig.25D Involving WatersSynapt G2-Si (RTM) mass spectrometer, Fig.25C and Fig.25EA Waters Xevo G2-XS (RTM) mass spectrometer is involved. The average intensity of selected fatty acids (FA), phosphatidyl ethanolamines (PE) and the most abundant phosphatidylinositol (PI) in pig liver sections is shown.

[0961] from Fig.25B -E It is apparent that increasing the temperature of the ion transfer capillary or transfer device 330 can increase the observed phospholipid intensity by almost two orders of magnitude.

[0962] Open Ionization Analysis of Biopsy Samples

[0963] Various other embodiments relate to analyzing biopsy samples using open ionization mass spectrometry, and in particular rapid evaporative ionization mass spectrometry ("REIMS") and desorption electrospray ionization ("DESI") mass spectrometry.

[0964] A biopsy is a sample of cells or tissue, usually removed from a living subject, and used to determine the presence or extent of disease.

[0965] Biopsy samples can be provided using, for example, (i) fine needle aspiration biopsy, in which a small amount of tissue is aspirated using a fine needle attached to a syringe; (ii) core needle biopsy, in which a cylinder (or core) of tissue is extracted using a hollow needle; and (iii) surgical (or incisional) biopsy, in which tissue is surgically cut, for example using a scalpel. A biopsy may optionally be excisional, excised, or retrieved from a surgical resection. A biopsy specimen comprises cells and may optionally be a tissue specimen, e.g., comprising or consisting of diseased tissue and / or non-diseased tissue.

[0966] Fig.26 A typical core needle biopsy is illustrated in which a cylinder (or core) 241 of tissue, such as including a tumor 242 or other area of ​​medical interest, is extracted using a biopsy needle 240 comprising a hollow needle.

[0967] Conventional histopathological analysis of biopsy samples involves sending the sample to a specialized laboratory where it is prepared for examination and examined under a microscope. Thus, conventional biopsy analysis involves a time-consuming and costly workflow.

[0968] According to various embodiments, the biopsy sample is analyzed using open ionization mass spectrometry, and in particular rapid evaporation ionization mass spectrometry ("REIMS") and / or desorption electrospray ionization mass spectrometry ("DESI-MS").

[0969] Applicants have discovered that open ionization mass spectrometry, and in particular rapid evaporation ionization mass spectrometry ("REIMS") analysis and desorption electrospray ionization ("DESI") mass spectrometry of biopsy samples can yield pathologically relevant information. In addition, open ionization mass spectrometry, and in particular rapid evaporation ionization mass spectrometry ("REIMS") analysis and desorption electrospray ionization ("DESI") mass spectrometry can provide rapid, real-time, point-of-care information in a particularly convenient and effective manner.

[0970] A particularly useful feature of rapid vaporization ionization mass spectrometry ("REIMS") and desorption electrospray ionization ("DESI") mass spectrometry analysis of biopsy samples is the relative ease with which spatially resolved data can be acquired using these techniques.

[0971] In particular, spatially resolved information from a biopsy sample can be used to determine the presence, location, and / or extent or size of diseased tissue, such as tumor and / or necrotic tissue, in the biopsy sample.

[0972] In this regard, the applicant has found that the methods according to various embodiments can provide more accurate data when compared with, for example, tumor magnetic resonance imaging ("MRI"). In one particular embodiment, this can be used, for example, to accurately determine whether the tumor location (e.g., proximity to a vein or other situation) makes the tumor inoperable before operation. In another embodiment, this can be used to determine how much diseased tissue, such as necrotic tissue or cancerous tissue, needs to be removed or can be safely removed.

[0973] Spatially resolved information from a biopsy sample can additionally or alternatively be used to determine the aggressiveness of a tumor and / or the likelihood that a tumor will respond to a particular treatment or the extent to which it will respond. For example, by analyzing a portion of a biopsy sample adjacent to a tumor, information about the body's natural response to the tumor can be determined, for example by identifying relevant biomarkers. This information can be directly correlated to the aggressiveness of a tumor and / or the likelihood that a tumor will respond to a particular treatment or the extent to which it will respond.

[0974] The analysis may optionally be used to identify disease margins.Disease margins may optionally be analyzed, for example, by analyzing the concentration of specific cell types in the target area, such as diseased cell types, cancerous cell types, and / or necrotic cell types.

[0975] According to various other embodiments, information obtained from analysis of biopsy samples may be used in real time to determine the course of action, for example, while the patient is under anesthesia and / or during surgery.

[0976] Biopsy Analyzer

[0977] According to one embodiment, a biopsy needle may be inserted into a patient and the resulting biopsy sample (eg, a biopsy core) may then be inserted into a dedicated channel of a mass spectrometer and / or an ion mobility spectrometer.

[0978] The biopsy sample may be inserted into the channel of a mass spectrometer and / or ion mobility spectrometer together with the biopsy needle itself (ie, the biopsy needle used to extract the sample) or together with some other device for fixing and / or supporting the biopsy sample.

[0979] A mass spectrometer and / or an ion mobility spectrometer may then analyse the sample.A mass spectrometer and / or an ion mobility spectrometer may, for example, analyse the biopsy needle or sample longitudinally, ie along the length of the biopsy needle or sample.

[0980] According to various embodiments, a one-dimensional (eg, longitudinal) mass spectrometry image or ion image of a biopsy sample may be provided.

[0981] In these embodiments, rapid evaporation ionization mass spectrometry ("REIMS") and desorption electrospray ionization mass spectrometry ("DESI-MS") are both particularly suitable for analyzing biopsy samples because they can be easily used to provide spatially resolved mass spectral data, i.e., to provide a one-dimensional (longitudinal) mass spectral image or ion image of a biopsy sample or core.

[0982] In various embodiments, mass spectrometry analysis, including diagnostics, can be performed in real time and / or at the point of care ("POC"). This represents a rapid and convenient method for analyzing biopsy samples.

[0983] If any problems arise from the quality analysis of the biopsy needle, a second biopsy can be performed immediately, thereby advantageously saving the patient from having to undergo a second biopsy procedure at a later date.

[0984] These embodiments are particularly relevant to, for example, liver and kidney biopsies. In these cases, current medical practice is to insert a catheter through the neck and use a snare to (hopefully) capture a piece of the liver or kidney. Therefore, the various embodiments represent an improved approach to liver and kidney biopsies.

[0985] Biopsy needle

[0986] According to various embodiments, a biopsy needle can be provided that is arranged to simultaneously collect two (or more) separate samples or portions (e.g., two (or more) biopsy cores or cylinders) of a biopsy sample. The biopsy needle can be configured so that when it is inserted into tissue, two (or more) separate tissue samples or portions (e.g., two (or more) biopsy cores or cylinders) are produced.

[0987] The biopsy needle may include, for example, a needle comprising a first hollow tube or barrel and a second hollow tube or barrel. The first and second hollow tubes or barrels may be connected together, for example, along a portion, a majority or the entirety of the axial length of the first and / or second hollow tube or barrel.

[0988] The two separate tissue samples or portions (e.g., two biopsy cores or cylinders) produced by the biopsy needle can include adjacent tissue portions. For example, two biopsy cores or cylinders can be produced where a first biopsy core or cylinder includes tissue that is initially adjacent to and / or connected to tissue of a second biopsy core or cylinder, i.e., along a portion, a majority, or the entirety of the axial length of the first and / or second biopsy cores or cylinders.

[0989] According to one embodiment, one of the two samples may be sent for conventional histopathology analysis and the second sample may (e.g., substantially immediately thereafter) be subjected to open ionization mass spectrometry, and in particular rapid evaporative ionization mass spectrometry ("REIMS") analysis and / or desorption electrospray ionization ("DESI") analysis.

[0990] Advantageously, open ionization mass spectrometry, and in particular rapid evaporation ionization mass spectrometry ("REIMS") and / or desorption electrospray ionization ("DESI") analysis can provide additional information relative to that provided by histopathology. Specifically, rapid evaporation ionization mass spectrometry ("REIMS") can potentially identify the underlying disease, whereas histopathology can only provide information related to cell chemistry.

[0991] An exemplary application according to one embodiment is the diagnosis of non-alcoholic fatty liver disease (NAFLD).

[0992] Rapid Evaporative Ionization Mass Spectrometry ("REIMS") Biopsy

[0993] According to various embodiments, a biopsy needle may be provided that includes a device configured to generate an aerosol, mist or vapor from a target. The device may include or form part of an open ion or ionization source, or the device may generate an aerosol, mist or vapor for subsequent ionization via an open ion or ionization source or other ionization source.

[0994] According to a particular embodiment, a biopsy needle may be provided that includes one or more electrodes, and in particular one or more rapid vapor ionization mass spectrometry ("REIMS") electrodes.

[0995] According to another embodiment, a biopsy needle may be provided that includes a laser ionization ion source, such as described above. According to another embodiment, a biopsy needle may be provided that includes an ultrasound ablation ion source, such as described above.

[0996] The device or electrodes may be activated while a biopsy needle is inserted into a patient to provide mass spectrometry data associated with a tissue sample, for example, from the biopsy needle.

[0997] Patients using needle biopsies including open ion sources, such as rapid evaporative ionization mass spectrometry ("REIMS") electrodes will require anesthetic, but the various embodiments have multiple applications, including real-time diagnosis and analysis of diseases, such as any of the diseases mentioned elsewhere in this document, such as hepatitis, cirrhosis, and cancerous tissue.

[0998] According to another embodiment, the device or electrodes may be activated after the biopsy needle containing the biopsy sample (core) has been removed from the patient.

[0999] According to various embodiments, the biopsy needle may include a biopsy needle arranged to simultaneously collect two (or more) independent samples or portions (e.g., two (or more) biopsy cores or cylinders) of a biopsy sample, such as described above. In these embodiments, the device or electrode may be configured to generate an aerosol, smoke, or vapor from one or both (e.g., selectively) of the biopsy samples. In the case where the device or electrode is configured to generate an aerosol, smoke, or vapor from only one of the biopsy samples, the other biopsy sample may be sent for conventional histopathological analysis, such as described above.

[1000] Fig. 27 An embodiment is shown in which a biopsy needle 250 is provided with an electrode 251, which may be at the distal end of the needle 250. Fig.26 In the embodiment illustrated in FIG. 2 , a biopsy needle has been inserted into a tumor 252 present within an internal organ 253 of a patient.

[1001] The electrode 251 is connected to an RF voltage generator (not shown). When an RF voltage is applied to the electrode 251, the electrode 251 acts as an electrosurgical tool and effectively cuts the tumor 252. This causes the generation of surgical smoke or aerosol.

[1002] Surgical smoke or aerosol is drawn into tube 254, for example, through one or more perforations or suction ports (not shown), and is passed along the length of tube 254 through atmospheric pressure inlet 256 to the vacuum chamber of mass spectrometer and / or ion mobility spectrometer 255. A venturi pump may be used to assist in the suction of surgical smoke or aerosol, for example, driven by standard medical air or nitrogen.

[1003] The surgical smoke or aerosol can then be ionized, for example by impacting a collision surface which can be heated.

[1004] The resulting analyte ions may then be analyzed, for example mass analyzed and / or subjected to ion mobility analysis or separation, and real-time information related to the tissue or tumor 252 may be provided to the user.

[1005] The mass spectrometer and / or ion mobility spectrometer 255 may include a modified atmospheric pressure interface 256, which includes a collision surface that can be placed along and near the central axis of the large opening of the StepWave (RTM) ion guide. As will be appreciated by those of ordinary skill in the art, the StepWave (RTM) ion guide comprises two ion tunnel ion guides connected together. Each ion guide comprises a plurality of rings or other electrodes, wherein ions pass through the central orifice provided by the rings or other electrodes. A transient DC voltage or potential is applied to the electrodes. The StepWave (RTM) ion guide is based on stacked ring ion guide technology and is designed to maximize ion transmission from an ion source to a mass analyzer or filter. The device allows active removal of neutral contaminants, thereby enhancing the overall signal-to-noise ratio. The design can effectively capture the diffuse ion cloud entering the first lower platform, and then focus the ion cloud into the upper ion guide for transfer to the mass analyzer or filter.

[1006] The collision surface that may be located within the vacuum chamber of the mass spectrometer and / or ion mobility spectrometer 255 helps to effectively fragment the molecular clusters formed in the free jet region of the atmospheric pressure interface 256 because the gas entering the vacuum chamber undergoes adiabatic expansion, causing a temperature drop. The surface-induced dissociation of the supramolecular clusters improves signal intensity and also alleviates problems associated with ion optics contamination.

[1007] The biopsy needle can be used in any body part or organ, such as the lungs, liver, and breast.

[1008] The biopsy needle may comprise a monopolar device, and a relatively large pad that acts as a return electrode may be placed under the patient so that current flows from electrode 251 through the patient to the return electrode. Alternatively, the biopsy needle may comprise a bipolar device, e.g., comprising two electrodes, so that current does not flow through the patient's body. A bipolar biopsy needle may be used, e.g., in situations where it is undesirable for current to flow through surrounding tissue.

[1009] While monopolar or bipolar electrode arrangements are particularly advantageous, other embodiments are also contemplated in which the biopsy needle may comprise a multi-phase or 3-phase device and may comprise, for example, three or more independent electrodes.

[1010] The substrate can be added or mixed with the surgical smoke or aerosol before the surgical smoke or aerosol hits the collision surface. The substrate can include a solvent for the surgical smoke or aerosol, and can include an organic solvent and / or a volatile compound. The substrate can include polar molecules, water, one or more alcohols, methanol, ethanol, isopropanol, acetone or acetonitrile. The use of isopropanol is particularly advantageous.

[1011] The added matrix may additionally or alternatively contain lock masses, lock mobilities or calibration compounds.

[1012] A particular advantage of adding a matrix is ​​that the analyte is dissolved in the matrix, eliminating the intermolecular bonds between the analyte molecules. Thus, when the dissolved analyte collides with the collision surface, the dissolved analyte will fragment into droplets and any given droplet may contain fewer analyte molecules than if the matrix were not present. This in turn results in more efficient ion generation as the matrix in each droplet evaporates.

[1013] According to various embodiments, data obtained by rapid vapor ionization mass spectrometry (“REIMS”) analysis of a biopsy sample within biopsy needle 250 may be used to ensure that the biopsy needle has properly sampled a portion of tissue of interest, such as a portion of tumor 252, thereby ensuring, for example, that the needle has been inserted to the proper depth within the patient's body and then removing biopsy needle 250 from the patient.

[1014] Additionally or alternatively, data obtained from rapid vapor ionization mass spectrometry (“REIMS”) analysis of a biopsy sample within biopsy needle 250 may be used to make a diagnosis or characterization of tissue or tumor 252 , for example.

[1015] Data obtained from REI rapid vapor ionization mass spectrometry ("REIMS") analysis of a biopsy sample within biopsy needle 250 may be used by itself, or may be used to supplement subsequent analysis (eg, histopathology analysis) of a (eg, remaining) biopsy sample (core).

[1016] Use biopsy data to improve surgical findings obtained during subsequent surgical procedures using open ionization surgical tools Subsequent real-time analysis of data

[1017] According to various embodiments, pre-operative characterization of biopsy data may be used to refine a surgical library, which may then be subsequently interrogated or used by an open ionization surgical tool, such as during a subsequent surgical procedure.

[1018] For example, subsequent mass spectrometry data obtained during the surgical procedure may be acquired and / or analyzed in an improved or optimized manner based on the results of the biopsy analysis.

[1019] For example, if a biopsy reveals that a patient has cirrhosis of the liver, a subsequent surgery may be performed on a portion of the liver, wherein the mass spectrometry analysis of the sample liver tissue is optimized to distinguish between healthy liver tissue and cirrhotic liver tissue. This applies, mutatis mutandis, to any other suitable disease, such as those mentioned elsewhere herein, particularly cancer, necrosis, etc.

[1020] It is contemplated that a pre-existing surgical mass spectrometry (and / or ion mobility) database, such as a dedicated surgical mass spectrometry (and / or ion mobility) database, may be provided, and prior to performing a surgical procedure, the appropriate surgical database may be pre-loaded into, for example, a mass spectrometer (and / or ion mobility analyzer) connected to an open ionization surgical tool. The database may then be improved or optimized using biopsy data according to various embodiments.

[1021] The optimized operating parameters of an open ionization surgical tool may depend on previously acquired data such as biopsy data. Data is programmed or set

[1022] According to various embodiments, one or more operating parameters of an open ionization surgical or diagnostic tool may be arranged to be varied or otherwise optimized during a surgical or diagnostic procedure. This may be done based on previously acquired data such as biopsy data.

[1023] For example, according to one embodiment, energy dissipated into surrounding tissue may be arranged to be reduced when a surgical or diagnostic device is in proximity to a living organ.

[1024] According to various embodiments, one or more operating parameters of an open ionization surgical tool may be set based on pre-acquired data, such as biopsy data.

[1025] For example, one or more operating parameters of an open ionization surgical tool may be set based on the type or grade of cancerous tissue identified during a biopsy or based on the nature of diseased tissue identified during a biopsy.

[1026] In these embodiments, the cancerous biological tissue or tumor can include, for example: (i) grade I, grade II, grade III, or grade IV cancer tissue; (ii) metastatic cancer tissue; (iii) mixed grade cancer tissue; or (iv) subgrade cancer tissue.

[1027] Different operating parameters may be used depending on the type of tissue being manipulated, such as whether healthy tissue, significantly diseased (eg, cancerous) tissue, or tissue at the edge of disease (eg, cancer) is being manipulated.

[1028] According to various embodiments, the biopsy data may include spatial information and thus tissue changes as a function of depth within an organ may be determined. Thus, for example, various operating parameters of an open ionization surgical tool may be set using previously acquired biopsy data as the surgical tool is moved deeper into the organ.

[1029] Furthermore, various ionization parameters may be varied, for example, as an open ionization surgical tool is moved deeper into an organ.

[1030] For example, when an open ionization surgical tool is initially inserted into an organ, one or more ionization parameters (e.g., the composition of the substrate added to the aerosol, smoke or vapor released by the tissue, the temperature of the ionization collision surface, the voltage applied to the ionization collision surface, etc.) can be optimized for the surgical conditions encountered when the open ionization surgical tool is initially inserted into the organ (e.g., initial blood loss, tissue composition, etc.). As the open ionization surgical tool moves deeper into the organ, the optimal ionization parameters of the surgical tool can change, reflecting, for example, different blood levels and different tissue compositions. Therefore, one or more ionization parameters (e.g., the composition of the substrate added to the aerosol, smoke or vapor released by the tissue, the temperature of the ionization collision surface, the voltage applied to the ionization collision surface, etc.) can be arranged to also change to match the changed surgical conditions.

[1031] Many different embodiments are contemplated in which various operating parameters of a surgical device, such as an open ionization ion source (e.g., a rapid evaporative ionization mass spectrometry ("REIMS") ion source), can be varied based on pre-acquired data, such as biopsy data.

[1032] Analyze sample spectra

[1033] A list of analytical techniques that may be used according to various embodiments is given in the following table:

[1034]

[1035]

[1036] The above analysis methods can also be used in combination, such as PCA-LDA, PCA-MMC, PLS-LDA, etc.

[1037] Analyzing sample spectra can include unsupervised analysis for dimensionality reduction followed by supervised analysis for classification.

[1038] A number of different analytical techniques will now be described in more detail by way of example.

[1039] Multivariate Analysis - Developing Classification Models

[1040] The method of constructing a classification model using multivariate analysis of multiple reference sample spectra will now be described by way of example.

[1041] Fig.28 A method 1500 of building a classification model using multivariate analysis is shown. In this example, the method comprises a step 1502 of obtaining multiple sets of intensity values ​​of a reference sample spectrogram. The method then comprises an unsupervised principal component analysis (PCA) step 1504, followed by a supervised linear discriminant analysis (LDA) step 1506. This method may be referred to herein as PCA-LDA. Other multivariate analysis methods may be used, such as PCA-MMC. The PCA-LDA model is then output to, for example, a memory in step 1508.

[1042] Multivariate analysis like this can provide a classification model that can classify samples using one or more sample spectra obtained from samples such as aerosol, smoke or vapor samples, biological samples, etc. Multivariate analysis will now be described in more detail with reference to a simple example.

[1043] Fig.29 A set of reference sample spectra obtained from two classes of known reference samples is shown. The classes may be any one or more of the target classes described herein. However, for simplicity, in this example, the two classes are referred to as the left class and the right class.

[1044] Each of the reference sample spectra has been preprocessed to obtain a set of three reference peak intensity values ​​for the corresponding mass-to-charge ratios in the reference sample spectra. Although only three reference peak intensity values ​​are shown, it should be understood that more reference peak intensity values ​​(e.g., about 100 reference peak intensity values) can be obtained for the corresponding number of mass-to-charge ratios in each reference sample spectrum. In other embodiments, the reference peak intensity values ​​may correspond to: mass; mass-to-charge ratio; ion mobility (drift time); and / or operating parameters.

[1045] Fig.30 A multivariate space having three dimensions defined by intensity axes is shown. Each dimension or intensity axis corresponds to a peak intensity at a specific mass-to-charge ratio. It should also be appreciated that there may be more dimensions or intensity axes (e.g., about 100 dimensions or intensity axes) in the multivariate space. The multivariate space comprises a plurality of reference points, wherein each reference point corresponds to a reference sample spectrum, i.e., the peak intensity value of each reference sample spectrum provides the coordinates of the reference point in the multivariate space.

[1046] This set of reference sample spectra can be represented by a reference matrix D, which has rows associated with corresponding reference sample spectra, columns associated with corresponding mass-to-charge ratios, and the matrix elements are peak intensity values ​​of corresponding mass-to-charge ratios of the corresponding reference sample spectra.

[1047] In many cases, the large number of dimensions in the multivariate space and matrix D can make the reference sample spectra difficult to classify. Therefore, PCA can be performed on matrix D to calculate a PCA model that defines a PCA space with a reduced number of one or more dimensions defined by the principal component axes. The principal components can be selected as those components that contain or "explain" the maximum variance of matrix D and cumulatively explain a threshold amount of variance of matrix D.

[1048] Fig.31 It is shown how the cumulative variance may increase with the number of principal components n in a PCA model. The threshold amount of variance may be selected as desired.

[1049] The PCA model can be calculated from the matrix D using a nonlinear iterative partial least squares (NIPALS) algorithm or a singular value decomposition method, the details of which are known to the skilled person and are therefore not described in detail herein. Other methods of calculating the PCA model can be used.

[1050] The resulting PCA model can be defined by the PCA score matrix S and the PCA loading matrix L. PCA also produces an error matrix E, which contains the variance that cannot be explained by the PCA model. The relationship between D, S, L, and E can be:

[1051] D=SL T +E (1)

[1052] Fig.32 Shows Fig.29 and Fig.30 The resulting PCA space of the reference sample spectra. In this example, the PCA model has two principal components PC 0 and PC 1 , so the PCA space has two dimensions defined by the two principal component axes. However, a smaller or larger number of principal components can be included in the PCA model as desired. It is usually desirable that the number of principal components is at least one less than the number of dimensions in the multivariate space.

[1053] The PCA space contains multiple transformed reference points or PCA scores, where each transformed reference point or PCA score corresponds to Fig.29 The reference sample spectrum and therefore corresponds to Fig.30 reference point.

[1054] like Fig.32 As shown in , the reduction in the dimensionality of the PCA space makes it easier to classify the reference sample spectra into two classes. At this stage, any outliers can also be identified and removed from the classification model.

[1055] Further supervised multivariate analysis, such as multi-class LDA or maximum margin criterion (MMC), can then be performed in the PCA space to define the classes and optionally further reduce the dimensionality.

[1056] As the skilled person will appreciate, multi-class LDA attempts to maximize the ratio of the variance between classes to the variance within a class (ie, to maximize the possible distance between the most compact possible classes). The details of LDA are known to the skilled person and will not be described in detail herein.

[1057] The resulting PCA-LDA model can be defined by the transformation matrix U, which can be derived from the PCA score matrix S and the class assignments of each transformed spectra contained therein by solving the generalized eigenvalue problem.

[1058] Then the transformation of the score S from the original PCA space to the new LDA space can be obtained as follows:

[1059] Z=SU (2)

[1060] where the matrix Z contains the scores transformed into LDA space.

[1061] Fig.33 shows a PCA-LDA space with a single dimension or axis, where LDA is Fig.32 The PCA space is carried out. Fig.33 As shown in , the LDA space contains multiple other transformed reference points or PCA-LDA scores, where each other transformed reference point corresponds to Fig.32 The transformed reference point or PCA score.

[1062] In this example, further reduction of the dimensionality of the PCA-LDA space makes it even easier to classify the reference sample spectra into two classes. Each class in the PCA-LDA model can be defined by its class mean and covariance matrix transformed in the PCA-LDA space or one or more hyperplanes (including points, lines, planes or higher order hyperplanes) or hypersurfaces or Voronoi units.

[1063] The PCA loading matrix L, the LDA matrix U and the transformed class means and covariance matrices or hyperplanes or hypersurfaces or Voronoi cells may be output to a database for subsequent use in classifying aerosol, smoke or vapor samples.

[1064] The transformed covariance matrix V' of category g in LDA space g It can be given by:

[1065] V' g =U T V g U (3)

[1066] Where V g is the class covariance matrix in PCA space.

[1067] The transformed category average position z of category g g This can be given by:

[1068] s g U=z g (4)

[1069] where s g is the class average position in PCA space.

[1070] Multivariate Analysis - Using Classification Models

[1071] A method for classifying samples (such as aerosol, smoke or steam samples) using a classification model will now be described by way of example.

[1072] Fig.34 A method 2100 for using a classification model is shown. In this example, the method comprises a step 2102 of obtaining a set of intensity values ​​for a sample spectrogram. The method then comprises a step 2104 of projecting the set of intensity values ​​for the sample spectrogram into a PCA-LDA model space. Other classification model spaces may be used, such as PCA-MMC. The sample spectrogram is then classified based on the projected position in step 2106, and the classification is then output in step 2108.

[1073] The classification of samples (eg, aerosol, smoke or vapor samples) will now be described in more detail with reference to the simple PCA-LDA model described above.

[1074] Fig.35 A sample spectrum obtained from an unknown aerosol, smoke or vapor sample is shown. The sample spectrum has been preprocessed to obtain a set of three sample peak intensity values ​​for corresponding mass-to-charge ratios. As mentioned above, although only three sample peak intensity values ​​are shown, it should be understood that more sample peak intensity values ​​(e.g., about 100 sample peak intensity values) can be obtained at more corresponding mass-to-charge ratios of the sample spectrum. In addition, as mentioned above, in other embodiments, the sample peak intensity values ​​can correspond to: mass; mass-to-charge ratio; ion mobility (drift time); and / or operating parameters.

[1075] The sample spectrum can be represented by the sample vector d X Represented by , where the vector elements are the peak intensity values ​​of the corresponding mass-to-charge ratio. The transformed PCA vector s of the sample spectrum can be obtained as follows X :

[1076] d X L=s X(5)

[1077] Then the transformed PCA-LDA vector z of the sample spectrum can be obtained as follows X :

[1078] s X U=z X (6)

[1079] Fig.36 Again, it shows Fig.33 PCA-LDA space. However, Fig.36 The PCA-LDA space further contains Fig.35 The peak intensity values ​​of the sample spectra are derived corresponding to the transformed PCA-LDA vector z X The projected sample points.

[1080] In this example, the projected sample point is on one side of the hyperplane between the categories, which side refers to the right category, so the sample (aerosol, smoke or steam) can be classified as belonging to the right category.

[1081] Alternatively, one can use the Mahalanobis distance from the cluster center in LDA space, where point z x The Mahalanobis distance to the center of class g can be obtained by the square root of:

[1082] (z x -z g ) T (V' g ) -1 (z x -z g ) (8)

[1083] And the data vector d x Assigned to the class with the smallest distance.

[1084] In addition, by treating each class as a multivariate Gaussian, the probability of a data vector being a member of each class can be calculated.

[1085] Library-based analysis - developing a classification library

[1086] A method for constructing a classification library using multiple input reference sample spectra will now be described by way of example.

[1087] Fig.37A method 2400 for constructing a classification library is shown. In this example, the method includes a step 2402 of obtaining a plurality of input reference sample spectra and a step 2404 of deriving metadata based on the plurality of input reference sample spectra for each sample class. The method then includes a step 2404 of storing the metadata for each sample class as a separate library entry. The classification library is then output to, for example, an electronic storage at step 2406.

[1088] Classification libraries like this are able to classify samples using one or more sample spectra obtained from the samples (eg aerosol, smoke or vapour samples).The library based analysis will now be described in more detail with reference to examples.

[1089] In this example, each entry of the classification library is created based on multiple preprocessed reference sample spectra representing one class. In this example, the reference sample spectra of one class are preprocessed according to the following procedure:

[1090] First, a re-binning process is performed. In this embodiment, the data is resampled to a logarithmic grid with the following abscissas:

[1091]

[1092] Where N chan is the selected value, represents the nearest integer less than x. In one example, N chan Serves 2 12 or 4096.

[1093] The background subtraction process is then performed. In this embodiment, a cubic spline with k knots is then constructed so that p% of the data between each pair of knots is below the curve. This curve is then subtracted from the data. In one example, k is 32. In one example, p is 5.

[1094] A constant value corresponding to the q% quantile of the data whose intensity is subtracted is then subtracted from each intensity. Positive and negative values ​​are retained. In one example, q is 45.

[1095] Then the data is normalized. In this example, the data is normalized to have an average value of In one instance,

[1096] The entries in the library are then generated by each N chan The metadata of a point is composed of the following form: median spectrum value μ i and deviation value D i .

[1097] The likelihood of the ith channel is given by:

[1098]

[1099] Where 1 / 2≤C<∞ and where Γ(C) is the gamma function.

[1100] The above equation is a generalised Cauchy distribution. When C = 1, it simplifies to a standard Cauchy distribution, and as C → ∞, it becomes a Gaussian (normal) distribution. Parameter D i Controls the width of the distribution (in the Gaussian limit, D i =σ i is just the standard deviation), while a global value C controls the size of the tail.

[1101] In one example, C is 3 / 2, which is between the Cauchy and Gaussian, so the likelihood becomes:

[1102]

[1103] For each library entry, the parameter μ i is set to the median of the list of values ​​in the ith channel of the input reference sample spectrum, and the deviation D i is taken as the interquartile range of these values ​​divided by √ 2. This choice ensures that the likelihood of the ith channel has the same interquartile range as the input data, where the quantiles are used to provide some protection against outliers.

[1104] Library-based analysis - using the classification library

[1105] A method of classifying a sample (eg, an aerosol, smoke, or vapor sample) using a classification library will now be described by way of example.

[1106] Fig.38 A method 2500 for using a classification library is shown. In this example, the method includes a step 2502 of obtaining a set of multiple sample spectra. The method then includes a step 2504 of calculating a probability or classification score for the set of multiple sample spectra for each sample class using metadata for the class entries in the classification library. The sample spectra are then classified in step 2506, and the classification is then output in step 2508.

[1107] The classification of samples (eg, aerosol, smoke or vapor samples) will now be described in more detail with reference to the classification library described above.

[1108] In this example, the unknown sample spectrum y is the median spectrum of a set of multiple sample spectra. Obtaining the median spectrum y can prevent outlier data from appearing in each channel.

[1109] The likelihood L of a given library entry s for the input data is then given by s :

[1110]

[1111] where μ i and D i are the median value and deviation value of the library of channel i respectively. For numerical safety, the likelihood L s It can be calculated by log likelihood.

[1112] Then for all candidate categories 's', the likelihood L s Normalization is performed to obtain probabilities, assuming that the prior probabilities of these classes are uniform. The classes are given by The probability of getting:

[1113]

[1114] The exponent (1 / F) can soften probabilities that might otherwise be too certain. In one example, F = 100. These probabilities can be expressed as percentages, for example in a user interface.

[1115] Alternatively, the RMS classification score R s The same median sample and derived values ​​from the library can be used to calculate:

[1116]

[1117] Score R s The normalization is also performed for all candidate classes 'S'.

[1118] The sample (eg, an aerosol, smoke, or vapor sample) may then be classified as belonging to the class with the highest probability and / or the highest RMS classification score.

[1119] Analytical methods, such as medications, surgical and diagnostic methods, and non-medical methods

[1120] Various different embodiments are contemplated. According to some embodiments, the methods disclosed above may be performed on tissue in vivo, ex vivo, or in vitro. The tissue may comprise human or non-human animal tissue. Embodiments are contemplated where the target may comprise biological tissue, bacterial or fungal colonies, or more generally, organic targets, such as plastics.

[1121] The following embodiments are covered, wherein the analyte ions produced by the open ionization ion source are subsequently subjected to: (i) mass analysis by a mass analyzer or filter, such as a quadrupole mass analyzer or a time-of-flight mass analyzer; (ii) ion mobility analysis (IMS) and / or differential ion mobility analysis (DMA) and / or field asymmetric ion mobility spectrometry (FAIMS) analysis; and / or (iii) the following combination: first (or vice versa) ion mobility analysis (IMS) and / or differential ion mobility analysis (DMA) and / or field asymmetric ion mobility spectrometry (FAIMS) analysis is performed, and secondly (or vice versa) mass analysis is then performed by a mass analyzer or filter, such as a quadrupole mass analyzer or a time-of-flight mass analyzer. Each embodiment also relates to an ion mobility spectrometer and / or a mass analyzer and an ion mobility spectrometry method and / or a mass analysis method. The ion mobility analysis can be performed before mass-to-charge ratio analysis, or vice versa.

[1122] Mention mass analysis, mass analyzer, mass analysis, mass spectral data, mass spectrometer and other related terms from time to time in the application, refer to the equipment and method for measuring the mass or mass-to-charge ratio of analyte ions.It should be understood that it is also conceivable that the present invention can be extended to ion mobility analysis, ion mobility analyzer, ion mobility analysis, ion mobility data, ion mobility spectrometer, ion mobility separator and other related terms, refer to the equipment and method for measuring the ion mobility, differential ion mobility, collision cross section or interaction cross section of analyte ions.It should also be understood that such embodiments are contained, wherein analyte ions can experience the combination of ion mobility analysis and mass analysis, i.e., measure the ion mobility, differential ion mobility, collision cross section or interaction cross section of (a) analyte ions and the mass-to-charge ratio of (b) analyte ions.Therefore, hybrid ion mobility-mass spectrum (IMS-MS) and mass spectrum-ion mobility (MS-IMS) embodiments are contained, wherein the ion mobility and the mass-to-charge ratio of the analyte ions such as produced by an open ionization ion source are measured. Ion mobility analysis can be performed before mass-to-charge ratio analysis, or vice versa. It should also be understood that embodiments are encompassed in which reference to mass spectral data and a database comprising mass spectral data is also understood to encompass ion mobility data and differential ion mobility data, etc., and a database comprising ion mobility data and differential ion mobility data, etc. (alone or in combination with mass spectral data).

[1123] Covers a wide range of surgical, therapeutic, medical, and diagnostic methods.

[1124] However, other embodiments involving non-surgical and non-therapeutic mass spectrometry methods that are not performed on in vivo tissue are also contemplated.Other related embodiments are contemplated that are performed in an ex vivo manner such that they are performed outside of the human or animal body.

[1125] Other embodiments are contemplated in which the methods are performed on a non-living human or animal, such as as part of an autopsy procedure.

[1126] According to some embodiments, the methods disclosed above may be performed on a "target", which may optionally be a subject or a specimen obtained from a subject, such as biological material on a swab or a biopsy specimen.

[1127] According to various embodiments, the target may contain biological matter or organic matter (including plastics). According to various embodiments, the target may contain one or more bacterial colonies and / or one or more fungal colonies.

[1128] A "subject" may be a human or non-human animal. A subject may be alive or dead. If the method is performed on a living subject, it may be referred to as an in vivo method. If the method is performed on a specimen, it may be referred to as an in vitro or ex vivo method.

[1129] Optionally, the non-human animal can be a mammal, optionally selected from, for example, any livestock, domestic animal or laboratory animal, such as mice, guinea pigs, hamsters, rats, goats, pigs, cats, dogs, sheep, rabbits, cows, horses and / or monkeys. Optionally, it can be an insect, bird or fish, such ...

Claims

1. A method, include: Provide a sample on a swab; Wetting the swab with a first liquid; contacting the wetted swab with one or more electrodes and applying an AC or RF voltage to the one or more electrodes to generate an aerosol, mist or vapor, wherein the one or more electrodes comprise a fast vapor ionization mass spectrometry device; A plurality of analyte ions are generated from the aerosol, mist or vapor by: directing or drawing at least some of the aerosol, mist or vapor into a vacuum chamber of a mass spectrometer and / or an ion mobility spectrometer, mixing the aerosol, mist or vapor with a substrate before causing the aerosol, mist or vapor to impinge on a collision surface, and causing at least some of the aerosol, mist or vapor to impact the collision surface to form the analyte ions; and The analyte ions are analyzed. The method of claim 1 , wherein the first liquid comprises water.

3. The method of claim 1, wherein the analyte ions are analyzed include: (i) performing mass analysis and / or ion mobility analysis on the analyte ions and / or ions derived from the analyte ions; and / or (ii) determining the ion mobility, collision cross section or interaction cross section of the analyte ion and / or ions derived from the analyte ion.

4. A mass spectrometry and / or ion mobility spectrometry method comprising the method according to claim 1.

5. A device, include: one or more electrodes arranged and adapted to contact a sample on a swab moistened with a first liquid so as to generate an aerosol, mist or vapor; means arranged and adapted to apply an AC or RF voltage to said one or more electrodes so as to generate said aerosol, mist or vapor, wherein said one or more electrodes comprises a fast vaporization ionization mass spectrometry device; A mass spectrometer and / or an ion mobility spectrometer having a vacuum chamber and a collision surface; means for directing or drawing at least some of said aerosol, mist or vapor into said vacuum chamber; means for mixing said aerosol, mist or vapor with a matrix prior to causing said aerosol, mist or vapor to impinge upon said collision surface, wherein said apparatus is arranged and adapted to cause at least some of said aerosol, mist or vapor to impinge upon said collision surface so as to produce analyte ions; and An analyzer for analyzing the analyte ions.

6. The apparatus of claim 5, wherein the first liquid comprises water.

7. A mass spectrometer and / or ion mobility spectrometer comprising the device according to claim 5.

8. A method, include: providing biological samples on swabs; directing a spray of charged droplets onto a surface of the swab to produce a plurality of analyte ions, wherein directing the spray of charged droplets onto the swab comprises ionizing the sample using desorption electrospray ionization or desorption electrodynamic focusing ionization; substantially continuously rotating the swab on the axis of the swab using an automatically rotatable swab holding device while directing the spray of charged droplets onto the surface of the swab, and / or substantially continuously translating and / or vibrating the swab substantially along the axial length of the swab while directing the spray of charged droplets onto the surface of the swab; performing mass analysis and / or ion mobility analysis on the analyte ions or ions derived from the analyte ions to obtain mass spectrum data and / or ion mobility data; and The mass spectrometry data and / or ion mobility data are analyzed.

9. The method of claim 8, wherein the biological sample is a mucosal sample.

10. The method of claim 9, wherein the mucosa comprises vaginal mucosa, nasal mucosa, or oral mucosa.

11. The method of claim 8, wherein the sample is provided on the swab in its native or unmodified state.

12. The method of claim 8, wherein the swab is a standard medical swab.

13. The method according to claim 8, further comprising: include: analyzing the biological sample on the swab in a first operating mode; and determining whether the analyte ions include one or more ions of interest; Wherein if it is determined that the analyte ions include the one or more ions of interest, the method further comprises: The sample on the swab is analyzed in a second, different mode of operation.

14. The method of claim 13, wherein the second operating mode comprises directing the spray of charged droplets onto the surface of the swab in a different second operating mode.

15. The method according to claim 14, in: (i) the first operating mode comprises a positive ion operating mode and the second operating mode comprises a negative ion operating mode; or (ii) the first operating mode comprises a negative ion operating mode and the second operating mode comprises a positive ion operating mode.

16. The method according to claim 14, in: The first mode of operation comprises directing a spray of the charged droplets onto the surface of the swab, wherein the charged droplets comprise a first solvent or solvent combination; and The second mode of operation comprises directing a spray of the charged droplets onto the surface of the swab, wherein the charged droplets comprise a second, different solvent or solvent combination.

17. The method of claim 13, wherein the second operating mode comprises generating a plurality of analyte ions from the sample using a different second open ionization analysis method, wherein the different second open ionization analysis method is selected from the group consisting of: (i) rapid evaporation ionization mass spectrometry method; (ii) laser desorption ionization method; (iii) thermal desorption ionization method; (iv) laser diode thermal desorption ionization method; (v) dielectric barrier discharge plasma ionization method; (vi) atmospheric pressure solid analytical probe ionization method; (vii) ultrasound assisted spray ionization method; (vii) i) simple open acoustic spray ionization method; (ix) desorption atmospheric pressure photoionization method; (x) paper spray ionization method; (xi) jet desorption ionization method; (xii) touch spray ionization method; (xiii) laser ablation electrospray ionization method; (xiv) real-time direct analysis ionization method; (xv) probe electrospray ionization method; (xvi) solid probe assisted electrospray ionization method; (xvii) ultrasonic surgical aspirator method; (xviii) focused or unfocused ultrasound ablation method; (xix) microwave resonance method; and (xx) pulsed plasma RF dissection method.

18. The method according to claim 13, in: The first operating mode includes analyzing the analyte ions using first operating parameters; and The second operating mode includes analyzing analyte ions from the sample using second, different operating parameters.

19. The method of claim 13, wherein the first operating mode and / or the second operating mode comprises (i) an operating mode for performing mass analysis and / or ion mobility analysis on the analyte ions or ions derived from the analyte ions; (ii) an operating mode for determining the ion mobility, collision cross section or interaction cross section of the analyte ions or ions derived from the analyte ions; (iii) an operating mode for further fragmenting the analyte ions; and / or (iv) an operating mode for reacting, exciting, fragmenting or fractionating the analyte ions.

20. The method of claim 13, wherein the second operating mode is different include: (i) Cultivate the operating model; (ii) Gene sequencing operation mode; or (iii) matrix-assisted laser desorption ionization mode of operation.

21. The method of claim 13, further comprising selecting and / or optimizing the second operating mode based on information gathered during the first operating mode.

22. The method of claim 8, comprising directing the spray of charged droplets from a nebulizer onto the swab; and The nebulizer was supplied with solvent at a flow rate of 10 μL / min.

23. A device, include: a first device arranged and adapted to direct a spray of charged droplets onto a biological sample provided on a swab so as to produce a plurality of analyte ions, wherein the first device comprises a desorption electrospray ionization ion source or a desorption electrodynamic focusing ionization ion source; an automatically rotatable swab holding device arranged and adapted to substantially continuously rotate the swab on the axis of the swab while directing the spray of charged droplets onto the surface of the swab, and / or a device arranged and adapted to substantially continuously translate and / or vibrate the swab substantially along the axial length of the swab while directing the spray of charged droplets onto the surface of the swab; and A mass analyzer and / or an ion mobility analyzer is used to perform mass analysis and / or ion mobility analysis on the analyte ions or ions derived from the analyte ions to obtain mass spectrum data and / or ion mobility data.

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