System & method for collecting particles in exhaled air

Abstract

A system (100) for collecting particles (P) contained in air (16) exhaled by a subject for analysis comprising: an inertial impactor (10) configured to sort and collect particles (P) contained in air (16) exhaled by a subject according to their mass. The inertial impactor (10) comprises at least one substrate (33, 43, 53) for collecting particles (P), and at least one holding device (24) for securing the at least one substrate (33, 43, 53) in the inertial impactor (10) during the collection of particles (P). The at least one substrate (33, 43, 53) comprises or consists of a material that is at least partially water-soluble at a temperature greater than 0°C and less than 100°C and / or least partially transparent.

Description

[0001] SYSTEM & METHOD FOR COLLECTI NG PARTI CLES I N EXHALED Al R

[0002] Field of I nvention

[0003] The present disclosure pertains to a system and method for collecting a biological sample constituted by particles contained in air exhaled by a subject, such as a human or other animal subject, for analysis, such as biochemical analysis, the analysis of bacterial or virus growth-based assays, or any other physical or chemical analysis or characterisation. The present disclosure further pertains to the use of at least one substrate with properties that simplifies sample preparation and facilitates the quantification, characterisation, and / or other type of analysis of particles impacted onto the at least one substrate in such a system or method.

[0004] Backg rou nd of th e I nvention

[0005] The lungs of a human being or animal have a considerable excess capacity, and their function declines gradually with age, with a noticeable reduction typically occurring only in old age. Even if a pathological process is initiated and certain airway segments cease to function, reduced lung function is not always noticed due to the compensatory function of other intact airways in the lungs. While this redundancy may initially seem advantageous, it means that lung diseases are often detected too late at an irreversible stage. Moreover, when a subject starts to experience problems with breathing, primary healthcare practitioners usually evaluate the condition using indirect measurements that can confirm reduced lung function but that cannot determine the type of pathological process that may be causing the subject’s symptoms. A severe lung disease state is therefore usually not confirmed until a subject’s symptoms get so bad that a more thorough and invasive investigation is motivated. However, as mentioned above, at that stage it may be too late for efficient treatment. This is a major problem with today’s respiratory medicine.

[0006] This problem has been addressed by an innovation entitled PExA which uses non-invasive, yet local sampling technology in which small droplets, or particles, that arise from lung fluid when the small airways of a subject’s lungs close and re-open, are collected from the exhaled air of a subject, and sorted according to their mass and collected using an inertial impactor. Various developmental stages and versions of the PExA technology are described in the international publication numbers WO 2009 / 045163, WO 2019 / 01 1750 and WO 2022 / 223420. PExA technology thereby enables the non-invasive collection of a biological sample precisely from the region where many lung diseases initiate and develop. This non- invasiveness, coupled with the local and well-defined origin of the sample, offers unique opportunities for precise and early detection of lung diseases.

[0007] Since PExA technology involves the collection of material from exhaled air, the technology is often considered to be sim ilar to other breath collection technologies such as Exhaled Breath Condensate (EBC) or electronic noses, which detect volatile organic compounds (VOC) in air. This is however incorrect. The so called PEx sample that is collected by PExA technology consists of an aerosol that originates directly from the respiratory lining fluid (RTLF) and the surfactant fluid that is present in the most distal part of a subject’s airways. Moreover, in contrast to EBC, a PEx sample is not contam inated with material from the upper airways or the oral cavity of a subject, and it is undiluted. The fact that a PEx sample originates exclusively from the deepest parts of a subject’s lungs as opposed to the subject’s blood, or in the case of a EBC or VOC sample, from substantially the whole body of a subject in undefined proportions, it is believed that PExA technology allows more relevant, more accurate, and less confounded biomarker data to be obtained.

[0008] The PExA sampling technology is inherently dependent on impaction of the exhaled particles onto a solid surface, e.g., a substrate made of a material with specific physical and chem ical properties. By extracting and solubilising biomolecules in or associated with the particles impacted on the substrate in an extraction solution, it is possible to analyse the molecular content of the PEx sample and thereby screen for molecular biomarkers from the most distal airways in a subject’s lungs in a non-invasive way. This facilitates the development of more precise and early diagnostics in the field of respiratory medicine.

[0009] The particles are collected by impaction on defined areas of a substrate. After the collection of particles, the substrate or parts of the substrate is / are removed and prepared for analysis. I n one analysis strategy, the particles are analysed whilst still on the substrate. I n another analysis strategy, the substrate or parts of the substrate is transferred to a sample tube or other type of container and brought into contact with an extraction solution such that particles become suspended and disintegrated so that biomolecules in or associated with the particles can become solubilised or suspended and thereby become accessible for an analysis technique of choice.

[0010] To allow the concentration of biomolecules for subsequent biochemical analyses to be increased, the tiny and specific areas of the substrate that hold the collected particles, which are invisible to the naked eye, are advantageously individually processed by means of a specially developed sample preparation kit, such as the sample preparation kit disclosed in international publication number WO 2022 / 223420. The sample preparation kit comprises a punch guide with precisely positioned guiding bores which is attached to a holding device that holds the substrate at a defined position.

[0011] A punch tool is then, sequentially pushed through each of the guiding bores of the punch guiding device to excise precisely the tiny circular areas of the substrate that hold the collected particles. The excised substrate plugs or discs holding the collected particles may then be subjected to extraction using a significantly smaller volume of extraction solution than otherwise needed, for solubilisation of biomolecules subsequent to biochemical analysis.

[0012] An inertial impactor usually comprises a particle counter to measure the number of particles collected on a substrate to help quantify the size distribution of collected particles.

[0013] However, since some of the solubilised biomolecules, such as certain proteins may have an affinity for the surface of the substrate on which they were collected, some biomolecules may not remain dissolved exclusively in the free solution. This problem , which is often referred to as non-specific binding or adsorption, or matrix effect, means that only a subpopulation of the collected biomolecules become available for chemical reaction in the biochemical assay in use. This circumstance may result in inaccurate measurements of the concentrations or properties of the biomolecules being studied. Mitigating non-specific binding is therefore crucial for obtaining accurate and reliable results.

[0014] Non-specific binding is usually m inim ized by using blocking agents to cover surfaces that biomolecules or analytes may come into contact with, or chemically modifying or coating the surfaces to make them less prone to non-specific binding. A complementary approach is to keep the area of surfaces that the analytes come into contact with before and during the biochem ical analysis to an absolute m inim um .

[0015] Description of th e I nvention

[0016] I n a first aspect of the invention there is provided a system for collecting particles contained in air exhaled by a subject, such as a human or other animal, for analysis, which system comprises the features recited in claim 1 . The system comprises an inertial impactor configured to sort and collect particles contained in air exhaled by a subject according to their mass. The inertial impactor comprises at least one substrate for collecting particles, and at least one holding device for securing the at least one substrate in the inertial impactor during the collection of particles. The at least one substrate comprises or consists of a material that is water- soluble at a temperature greater than 0°C and less than 100°C and / or at least partially at least partially transparent.

[0017] A partially or fully transparent substrate facilitates the quantification, characterisation, and / or other type of analysis of particles impacted onto the at least partially transparent substrate since the substrate may be analysed using an optical method, such as imagebased densitometry, light scattering technologies, spectroscopy and confocal m icroscopy or a combination thereof.

[0018] The system does not therefore necessarily have to include a particle counter, which reduces the cost, complexity, and space requirements of the system .

[0019] An entire substrate may be partially or fully transparent. Alternatively, a substrate may comprise one or more areas of partially or fully transparent material. A substrate may comprise or consist of organic material, such as plastic, or inorganic material, such as glass.

[0020] Advantageous implications

[0021] The inventors have found that, even though a water-soluble material’s potential for deformation and dissolution would make it seem unsuitable for use as a substrate in an inertial impactor, where substrate stability and inertness are crucial for impaction, and where particles that originate from an body fluid come into contact with the at least partially water-soluble substrate during the collection of particles, a substrate comprising or consisting of water-soluble material may be used in an inertial impactor since a substrate as claimed is not adversely affected by the particles that impact the surface of the at least partially water-soluble substrate. Moreover, the inventors have found that the at least partially water-soluble substrate is rigid and stable enough after the collection of particles, to allow at least one part of it to be excised, such as by suing a punch tool as described in WO 2022 / 223420. Utilizing an at least partially water-soluble substrate in lieu of a non-dissolvable counterpart within an inertial impactor presents numerous advantages. For example, it means that the particles, initially collected by impaction on the solid surface of the at least partially water-soluble substrate can be fully suspended with no solid residues of the substrate left behind, simply by letting the substrate come into contact with water. This obviates the need for labour-intensive processes wherein the impacted particles otherwise would require suspension through substrate washing and subsequent separation of the suspension from the solid substrate rem nants. Such stream lining significantly simplifies the workload and logistical aspects of sample preparation and sample analysis. Consequently, the present innovation allows for sample preparation to be executed by personnel lacking prior laboratory experience, thus markedly enhancing the system's usability and practicality.

[0022] Utilizing an at least partially transparent and an at least partially water-soluble substrate in lieu of an at least partially transparent but non-dissolvable counterpart within an inertial impactor presents numerous advantages. For example, it means that the particles, initially collected by impaction on the solid surface of the at least partially transparent and the at least partially water-soluble substrate can be fully suspended with no solid residues of the substrate left behind, simply by letting the substrate come into contact with water. This obviates the need for labour-intensive processes wherein the impacted particles otherwise would require suspension through substrate washing and subsequent separation of the suspension from the solid substrate remnants. Such streamlining significantly simplifies the workload and logistical aspects of sample preparation and sample analysis. Consequently, the present innovation allows for sample preparation to be executed by personnel lacking prior laboratory experience, thus markedly enhancing the system's usability and practicality.

[0023] Another significant facet of the lack of solid residues post-solubilization of an at least partially transparent and / or the at least partially water-soluble substrate is the prevention of biomolecules binding to the substrate surface, a problem commonly referred to as unwanted absorption or matrix effect. By circumventing excess surfaces prone to inducing physical-chem ical interference with solubilized biomolecules, the analysis of biomolecules in the sample stands to yield more reproducible data outputs. This reproducibility is a pivotal characteristic of a system tailored for biomarker discovery. Furthermore, since an at least partially transparent and / or an at least partially water- soluble substrate, as described herein exhibits water solubility and one or more parts of the substrate that hold(s) the impacted particles can be excised and transferred into a suitable sample tube, the biomolecules in or associated with the collected particles can be solubilised in a m inimal volume of aqueous solution. This maximizes the concentration of biomolecules in the resulting sample. Moreover, since an at least partially transparent but non-water dissolvable substrate suitable for impaction may contain pores or fibres, leading to absorption and retention of a significant portion of the biomolecule solution postsolubilization, the use of an at least partially transparent and / or an at least partially a water-soluble substrate enables complete volume recovery of the biomolecule solution, thereby avoiding potential waste of the sample.

[0024] Another significant facet of the lack of solid residues post-solubilization of an at least partially water-soluble and / or an at least partially transparent substrate is the prevention of biomolecules binding to the substrate surface, a problem commonly referred to as unwanted absorption or matrix effect. By circumventing excess surfaces prone to inducing physical-chem ical interference with solubilized biomolecules, the analysis of biomolecules in the sample stands to yield more reproducible data outputs. This reproducibility is a pivotal characteristic of a system tailored for biomarker discovery.

[0025] Furthermore, since the at least partially water-soluble substrate and / or an at least partiallt transparent substrate, as described herein exhibits water solubility and one or more parts of the substrate that hold(s) the impacted particles can be excised and transferred into a suitable sample tube, the biomolecules in or associated with the collected particles can be solubilised in a m inimal volume of aqueous solution. This maximizes the concentration of biomolecules in the resulting sample. Moreover, since a non-water dissolvable substrate suitable for impaction may contain pores or fibres, leading to absorption and retention of a significant portion of the biomolecule solution post-solubilization, the use of an at least partially water-soluble substrate, as claimed, enables complete volume recovery of the biomolecule solution, thereby avoiding potential waste of the sample.

[0026] This is an advantage as the increased total amount and concentration of solubilized biomolecules in the resulting sample enables detection of a greater number of different biomolecules, thereby enhancing the likelihood of discovering novel biomarkers. Alternatively, the opportunity offered by the usage of an at least partially water-soluble and / or at least partially transparent substrate to make better use of all biomolecules in the sample can instead translate into a shorter sampling time. The latter is of particular importance in clinical trials and / or in clinical use where time for sampling is often a critical factor. Additionally, or alternatively, a sample can be split into a so-called primary run and a back-up sample, without sacrificing too m uch detectability.

[0027] Furthermore, the ability to dissolve the at least partially water-soluble and / or at least partially transparent substrate by simply adding a sample buffer of choice and a customizable volume j ust before the biochem ical analysis is conducted allows for analysis of the sample by laboratories that specialize in analysing standard body fluids and do not offer sample preparation bench work. Again, this significantly enhances the applicability of the system .

[0028] Other potential advantages arising from the utilization of an at least partially water-soluble and / or at least partially transparent substrate which enables efficient solubilization of biomolecules in a rapid and straightforward manner, is the option to solubilize the sample directly after collection with minimal effort. For instance, this provides the user of the described system the ability, if desired, to store the sample in a liquid state, thereby preventing or minimizing oxidation of biomolecules, such as lipids and proteins present in the particles, thus potentially reducing the risk of obscured data. Another example of how an immediate conversion of a solid sample into a liquefied and small volume sample creates new opportunities is that it opens up for utilization of a biosensor that consumes j ust a drop of sample and provides biomarker data almost directly after sample collection, which is a crucial feature for the further development of the described system .

[0029] Following the extraction and solubilization of biomolecules among a series of samples containing varying amounts of particles collected on solid non-soluble substrates, the concentration of solubilised biomolecules can be standardized by applying a volume of the extraction solution which is proportional to the amount of collected particle material (whereby the mass of collected particles may be obtained from a particle counter integrated to the system , herein described) . However, the amount of the material that constitutes the non-soluble substrate and that is present during the extraction and solubilisation of biomolecules in the sample cannot be adj usted for proportionally. This discrepancy means that the ratio between the concentration of biomolecules and the surface area of the solid non-soluble substrate will differ from one sample to another, potentially leading to unequal surface chem istry equilibrium conditions among the samples and, consequently, undesired variability in data output. However, with an at least partially water-soluble and / or at least partially transparent substrate, as herein claimed, the mass of particles collected on the at least partially water-soluble and / or at least partially transparent substrate as well as the amount of molecules that constitute the at least partially water-soluble and / or at least partially transparent substrate can itself be adjusted to equal concentrations among the solubilised samples by proportional addition of the water-soluble chem ical that constitutes the at least partially water-soluble and / or at least partially transparent substrate.

[0030] Since the manufacturing of the at least partially water-soluble and / or at least partially transparent substrate, as exemplified herein, may involve casting a film from a waterbased solution containing the polymer molecules constituting the at least partially water- soluble substrate material, it allows for the incorporation of various additives into the at least partially water-soluble substrate. This can for instance be protease and phosphatase inhibitors or RNA stabilizing compounds like chaotropic salts, or other components com monly utilized in buffers for nucleic acid or protein extraction. This aspect of the claimed system holds particular significance, especially in scenarios where particles are collected from lung fluid using an impactor that maintains a temperature conducive to biochemical reactions, such as the degradation of proteins or nucleic acids by endogenous enzymes. I n such instances, the possibility to add reagents that stabilise biomolecules of interest to the substrate has the potential to become invaluable, opening up possibilities for versatile and more efficient utilization of the described system . As a final note, in certain cases, it is desirable to analyse various types of analytes present in a sample like the one described herein. However, the surface chemistry of a particular substrate may be optimal for one type of analyte and suboptimal for another, placing the system operator in a quandary. On the contrary, if the particle sample is impacted onto the surface of an at least partially water-soluble substrate, the issue of incompatible surface chem istry can be circumvented. I n such instances, the water-solubility of the at least partially water-soluble substrate according to the invention holds the potential to become invaluable, unlocking possibilities for more versatile and efficient utilization of the described system .

[0031] Em bodim ents of th e invention

[0032] The at least one substrate may be at least partially or fully water-soluble at a temperature greater than 0°C and less than 100°C, i.e. the at least one substrate may be at least partially water-soluble.

[0033] Alternatively, the at least one substrate may be at least partially or fully non-dissolvable, i.e. the at least one substrate may comprise or consist of a material that is not water- soluble at a temperature greater than 0°C and less than 100°C, such as glass, for example borosilicate glass, or plastic. The at least one substrate may comprise both at least one material that is water-soluble at a temperature greater than 0°C and less than 100°C, and at least one material that is non-dissolvable.

[0034] Even though a water-soluble material’s potential for deformation and dissolution would make it seem unsuitable for use as a substrate in an inertial impactor, where substrate stability and inertness are crucial for impaction, and where particles that originate from an body fluid come into contact with the at least partially water-soluble substrate during the collection of particles.

[0035] Additionally, or alternatively, an at least partially transparent substrate comprising or consisting of water-soluble material may be used in an inertial impactor since an at least partially transparent and an at least partially water-soluble substrate as described herein is not adversely affected by the particles that impact the surface of the at least partially transparent and the at least partially water-soluble substrate. Moreover, the inventor has found that an at least partially transparent and water-soluble substrate is rigid and stable enough after the collection of particles, to allow at least one part of it to be excised, such as by suing a punch tool as described in WO 2022 / 223420.

[0036] The substrate may comprise or consist of a material that has a water solubility of at least 0.1 g / L, at least 1 g / L, at least 5 g / L, or at least 10 g / L, or at least 20 g / L, or at least 30 g / L, or at least 40 g / L, or at least 50 g / L, or at least 60 g / L, or at least 70 g / L, or at least 80 g / L, or at least 90 g / L, or at least 100 g / L, or at least 150 g / L, or at least 200 g / L or more at a temperature greater than 0°C and less than 100°C. For example, the at least one substrate may comprise or consist of a material that has a water solubility of 0.1 -1 .0 g / L, 1 .0-5 g / L, 5-10 g / L, 10-20 g / L, 30-40 g / L, 40-50 g / L, 50-60 g / L, 60-70 g / L, or 70- 80 g / L, or 80-90 g / L, 90-100 g / L, 100-150 g / L, or 150-200 g / L or more. a) The at least one substrate may comprise or consist of at least one of the following substances: Poly(2-ethyl-2-oxazoline) , Poly(2-hydroxyethyl methacrylate / methacrylic acid) , Poly (2- vinyl- 1 -methylpyridinium bromide) , Poly(2- viny Ipy ridine N-oxide) , Poly(4- vinylpyridine) , Poly(Acrylic Acid) PAA, Poly(allylamine) , Poly(ethylene oxide) PEO, Poly(propylene oxide) PPO, Poly(ethylene oxide-b-propylene oxide) , Poly ( I- lysine hydrobromide) , Poly(maleic acid) , Poly(methacrylic acid) , Poly(N-isopropylacrylamide) , Poly(N-vinylpyrrolidone) , Poly(N-vinylpyrrolidone / vinyl acetate) , Poly(oxyethylene) , Poly(styrenesulfonic acid) , Poly(vinyl acetate), Poly(vinyl alcohol) PVA, Poly(vinyl methyl ether) , Poly(vinyl phosphoric acid) , Poly(vinylam ine) , Poly(vinylphosphonic acid) , Poly(vinylsulfonic acid) , Polyacrylamide, polyacrylic-acid-63-soln-in-water, Poly-ethylene- oxide, Poly-l-lysine-hydrobromide, Poly-methacrylic-acid, collagen, gelatine, salt, polysaccharides, or any other organic or non-organic, synthetic or non-synthetic, at least partially water-soluble and / or at least partially transparent polymer or substance. b) Optionally, the least partially transparent and / or the at least partially water-soluble substrate may contain one or more additives, such as additives that help to stabilize labile molecules like RNA and DNA and / or post translational modifications in proteins (PTMs) .

[0037] Preferably, the at least partially water-based substrate and / or at least partially transparent described herein does not contain molecules present in or associated with the particles that are to be collected by the system and suspected to interfere in a negative way with the method for analysis of the particles or biomolecules in, or associated with the particles that are to be collected by the system .

[0038] For example, the water-based and / or the at least partially at least partially transparent substrate described herein does not contain proteins, lipids or metabolites expected to be found in the particles that are to be collected by the system .

[0039] Alternatively, or additionally, the at least one substrate has a maxim um thickness of 1 mm , or 0.9 mm , or 0.8 m m , or 0.7 m m , or 0.8 m m , or 0.5 m m , or 0.4 m m , or 0.3 mm , or 0.2 mm , or 0.1 mm , or 0.09 m m , or 0.08 mm , or 0.07 m m , or 0.06 mm , or 0.05 m m , or 0.04 mm , or 0.03 m m , or 0.02 m m , or 0.01 mm . The thickness of a substrate need not necessarily be uniform . An average thickness of a substrate may be determined by stacking a plurality of substrates, such as ten or more substrates, measuring the total thickness of the stack of substrates using any suitable method, and dividing the total thickness by the number of substrates in the stack.

[0040] Alternatively, or additionally, an at least one at least partially water-soluble substrate is non-transparent or at least partially transparent. A partially or fully transparent and an at least partially water-soluble substrate facilitates the quantification, characterisation, and / or other type of analysis of particles impacted onto the at least partially water-soluble substrate since it may be analysed using an optical method, such as image-based densitometry, light scattering technologies, spectroscopy and confocal microscopy or a combination thereof. A particle counter would therefore not be necessary. An entire substrate may be non-transparent, partially or fully transparent. Alternatively, a substrate may comprise one or more areas of non-transparent, partially or fully transparent material.

[0041] A substrate comprising one or more of the features outlined above may be manufactured relatively inexpensively by casting thin film , for example, by casting for example. The substrate is preferably manufactured under clean conditions, such as in a standard laboratory clean room or lam inar flow hood, with no contamination of unknown material from ambient air. The manufacturing process can thereby be fully controlled, for example to ensure that no unwanted material or molecules will be introduced into the at least one substrate during its production.

[0042] The precise vertical position of the substrate in the at least one holding device, including the vertical position of the at least one holding device in the inertial impactor, i.e., the vertical level of the surface of the substrate, is critical in order to allow for the collection of a specific size range of particles. The at least one holding device may comprise at least one spacer configured to adj ust, for example to increase, the vertical level of the surface of the at least one substrate in the at least one holding device. The at least partially water- soluble and / or the at least partially transparent substrate described herein may thereby be used in the holding devices of existing systems since at least one spacer may be used to compensate for the reduced thickness of a substrate that may not be as thick as a conventional substrate in the at least one holding device.

[0043] The system may comprise a sample preparation kit comprising an excision tool, such as a punch tool, for excising and transferring parts of an at least partially water-soluble and / or the at least partially transparent substrate with exhaled particles to a sample tube or other container suitable for extraction, solubilisation and / or analysis of biomolecules in and / or associated with the collected particles. The sample preparation kit may comprise at least one excision tool guide, such as a punch tool guide, for steering the excision tool tip to the exact area or areas where the particles are impacted on the substrate. The sample preparation kit may further comprise at least one holding device for securing a substrate before and during the collection of particles and excising of one or more parts of the substrate.

[0044] The sample preparation system described above, may comprise a robotic mechanism and / or an automated design to reduce hands on time and increase reproducibility, which can in turn facilitate a high throughput and more cost-effective sample preparation, all of which is facilitated by the use of an at least partially water-soluble and / or the at least partially transparent substrate for impaction of the exhaled particles. Dissolving one or more excised parts of an at least partially transparent and / or an at least partially water-soluble substrate in an aqueous solution or other solvent rather than dissolving the whole substrate, will reduce the amount of substrate material that is to be dissolved in the aqueous solution or other solvent. This consequently m inim izes the concentration of molecules that constitute the at least partially transparent and / or the at least partially water-soluble material and thereby reduces possible interference of molecules that constitute the at least partially transparent and / or the at least partially water-soluble material with chem ical reactions that take place in the subsequent analysis of the sample. Furthermore, since excess areas of the at least partially transparent and / or the at least partially water-soluble substrate can be avoided by using the sample preparation system described above a smaller volume of aqueous solution can be used to fully dissolve the biomolecules in or associated with the particles collected on the at least partially transparent and / or the at least partially water-soluble substrate, the sample preparation system described above, provides the means for increasing the concentration of the biomolecules in or associated with the particles collected on the at least partially transparent and / or the at least partially water-soluble substrate. Since analytical platforms are lim ited with regard to how high a concentration of the analyte is needed to generate a useful signal and can only make use of a limited sample volume, the use of an at least partially transparent and / or an at least partially water-soluble substrate, as herein described, helps to detect a higher number of analytes and thereby increases the chances of discovering novel biomarkers.

[0045] Excising at least one part of a substrate on which particles have been collected may also be carried out if one or more piles of particles that have been collected on a single substrate are to be analysed separately.

[0046] I n a second aspect of the invention there is provided a method for collecting particles contained in air exhaled by a subject, such as a human or an animal, for analysis, which method comprising the features recited in the independent method claim .

[0047] The method comprises collecting particles from the subject and sorting the particles according to their mass using an inertial impactor. The inertial compactor comprises at least one substrate for collecting particles, and at least one holding device for securing the at least one substrate in the inertial impactor during the collection of particles, and optionally before and after the collection of particles. The at least one substrate comprises or consists of a material that is water-soluble at a temperature greater than 0°C and less than 100°C and / or at least partially transparent. The at least one at least partially transparent substrate may also comprise or consist of a material that is water-soluble at a temperature greater than 0°C and less than 100°C.

[0048] The at least one at least partially transparent substrate may comprise or consist of a material that has a water solubility of at least 0.1 g / L, at least 1 g / L, at least 5 g / L, or at least 10 g / L, or at least 20 g / L, or at least 30 g / L, or at least 40 g / L, or at least 50 g / L, or at least 60 g / L, or at least 70 g / L, or at least 80 g / L, or at least 90 g / L, or at least 100 g / L, or at least 150 g / L, or at least 200 g / L or more at a temperature greater than 0°C and less than 100°C. For example, the at least one substrate may comprise or consist of a material that has a water solubility of 0.1 -1 .0 g / L, 1 .0-5 g / L, 5-10 g / L, 10-20 g / L, 30- 40 g / L, 40-50 g / L, 50-60 g / L, 60-70 g / L, or 70-80 g / L, or 80-90 g / L, 90-100 g / L, 100- 150 g / L, or 150-200 g / L or more.

[0049] Alternatively, or additionally, the at least one substrate comprises or consists of at least one of the following substances: The at least one substrate may comprise or consist of at least one of the following materials: Poly(2-ethyl-2-oxazoline) , Poly(2-hydroxyethyl methacrylate / methacrylic acid) , Poly (2- vinyl- 1 -methylpyridinium brom ide) , Poly(2- vinylpyridine N-oxide) , Poly (4- vinylpyridine) , Poly(Acrylic Acid) PAA, Poly(allylam ine) , Poly(ethylene oxide) PEO , Poly(propylene oxide) PPO, Poly(ethylene oxide-b-propylene oxide) , Poly ( I- lysine hydrobrom ide) , Poly(maleic acid) , Poly(methacrylic acid) , Poly(N- isopropylacrylam ide) , Poly(N-vinylpyrrolidone) , Poly(N-vinylpyrrolidone / vinyl acetate) , Poly(oxyethylene) , Poly(styrenesulfonic acid) , Poly(vinyl acetate) , Poly(vinyl alcohol) PVA, Poly(vinyl methyl ether) , Poly(vinyl phosphoric acid) , Poly(vinylamine) , Poly(vinylphosphonic acid) , Poly(vinylsulfonic acid) , Polyacrylamide, polyacrylic-acid-63- soln-in-water, Poly-ethylene-oxide, Poly-l-lysine-hydrobrom ide, Poly-methacrylic-acid, collagen, gelatine, salt, polysaccharides, or any other organic or non-organic, synthetic or non-synthetic, at least partially water-soluble and / or at least partially transparent polymer or substance.

[0050] Alternatively, or additionally, the at least one substrate has a maxim um thickness of 1 mm , or 0.9 mm , or 0.8 m m , or 0.7 m m , or 0.8 m m , or 0.5 m m , or 0.4 m m , or 0.3 mm , or 0.2 mm , or 0.1 mm , or 0.09 mm , or 0.08 m m , or 0.07 mm , or 0.06 m m , or 0.05 mm , or 0.04 m m , or 0.03 m m , or 0.02 mm , or 0.01 mm .

[0051] Alternatively, or additionally, the at least one substrate is non-transparent or at least partially or fully transparent. Alternatively, or additionally, the at least one substrate is non-dissolvable or at least partially or fully dissolvable. The method may comprise placing at least one spacer in the at least one holding device to adj ust the vertical level (i.e. the exact vertical position) of the surface of the at least one substrate in the at least one holding device in an inertial impactor.

[0052] The method may comprise removing a holding device from the inertial impactor after the collection of particles, and dissolving at least part of an at least partially transparent and / or the at least partially water-soluble substrate in an aqueous solution or other solvent, such as a water-based buffer, by either bringing the at least partially transparent and / or the at least partially water-soluble substrate into contact with the aqueous solution or other solvent, or by excising at least one part of the at least partially transparent and / or the at least partially water-soluble substrate, such as a sample buffer with or without non-polar solvent, and bringing the at least one excised part of the at least partially transparent and / or the at least partially water-soluble substrate into contact with the aqueous solution or other solvent.

[0053] Preferably, the method involves dissolving at least part of a substrate with exhaled particles in an aqueous solution or other solvent. The integrity of the biomolecules is thereby preserved, valuable biomarkers are not lost, the risk of inaccurate quantification is reduced, and the reliability of subsequent biochemical analyses is not compromised. Additionally, there is no variation in the extraction efficiency for different particles, such as for different proteins or other analytes.

[0054] The method may comprise removing a sample holder from the inertial impactor followed by excising and transferring at least one part of an at least partially water-soluble and / or at least partially transparent substrate with exhaled particles to a sample tube for disintegration of the particles deposited on the at least one part of the at least partially water-soluble and / or at least partially transparent substrate and solubilising and / or extracting biomolecules in or associated with the particles, in an aqueous solution or other solvent, such as a sample buffer with or without non-polar solvent. This allows collected particles to be stored and analysed in a similar manner to other types of body fluid samples. Collected particles solubilised in an aqueous solution or other solvent may be studied at any suitable time after the particles have been collected, such as several months or years after the particles have been collected.

[0055] Alternatively, the method comprises bringing a substrate with exhaled particles into contact with the aqueous solution or other solvent directly after the at least partially water- soluble and / or at least partially transparent substrate with exhaled particles has been removed from an inertial impactor, whereby solubilised biomolecules, will not come into contact with any additional surface before they come into contact with affinity reagents, thereby decreasing the risk of non-specific binding occurring before biochemical analysis.

[0056] Collected particles solubilised in an aqueous solution or other solvent may be analysed immediately using a suitable biochemical analysis technique. For example, the method comprises using a biosensor, such as a biosensor that consumes j ust a drop of sample, to provide biomarker data directly after particle collection. Alternatively, the method comprises sending collected particles solubilised in an aqueous solution or other solvent (or not solubilised) to a remote location, such as to a CRO, for biochem ical analysis.

[0057] The method may comprise quantifying and / or characterising and / or analysing exhaled particles impacted on the at least one substrate using any suitable optical method, such as image-based densitometry, light scattering technologies, spectroscopy and confocal m icroscopy or a combination thereof.

[0058] A system according to any of the embodiments described herein may be used to carry out a method according to any of the embodiments described herein.

[0059] I n a third aspect, the present invention concerns the use of at least one substrate comprising or consisting of a material that is at least partially transparent in a system or method comprising an inertial impactor configured to sort and collect particles contained in air exhaled by a subject according to their mass.

[0060] The present invention also concerns the use of a system according to any of the embodiments described herein for determining a biomarker associated with a medical condition for clinical applications, for scientific research, or research and development in the pharmaceutical industry.

[0061] Def in itions

[0062] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0063] As used herein, the term “sample” means one or more parts of, or the whole of an at least partially water-soluble and / or at least partially transparent substrate onto which particles have been impacted, and / or the biomolecules solubilised or dispersed in an aqueous solution or other solvent. A sample may be a “blank sample” which is a sample that has been collected without exposing an at least partially water-soluble and / or at least partially transparent substrate to air from a subject. A blank analysis may be carried out to check whether any kind of contam ination is present in the system .

[0064] As used herein, the term “substrate” refers to a layer or piece of material having a surface that is used for impaction of particles. The at least partially transparent substrate may be placed in a holding device which holds the at least partially transparent substrate at a specific position and vertical level in the inertial impactor. The at least partially transparent substrate or at least one piece thereof may then be detached from the holding device and processed for analysis of the particles or the biomolecules in or associated with the particles.

[0065] As used herein the term “material that is at least partially transparent” is intended to mean material that is optically transparent or translucent to electromagnetic waves, i.e. that allows the transm ission of electromagnetic waves of any frequency to pass through it, or a material that allows the transm ission of electromagnetic waves of at least one frequency to pass through it. The electromagnetic waves may be radio waves, m icrowaves, infrared, visible light, ultraviolet, X-rays or gam ma rays. The term “material that is transparent” thereby means any material that is suitable for quantification or characterisation of particles or molecules or biomolecules constituting the particles deposited on the surface of the material or any material that is not opaque, i.e. any material that does not absorb all portions of the electromagnetic spectrum .

[0066] As used herein, the term “optical method”, refers to any method that is suitable for quantification or characterisation of particles or molecules or biomolecules constituting the particles deposited on the surface of an at least partially transparent substrate, for example, image-based densitometry, light scattering technologies, spectroscopy and confocal m icroscopy or a combination thereof.

[0067] As used herein, the term “water-soluble material” is intended to mean material that has an ability to dissolve in an aqueous solution, such as water, or other solvent at a temperature greater than 0°C and less than 100°C, form ing a homogeneous solution or em ulsion or suspension, either with or without shaking. Preferably, the water-soluble material is fully dissolvable in water at a temperature greater than 0°C and less than 100°C, i.e. water-soluble material dissolves at a temperature of 0-10°C, 10-20°C, 20- 30°C, 30-40°C, 40-50°C, 50-60°C, 60-70°C, 70-80°C, 90-100°C, or water-soluble at room temperature, either with or without shaking. As used herein, the term “excising” refers to the usage of an excision tool, such as a punch tool or other means for excising at least one part of a substrate of any shape and size, followed by transferring the excised material to a surface, sample tube or other container suitable for extraction, solubilisation or dispersion of the substrate, particles and biomolecules.

[0068] As used herein, the term “extraction solution” or “sample buffer”, refers to pure water, pH buffered water, salt-based solution or organic solvent, or a m ixture of the afore mentioned solutions, suitable for suspension, disintegration of particles and / or solubilisation of biomolecules in or in association with the exhaled particles described elsewhere herein.

[0069] As used herein, the term “particles” or “collected particles, or “impacted particles” refers to aggregates of biomolecules formed from the RTLF and / or surfactant in a subject’s airways which are exhaled during a breathing manoeuvre, and which leave the lungs of a subject as an aerosol of droplets and end up on the surface of a substrate. When an at least partially transparent and / or an at least partially water-soluble substrate is brought into contact with an aqueous solution or other solvent, the at least partially water-soluble material dissolves at least partially, the impacted particles are released, disintegrate, or disperse to create an emulsion, or a suspension, in the aqueous solution or other solvent. A “particle” is intended to mean any solid, liquid, or liquid-coated solid object or other biological entity, such as a vesicle, m icelle or microorganism , which is suspended in air. Particle sizes may be in the range of 0.005 micrometres to 15 m icrometres, such as 0.005 to 0.010 m icrometres, 0.010 to 0.100 micrometres, 0.10 to 1 .00 micrometres, 1 -5 micrometres, 5-10 micrometres, or 10-15 m icrometres measured with or without any layer of liquid that adheres to the particle when it is formed or after it has been formed.

[0070] As used herein, the term “biomolecules” or “analytes” or “biological material” may refer to any type of RNA, DNA, proteins, lipids, phospholipids, metabolites, small molecules like drug substances and / or other types of endogenous substances that can be organic or inorganic substances. Biomolecules can include intact microbes or parts of m icrobes, such as bacteria, viruses and fungi, or eucaryotic cells.

[0071] As used herein, the terms “biomolecules in particles” and “biomolecules associated with particles” refer to biomolecules that constitute aggregates or part of the particle material collected from exhaled air from a subject.

[0072] As used herein, the term “solubilisation” of an at least partially transparent and / or an at least partially water-soluble substrate refers to bringing the one or more parts of the at least partially transparent and / or the at least partially water-soluble substrate into contact with an aqueous solution or other solvent to dissolve the material that constitutes the substrate matrix, thereby releasing, suspending and / or disintegrating the particles so that biomolecules in, or associated with the particles are dissolved together with the molecules constituting the at least partially transparent and / or the at least partially water-soluble substrate.

[0073] As used herein, the term “solubilisation” or “extraction” of the biomolecules or particles refers to the release, suspension and / or disintegration of particles impacted on an at least partially water-soluble and / or at least partially transparent substrate when the at least partially water-soluble and / or at least partially transparent substrate is brought into contact with an aqueous solution or other solvent whereby water-soluble biomolecules, in or associated with the particles are solubilised in the aqueous solution or other solvent, and / or non-water-soluble biomolecules are dispersed and form an emulsion or a suspension in the aqueous solution or non-water soluble biomolecules are partitioned into the organic solvent phase during a two-phase extraction process. An at least partially water-soluble and / or at least partially transparent substrate may be brought into contact with an aqueous solution or other solvent by, either placing the at least one part of the at least partially water-soluble and / or at least partially transparent substrate in a sample tube or other suitable container, or on a surface and subsequently adding an aqueous solution or other solvent or placing the at least one part of the at least partially water- soluble and / or at least partially transparent substrate in a sample tube or other suitable container, or on a surface previously prepared with a volume of a aqueous solution or other solvent.

[0074] As used herein, the term “aqueous solution or other solvent” is intended to mean any solution of water or any solution containing water, or other polar or non-polar solvent which is used in chem ical, biological or biochem ical laboratory techniques to facilitate the preparation and analysis of biological samples. Water serves as the primary solvent for the various components in the solution. Aqueous solution or other solvents are essential for preparing biological samples for analysis. They create an environment that promotes the separation of molecules based on size, charge, or other characteristics, allowing researchers to study and analyse the composition of biological samples.

[0075] As used herein, the term “aqueous solution or other solvent” can also be a two-phase extraction system constituting water and an organic non-polar solvent phase. An aqueous solution, buffer or other solvent may help to maintain a stable pH, and / or it may contain salts to provide the necessary ionic strength for optimal performance biochemical assay and / or it may comprise a detergent that is used to disintegrate particles and solubilise biomolecules, such as proteins, or nucleic acids, and to prevent their aggregation, and / or it may contain a reducing agent to break disulfide bonds in proteins, unfolding them for more accurate size separation. It may include a stabilising constituent, e.g. a chaotropic salt and / or another reagent for the stabilisation of RNA or DNA during sample collection and sample preparation.

[0076] As used herein, the term “analysis” refers to any type of analysis resulting in the generation of quantitative, and / or qualitative molecular data, including chem ical or physical characterisation of the particles and / or biomolecules, or viability and activity measures of virus, prokaryotic or eukaryotic cells

[0077] As used herein, the term “biomarkers” refers to one or a set of different biomolecules, by which a particular medical condition, such as a pathological or physiological process, disease, etc. can be identified. A biomarker may be one or several different biomolecules that, when detected, quantified or qualitatively characterised in a sample, convey information concerning a medical condition, the presence, absence, stage, severity or resolution of a disease for example, or a physiological response to a specific environmental challenge, or exposure to a drug, food or other substance. A biomarker may also include one or more different biomolecules that, when detected, quantified, or qualitatively characterised in a sample, convey information concerning a specific genotype, phenotype, endotype, treatable trait, or combination thereof. A biomarker may be an endogenous or exogenous molecule, or a residue from a biochemical reaction or other type of cellular activity. The term “biomarker” or “biomarkers” as herein used also encompass biomolecules that are detected, quantified and / or qualitatively characterised in a sample for the purpose of hypothesis or non-hypothesis-based research and basic science, as well applied research.

[0078] As used herein, the term “room temperature” is intended to mean a temperature such as 22°C, or any temperature in the range 20-25°C, or 20°-24°C, or 20°-23°C, or 21 -25°C, or 22-24°C. Preferably the water-soluble material is fully dissolvable in water within 60 minutes, or within 50 m inutes, or within 40 minutes, or within 30 m inutes, or within 20 minutes, or within 10 m inutes, or within 5 m inutes, or within 4 m inutes, or within 3 minutes, or within 2 m inutes, or within 1 m inute, or within 30 seconds, or within 20 seconds, or within 10 seconds, or within 5 seconds, or within 1 second at a temperature greater than 0°C and less than 100°C, either with or without shaking. As used herein, the term “biochem ical analysis” is intended to refer to any biological, chem ical or biochemical analysis technique that may be used to detect quantify and / or characterise substances or analytes such as proteins, lipids, carbohydrates, metabolites, or other biomolecules, including substances included in drug formulations, in a sample of particles in exhaled air from a subject, such as a human or animal.

[0079] As used herein, the term “air” is intended to mean any gas, or a m ixture of gases, which may be inhaled and / or exhaled by a subject. Exhaled air may contain a mixture of gases that differ from that in inhaled air. Optionally, inhaled or exhaled air may contain one or more additives intentionally added to the air. Furthermore, as used herein, the term “air” also includes aerosol particles that are carried by the inhaled or exhaled air.

[0080] As used herein, the term “clean air” is intended to mean air that is not m ixed or contam inated with biomolecules or particles that are to be collected by the system for analysis. “Clean air” may for example be air that has been cleaned in any suitable way, such as air that has been passed through at least one particle filter.

[0081] The term “one or more locations of collected particles on the or each substrate” is intended to mean the specific area or areas of a substrate on which the exhaled particles are collected, which may constitute a ring-shaped deposition pattern. The one or more locations of collected particles on a substrate will depend on the design and operation parameters of the inertial impactor.

[0082] The term “determ ining the medical condition of a subject” is intended to mean evaluating the presence (qualitative) and / or the extent (quantitative) of a medical condition or any predisposition a subject might have to acquire a given medical condition.

[0083] The term “medical condition” should not be understood as lim ited to diseases and disorders. It may be relevant to investigate the medical condition of healthy subjects, such as subjects who may be under the influence of medication or drugs (e.g., doping tests) , or otherwise exposed to chemical substances (e.g., pollutants, occupational hazards) .

[0084] As used herein, the term “holding device” refers to a device for holding one or more, substrates. The one or more substrates may be mounted on the “holding device” and fixed in place with a fixation ring, such as a magnetic fixation ring.

[0085] As used herein, the term “comprises” will take its usual meaning in the art, namely indicating that the component includes but is not limited to the relevant features (i.e., including, among other things) . As such, the term “comprises” will include references to the component consisting essentially of the relevant feature(s) . For the avoidance of doubt, the term “comprises” will also include references to the component “consisting essentially of” (and in particular “consisting of”) the relevant feature(s) .

[0086] As used herein, the term “constitute” will take its usual meaning in the art, namely indicating that the component includes but is not limited to the relevant features (i.e., including, among other things) . As such, the term “constitute” will include references to the component consisting essentially of the relevant feature(s) . For the avoidance of doubt, the term “comprises” will also include references to the component “consisting essentially of” (and in particular “consisting of”) the relevant feature(s) .

[0087] The invention is illustrated by way of the following examples, which are not intended to be limiting on the general scope of the invention.

[0088] Description of th e Fig u res

[0089] Fig u re 1 : shows a system for collecting particles exhaled by a subject,

[0090] Fig u re 2 : shows an inertial impactor that may be used in a system according to the present invention,

[0091] Fig u re 3 : shows an at least partially water-soluble and / or partially transparent substrate in a holding device of a system according to the present invention,

[0092] Fig u re 4 : shows an excision tool, namely a punch tool, that may be used with a system according to the present invention,

[0093] Fig u re 5 : shows part of a method according to an embodiment of the invention, and

[0094] Fig u re 6 : is a photograph show particles on an at least partially water-soluble and / or an at least partially transparent substrate of a system according to the present invention.

[0095] Table 1 : shows the results obtained in a feasibility test to determ ine the practicality and viability of using various PVA films as substrates in a system and method according to the present invention. The schematic drawings have not necessarily been drawn to scale and the dimensions of certain features may have been exaggerated for the sake of clarity.

[0096] Description of em bodim ents

[0097] Figure 1 shows a system 100 for collecting particles exhaled by a subject in which the claimed invention constitutes a significant improvement of the system . The system 100 comprises, optional a particle counter 1 17, an inertial impactor 10 and is arranged to maintain a constant flow of air exhaled through the inertial impactor 10 by means of a pump 1 15. Filtered air 18 inhaled by the subject passes through a unidirectional valve and mouthpiece 1 10. Air 16 from the subject’s distal airways, including generated particles is exhaled through a second unidirectional valve 1 16, and enters the inertial impactor 10. Optionally, the inertial impactor 10 may comprise a reservoir 1 14 if the inertial impactor capacity is insufficient for handling an entire exhalation at a time. Thus, any exhaled air 16 exceeding the impactor capacity enters the reservoir 1 14. Part of the system 100 may be located within a temperature regulated compartment 120. The system 100 may or may not comprise a particle counter.

[0098] The system 100 may comprise one or more valves 1 16. When the subject finishes his / her exhalation, a first valve 1 16 may be closed. The pump 1 15 will then draw the exhaled air stored in the reservoir 1 14 through the inertial impactor 10, thereby maintaining a continuous flow of exhaled air 16 through the inertial impactor 10.

[0099] The entire volume of each exhalation of a subject, or substantially the entire volume of each exhalation of a subject may be examined. The system 100 may be operated without requiring external air to be added to the reservoir 1 14 to maintain a flow through the system 100, thereby reducing a risk for contam ination present in the ambient air.

[0100] Such a system 100 may be used in the following way. A subject exhales, holds their breath for a period of time, inhales clean air 18, optionally via the mouthpiece 1 10 and then exhales through the mouthpiece 1 10 into the instrument. This will cause the small airways of the subject’s lungs to close and re-open, releasing particles. I n this way, biological material may be non-invasively collected from the distal airways of the subject’s lungs for analysis. The exhaled air 16 will flow through the inertial impactor 10. Figure 2 shows an inertial impactor 10 that may be used in a system 100 or method according to the present invention. The inertial impactor 10 may be any inertial impactor known in the art suitable for use in a medical application. The system 100 illustrated in Figure 2 includes an inertial impactor 10 comprising: an inlet 12 and an outlet 14, and a plurality of stages, 20, 30, 40, 50 arranged such that an air stream , i.e., exhaled air 16 from a subject, comprising particles P enters the inertial impactor 10 via the inlet 12 and passes through each stage in turn before exiting the inertial impactor 10 via the outlet 14.

[0101] Figure 2 illustrates four stages 20, 30, 40, 50 although inertial impactors with from two to fifteen stages are known. The flow through the inertial impactor 10 is caused by a pump 1 15 connected to the outlet 14 of the inertial impactor 10.

[0102] Each stage is separated from adjacent stages by a partition 21 , 31 , 41 , 51 , wherein each partition has at least one orifice 22, 32, 42, 52 (in practice, a plurality of orifices is present in each partition) which directs the air stream 16 towards substrates 33, 43, 53. The major face of each substrate 33, 43,53 is arranged substantially perpendicular to the direction of flow of the air stream 16, and when the particles P exit the at least one orifice 22, 32, 42, 52 they will impact onto the surface of the subsequent substrate 33, 43, 53 and form a neatly contained pile 34, 44, 54 of particles P thereon. This means that if for example the first partition 21 has ten orifices, ten separate piles of particles P will form onto the first substrate 33 in a pattern corresponding to the pattern of the orifices.

[0103] Particles with inertia such that they are unable to follow the air stream when it is deflected around the first substrate 33, will impact onto the first upper substrate 33 while particles with less inertia follow the air stream to the next stage 40. The inertia of a particle depends on its mass which, in turn, depends on its size and density. I n this way, mass-segregation of the particles is possible. Thus, by choosing the number of orifices, their diameter and the distance from orifice to substrate in each stage, mass segregation of the particles contained in the exhaled air of a subject is achieved.

[0104] The substrates 33, 43, 53 comprise or consist of a material that is at least partially water- soluble and / or at least partially transparent. While the exemplary substrates 33, 43, 53 described below may also comprise or consist of a material that is at least partially water- soluble at a temperature greater than 0°C and less than 100°C, it should be noted that at least one of the substrates 33, 43, 53 used in any embodiment of the claimed system or method may be non-dissolvable. Further while the exemplary substrates 33, 43, 53 described below may also comprise or consist of a material that is at least partially transparent, it should be noted that at least one of the substrates 33, 43, 53 used in any embodiment of the claimed system or method may be transparent.

[0105] The at least partially water-soluble and / or at least partially transparent substrates 33, 43, 53 may have a thickness of 0.01 -1 mm . substrate 33, 43, 53 may have any desired shape, such as a sym metrical or non-sym metrical shape. It may have any width from 0.1 m m to 2.5 cm or more.

[0106] A substrate 33, 43, 53 comprising or consisting of polyvinyl alcohol (PVA) , which is both transparent in its solid film form , and water soluble at a temperature greater than 0°C and less than 100°C, is advantageous since PVA is a substance that is generally considered to be safe for humans when used as intended, and it has been reported to be compatible with enzyme-linked im m unosorbent assays ( ELI SA) , wherein ELI SA is a laboratory technique used to detect and quantify specific substances, such as proteins, or other biomolecules, in a biological sample.

[0107] If an at least partially transparent substrates 33, 43, 53 having a relatively high watersolubility and / or a small volume of the at least partially water-soluble substrates 33, 43, 53is used, at least part of such a substrate 33, 43, 53 can be dissolved in a relatively small volume of aqueous solution or other solvent (such as in a volume of 10 microlitres) , which is advantageous since a high concentration of solubilised biomolecules may be needed for certain biochem ical assays.

[0108] A single breathing manoeuvre may provide a sufficient number of particles P, although typically, particles P are collected from repeated breathing manoeuvres. For the diagnosis of medical conditions in humans, particles P may be collected from repeated inhalations / exhalations for a period of time comprising up to several tens of minutes, e.g., between 1 second and 100 m inutes, such as between 1 second and 50 minutes, between 5 seconds and 20 m inutes or between 10 seconds and 5 m inutes.

[0109] A sample of particles P may be collected in the following way. The inertial impactor 10 is loaded with clean substrates 33, 43, 53, and the system 100, including the inertial impactor 10, is operated at a desired temperature. An optical method, such as light scattering or spectroscopy or an optical particle counter may be used to check that no spurious particles are present, e.g., indicating a leak into the system 100 and monitoring the amount of collected particles. Breathing manoeuvres and exhalation into the system 100 then begins. An optical method, such as light scattering or spectroscopy or a particle counter may be used to generate size distribution data at certain set time intervals, such as e.g., every 0.1 second while the inertial impactor 10 collects particles P for subsequent analysis. When a required amount of particles has been obtained, the collection is term inated. The flow through the inertial impactor 10 is turned off, the at least one holding device securing the at least partially water-soluble substrate and / or at least partially transparent substrates 33, 43, 53 inside the inertial impactor 10 is / are removed from the inertial impactor 10 and the at least one part of the at least partially water-soluble and / or at least partially transparent substrates 33, 43, 53 with exhaled particles are recovered from the at least one holding device.

[0110] The at least partially water-soluble substrate and / or at least partially transparent substrates 33, 43, 53 may be analysed using an optical method within the system , in a separate device, or in an online fashion. The analysis may be carried out in real time, during the collection of particles, after the collection of particles, or both during and after the collection of particles. Certain data may be obtained in real time or during the collection of particles, and additional and / or more detailed data may be obtained after the collection of particles.

[0111] Figure 3 shows an exemplary holding device 24 that may be used in the claimed system or method. The holding device system comprises a spacer 301 , a cutting mat 302, an at least partially water-soluble and / or an at least partially transparent substrate 33, 43, 53, a fixation ring 303, a dust cover 304, and an excision tool excision tool guide 305 with steering bores 306.

[0112] During the collection of particles contained in air 16 exhaled by a subject, particles will impact in specific areas on the at least partially water-soluble and / or at least partially transparent substrate 33, 43, 53. The at least partially water-soluble and / or at least partially transparent substrate 33, 43, 53 may be secured by means of the fixation ring 303. Advantageously the fixation ring 303 may be magnetic which facilitates loading and unloading of substrates 33, 43, 53. Advantageously the holding device 210 is made from e.g. , stainless steel, alum inium , or plastic to facilitate cleaning between samples. Alternatively, the holding device 210 can be made of single use but environmentally friendly plastic with a preloaded at least partially water-soluble and / or at least partially transparent substrate to enable the simplest possible usage of the system .

[0113] The holding device 24 illustrated in Figure 3 is configured to secure one at least partially water-soluble and / or at least partially transparent substrate 33, 43, 53 in the inertial impactor 10 during the collection of particles. I n the system according to the present invention one holding device 24 may however be configured to secure a plurality of at least partially water-soluble and / or at least partially transparent substrates.

[0114] If the substrate 33, 43, 53 is at least partly water-soluble, the collected particles do not need to be removed (washed off) from the at least partially water-soluble substrate 33, 43, 53, in order for the collected particles to be further processed for subsequent biochem ical analysis. I nstead, the particles are left on the at least partially water-soluble substrate 33, 43, 53 and particles together with at least one part of the at least partially water-soluble substrate 33, 43, 53 are brought into contact with an aqueous solution or other solvent for solubilisation of the at least partially water-soluble substrate and release / solubilisation of biomolecules for analysis using a suitable analysis technique known in the art.

[0115] Sub-samples of the collected particles P collected on the individual areas of an at least partially water-soluble and / or an at least partially transparent substrate 33, 43, 53 may be individually processed using a sample preparation kit comprising an excision tool guide 305 and an excision tool, such as the excision tool 230 illustrated in Figure 4.

[0116] The excision tool 230, which may be a punch tool, comprises a cutting tip 233 for cutting out and transferring one or more specific areas of the at least partially water-soluble and / or the at least partially transparent substrate 33, 43, 53 on which individual piles of particles P have been collected, to a sample tube or other suitable container or surface, for storage or further processing. The cutting tip 233 may be hollow and its foremost end may be open, and the open end may be provided with sharp cutting edge.

[0117] The diameter of the front end opening on the cutting tip 233 may be slightly larger than the diameter of the piles of particles P that have been collected on the at least partially water-soluble and / or the at least partially transparent substrate 33, 43, 53 (see example of visualisation of the material that is collected on an at least partially water-soluble and / or an at least partially transparent substrate shown in Figure 6) . The excision tool 230 is configured to push the cutting tip 233 through the at least partially water-soluble the at least partially transparent substrate 33, 43, 53 during the excision of the sample and hold the excised piece of the at least partially water-soluble and / or the at least partially transparent substrate with the pile of particles P inside the hollow space of the cutting tip 233. The exhaled particles P are deposited in neat piles on the at least partially water- soluble and / or the at least partially transparent substrate 33, 43, 53 in specific areas during particle collection in the inertial impactor 10. However, since the piles of particles P on the at least partially water-soluble and / or the at least partially transparent substrate 33, 43, 53 are invisible to the naked eye they are difficult to locate on surface of the substrate 33, 43, 53 after impaction. Thus, to facilitate sampling of the piles of particles P from the at least partially water-soluble and / or the at least partially transparent substrate 33, 43, 53, the sample preparation kit includes an excision tool guide 305 that is configured to steer the cutting tip 233 to the exact location where piles of particle P are impacted on the substrate 33, 43, 53.

[0118] The excision tool guide 305 is designed to be mounted in a specific position on the holding device 24 with high spatial precision. The excision tool guide 305, may be provided with one or more guiding bores 306 configured for steering the cutting tip 233 of the excision tool 230 to the exact location of the collected particles P on the at least partially water- soluble substrate and / or at least partially transparent substrate 33, 43, 53.

[0119] When the cutting tip 233 of the excision tool 230 is placed into a guiding bore 306 of the excision tool guide 305, the sharpened cutting edge of the cutting tip 233 is perfectly aligned for cutting and collecting the areas of the at least partially water-soluble and / or at least partially transparent substrate 33, 43, 53. that hold the collected particles P.

[0120] Any suitable device or method may be used to collect the particles P deposited on an at least partially water-soluble and / or at least partially transparent substrate 33, 43, 53. Each pile of particles P may be analysed individually or collectively depending on whether the excised piles are put in one or several test tubes.

[0121] If the at least one partially transparent substrate 33, 43, 53 is also water-soluble, once one or more excised parts of the at least partially water-soluble substrate with particles are placed in an aqueous solution or other solvent, the excised parts of the at least partially water-soluble substrate 33, 43, 53 will dissolve allowing the particle to be released from the substrate and biomolecules in or associated with the particles to be released and solubilised without solid remains of the at water-soluble part(s) of the at least partially water-soluble substrate. The released particles and / or solubilised biomolecules may then be physically, chemically or biochemically analysed.

[0122] The collected particles may be analysed to determine whether one or more biomolecules, which are potential biomarkers of medical conditions are present.

[0123] The holding device 24 also comprises a spacer 301 that fits at the bottom of the groove below the cutting mat 302 and is configured to adjust the vertical level of the surface of the at least partially transparent and / or at least partially water-soluble substrate 33, 43, 53 in the at least one holding device 24. At least one such spacer 30 may thereby be used to allow existing sample holders 24 to be used with at least partially water-soluble and / or at least partially transparent substrates 33, 43, 53 that may be thinner than other previously used substrate types, according to the present invention in inertial impactors 10 without the need of modification. An at least partially water-soluble and / or at least partially transparent substrate 33, 43, 53 may be firm ly secured on the substrate holding platform of the holding device 24 by any suitable means, such as a magnetic locking ring 218.

[0124] Figure 5 schematically shows a method which comprises removing an at least partially transparent and / or an at least partially water-soluble substrate 33, 43, 53 with exhaled particles from the inertial impactor 10. I n the illustrated example, the entire at least partially transparent and / or at least partially water-soluble substrate 33, 43, 53 with exhaled particles is brought into contact with a gel-like surface consisting of water and e.g. polysaccharides, an aqueous solution, or other solvent 32 to dissolve the at least partially transparent and / or at least partially water-soluble substrate 33, 43, 53. The aqueous solution or other solvent 32 may be optim ised for solubilisation of the biomolecules of interest and the biochemical analysis that is to be used.

[0125] The amount of aqueous solution or other solvent 32 that is required depends on the amount of substrate material that is to be dissolved and the water solubility of the substrate material. Collected articles, P, and the biomolecules or other analytes associated with the particles P, will solubilise in the aqueous solution or other solvent 32 and may then be analysed using any suitable biochem ical or other analysis technique.

[0126] Alternatively, the method comprises punching out at least one part of an at least partially water-soluble and / or an at least partially transparent substrate 33, 43, 53 with exhaled particles, using for example the sample preparation kit previously described. And bringing the at least one excised part into contact with the aqueous solution or other solvent 32.

[0127] Solubilisation can be made in an aqueous solution or other solvent 32 of choice, such as an aqueous solution or other solvent 32 that is optimised for the analysis technique in question. With an at least partially transparent and / or an at least partially water-soluble substrate 33, 43, 53, it not only become possible to adjust the concentration of collected particles P by dissolving the at least partially transparent and / or the at least partially water-soluble substrate 33, 43, 53 or excised or cut out pieces of the at least partially transparent and / or the at least partially water-soluble substrate 33, 43, 53 in a volume that is proportional to the amount of collected particles, P, but it is also possible to adj ust the concentration of the molecules that constitute the at least partially transparent and / or the at least partially water-soluble substrate 33, 43, 53, so that all samples in a study not only have the same concentration of collected particles, P, but also are equal with regard to the relation between amount of collected particles, P, and the amount of at least partially transparent and / or the at least partially water-soluble substrate 33, 43, 53 molecules. With a solid, non-water-soluble substrate this is not possible to achieve.

[0128] Figure 5 shows part of a method according to the present invention in which the at least partially transparent and / or the at least partially water-soluble substrate 33, 43, 52 is brought into contact with an aqueous solution or other solvent 32 in a sample tube 500 by placing the at least partially transparent and / or the at least partially water-soluble substrate 33, 43, 52 into a sample tube 500 and then adding an aqueous solution or other solvent 32 to solubilise the at least partially transparent and / or the at least partially water- soluble substrate 33, 43, 52, releasing and disintegrating the particles P, and thereby providing an efficient means for solubilising the biomolecules in, or associated with the particles P, in absence of any solid surfaces and without remains of the solid substrate that would otherwise need to be separated from the solution of biomolecules. The latter not only elim inates the problem of unwanted absorption of solubilised biomolecules to solid surfaces, but it also allows for a 100% recovery of the solubilisation volume as there is no solid material left behind that can withhold some of the liquid by capillary forces.

[0129] An at least partially transparent substrate may be analysed using an optical method within the claimed system , in a separate device, or in an online fashion. The analysis may be carried out in real time, during the collection of particles P, after the collection of particles P, or both during and after the collection of particles P. Certain data may be obtained in real time or during the collection of particles P, and additional and / or more detailed data may be obtained after the collection of particles P.

[0130] Pilot experiment

[0131] Table 1 shows the results obtained in a pilot experiment that was carried out to determ ine the practicality and viability of using various at least partially water-soluble and / or at least partially transparent films as substrates in a system and method according to the present invention. The following experiments were carried out.

[0132] Seven different commercially available PVA granular powder chemical products were tested for ease of production of thin films, detachment, durability, handling, transparency and solubility of the resulting polymer film product, each with a different combination of molecular weight and degree of hydrolysis. According to Safety Data Sheets provided by the manufacturer, none of the tested PVA products contained any substances which, at their given concentration, are considered to be hazardous to humans.

[0133] Ten millilitres of a 1 % and a 2% PVA solution was poured into levelled petri dishes (made from polystyrene and having a diameter of 87 mm) . The solution was allowed to dry and polymerise at room temperature for at least 72 hours in the petri dishes under a piece of clean paper towel. After complete drying, the resulting thin films were detached by peeling and dragging them from one edge of the petri dish.

[0134] The ease of handling the films was evaluated by detaching, stretching and bending the films. A film was considered to be suitable for the intended usage if it did not easily break or crack on being stretched or bent but was still rigid enough to allow discs having a diameter of 1 .2 m m to be excised therefrom by punching, the punching being performed on top of a standard cutting mat, using a standard punch tool such as the one described in the sample preparation kit disclosed in international publication number WO 2022 / 223420. For the at least partially transparent films, the transparency of the films was evaluated by visual inspection.

[0135] A small part of each at least partially water-soluble and / or at least partially transparent PVA film was placed on top of a filter paper containing a moisture colour indicator. A 5 pl drop of water was applied to the top of the film surface and allowed to penetrate the film under the force of gravity. The time it took until the water dissolved the at least partially water-soluble and / or at least partially transparent film and caused the moisture colour indicator to change colour was recorded. The test was performed at room temperature. Due to the difficulties of levelling the petri dishes with a spirit level, the petri dishes were not found to be completely level, resulting in some variations in the thickness of the at least partially water-soluble and / or at least partially transparent films for which the watersolubility was tested. All of the at least partially water-soluble and / or at least partially transparent films dissolved quickly enough for the intended application. Based on the short time that it took for the at least partially water-soluble and / or at least partially transparent films to dissolve and the ease with which the at least partially water-soluble and / or at least partially transparent films could be handled, films made from PVA sample # 4 in Table 1 were chosen for further optim isation and evaluation of the manufacturing process.

[0136] Since distal airways represent a very large luminal surface area and since the tissue temperature of a human subject is usually 37°C, dry air that enters the airways of a human subject quickly becomes saturated with gaseous water molecules, resulting in the human subject’s exhaled air being moist. It is therefore essential that a substrate comprising or consisting of an at least partially water-soluble material and / or an at least partially transparent substrate, such as PVA, does not dissolve or become gel-like during the collection of particles in an inertial impactor so that any excising and recovery of at least one part of the at least partially water-soluble and / or at least partially transparent substrate is not compromised.

[0137] A disc having a diameter of 25 mm was punched-out from the at least partially water- soluble and / or at least partially transparent film made from 2% PVA, sample # 4 in Table 1 to make a substrate suitable for use in the claimed system and method. The at least partially water-soluble and / or at least partially transparent substrate was loaded into a holding device of an inertial impactor and 120 ng of particles were collected from a healthy volunteer using the system and method described herein. The areas of the substrate on which the particles had impacted were punched out using a standard procedure but with the addition of three light turns of the punch tool to achieve a relatively forceless, gradual circumferential cutting of the at least partially water-soluble and / or the at least partially transparent substrate before a final more forceful punch through the at least partially water-soluble and / or the at least partially transparent substrate and into a cutting mat was carried out.

[0138] As can be seen in Table 1 , film produced from a 2% solution of PVA having a molecular weight from 31 -50 g / mol and a degree of hydrolysation between 87-89% , and which had been poured into a petri dish until the solution in the petri dish reached a volume of 0.168 m l / cm2, had the most favourable characteristics. Although several films met the requirements for use in a system or method according to the present invention, sample # 4 in Table 1 was found to be the most suitable of the products tested and was therefore subjected to further tests aiming to develop a feasible low scale manufacturing process.

[0139] Test of simple manufacturing method

[0140] Sixteen grams of PVA granular powder of sample # 4 was added to and dissolved in a one litre flask with 800 ml Milli-Q water under “clean room” conditions, to reach a concentration of 2% . The PVA powder was added to the Milli-Q water under vigorous magnetic stirring at room temperature. After the powder was dispersed, the flask was heated in a m icrowave oven for 4 m inutes avoiding overheating and boiling. When the flask was warm , it was moved to a heated magnetic stirring plate set at 100 °C until all of the powder was fully dissolved (as judged by visual inspection) . While still warm , the PVA solution was sterile- filtered through a Nalgene Rapid-Flow™ sterile disposable 0.2 pm filter unit. Six sterile Nunc™ Square BioAssay Dishes (245 x 245 x 25 m m) were positioned in a lam inar flow hood and 80 m l of the PVA solution was poured into each of the six dishes, reaching volume of 0.1 68 m l / cm2. To avoid contam ination from particles in am bient air, the BioAssay Dishes were covered by a Kim berly Clark KI MTECH Science Dry Cleanroom Wipe and m oved to a laboratory benchtop perfectly levelled with a professional spirit level. This process required less than a total of one hour of hands-on tim e.

[0141] The poured BioAssay dishes were allowed to rem ain on the benchtop for at least 72 hours, until the resulting films had becom e completely dry. Under a lam inar flow hood, the surface of standard polypropylene document folders was wiped with Kim berly Clark KI MTECH Science Dry Cleanroom Wipes wetted with RNase AWAY™ Surface Decontam inant and allowed to dry. The PVA films were detached from the BioAssay dishes under the lam inar flow hood by carefully loosening and dragging each film from one side and transferring the film into onto the inside surface of a clean polypropylene docum ent folder.

[0142] The polypropylene docum ent folders were sealed using adhesive tape at each open side of the docum ent folder. Sandwiched circular discs having a diam eter of 25 m m were punched out from the PVA film sandwiched between the polypropylene docum ent folder material using a pneum atic excision tool in such way that each disc sandwich fell directly into a sterile 50 m l falcon tube. All of the surfaces of the pneum atic puncher tool and the outside surfaces of the polypropylene folders were carefully wiped with Kim berly Clark KI MTECH Science Dry Cleanroom Wipe wetted with RNase AWAY™ Surface Decontam inant and allowed to dry before the 25 m m disc sandwiches were punched out.

[0143] Up to 58 disc sandwiches could be punched form each PVA film in less than 10 m inutes meaning that at least 340 PVA substrates could be produced in a total of approxim ately 2 hours hands-on tim e. The thickness of a stack of 30 pieces, cut from random ly selected locations of a sheet of the resulting PVA film was measured with a m icrom etre screw gauge and the average thickness of the substrates were found to be 45 pm . The film was too thin to allow for reliable measurem ents of each individual pieces with the m icrom etre screw gauge.

[0144] The concentration of PVA in a substrate punched from the m anufactured PVA film m ay be calculated as follows. Given that the film in one BioAssay dish (approx 60000 m m2) was cast using 1 .6 g of PVA, a single punched 1 .2 m m disc ( having an area of 1 .13 m m2) contains 0.03 mg of PVA. Dissolving ten of these discs in 100 pl of water means that the concentration of PVA will be 0.3 mg / 100 pl (W / V= 0.3% ) or, based on the m olecular weight of the PVA, (that has a mean molecular weight of 40 g / mol) a 100 pl sample would hold a molar concentration of 0.075 M.

[0145] This test, which constitutes an initial feasibility test including a method for manufacturing PVA films having an area of 245 x 245 m m , gave surprisingly good results. It was efficient enough to produce a sufficient number of substrates for conducting small scale pilot studies.

[0146] Furthermore, safety data sheets and published studies report that PVA is non-toxic to humans and compatible with affinity-based assays like ELISA at 0.5 %(W / V) . (See, for example, the article entitled “Enzyme-Linked I mm unosorbent Assay for Detection of Antibodies to Virus-Like Particles of Human Papillomavirus” by Yevgeniy et el. , which was published in the Journal of Clinical Microbiology 2002 May; 40(5): 1755-1760.) As the feasibility test showed that a PVA concentration of 0.3 % was enough to meet the required physical properties of an least partially water-soluble and / or an at least partially transparent substrate as claimed, and to provide a substrate which is likely to be suitable for collection of samples aimed for affinity-based protein analysis-based system .

[0147] The produced PVA films were found to be fully transparent giving rise to the following features of the invention, as described below.

[0148] A total of 100 ng of exhaled particles from a healthy subject were collected on one of the manufactured at least partially water-soluble and / or at least partially transparent substrates. The at least partially water-soluble and / or at least partially transparent substrate was placed on a dark background and illuminated from beneath and, when viewed at an angle, ten white dots emerged very clearly, five of which can be seen in the photograph shown in Figure 6. The optical effect can most likely be attributed a light scattering effect. To exclude that the light scattering based visualisation was not an effect of an alteration of structure in the PVA polymer film , the white dots were touched and scraped with a cotton bud during illum ination. I n doing so, it was observed that the white dots disappeared, proving that material was located on top of the surface of the substrate.

[0149] The implication of this discovery is that particles collected on an at least partially water- soluble and / or a at least partially transparent substrate according to the present invention may be quantified and / or characterised by for instance image-based densitometry, spectroscopic quantifications or confocal m icroscopy, or by other means without the need for a particle counter, which facilitates the manufacture of a smaller and cheaper system for collecting and measuring the amount of particles contained in exhaled air. I mportantly, the latter allows for a inter individual normalisation of biomarker data. Preferences and options for a given aspect, feature or parameter of the invention should, unless the context indicates otherwise, be regarded as having been disclosed in combination with any and all preferences and options for all other aspects, features and parameters of the invention. For example, any feature disclosed with respect to the system may be regarded as having been disclosed in combination with the method and vice versa.

[0150] The listing or discussion of an apparently prior-published document in this specification should not necessarily be taken as an acknowledgement that the document is part of the state of the art or is com mon general knowledge.

[0151]

Claims

CLAI MS1 . A system (100) for collecting particles (P) contained in air (16) exhaled by a subject for analysis comprising: an inertial impactor (10) configured to sort and collect particles (P) contained in air (16) exhaled by a subject according to their mass, the inertial impactor (10) comprising: o at least one substrate (33, 43, 53) for collecting particles (P) , and o at least one holding device (24) for securing the at least one substrate (33, 43, 53) in the inertial impactor (10) during the collection of particles (P) , wherein the at least one substrate (33, 43, 53) comprises or consists of a material that is at least partially water-soluble at a temperature greater than 0°C and less than 100°C and / or the at least one substrate (33, 43, 53) comprises or consists of a material that is least partially transparent.

2. The system (100) according to claim 1 , wherein the at least one substrate (33, 43, 53) :- comprises or consists of at least one of the following substances: The at least one substrate may comprise or consist of at least one of the following materials: Poly(2-ethyl- 2-oxazoline) , Poly(2-hydroxyethyl methacrylate / methacrylic acid) , Poly (2- vinyl- 1 - methylpyridinium bromide) , Poly(2- viny Ipy ridine N-oxide) , Poly (4- vinylpyridine) , Poly(Acrylic Acid) PAA, Poly(allylamine) , Poly(ethylene oxide) PEO , Poly(propylene oxide) PPO, Poly(ethylene oxide-b-propylene oxide) , Poly( I- lysine hydrobrom ide) , Poly(maleic acid) , Poly(methacrylic acid) , Poly(N-isopropylacrylam ide) , Poly(N- vinylpyrrolidone) , Poly(N-vinylpyrrolidone / vinyl acetate) , Poly(oxyethylene) , Poly(styrenesulfonic acid) , Poly(vinyl acetate) , Poly(vinyl alcohol) PVA, Poly(vinyl methyl ether) , Poly(vinyl phosphoric acid) , Poly(vinylam ine) , Poly(vinylphosphonic acid) , Poly(vinylsulfonic acid) , Polyacrylamide, polyacrylic-acid-63-soln-in-water, Poly-ethylene- oxide, Poly-l-lysine-hydrobrom ide, Poly-methacrylic-acid, collagen, gelatine, salt, polysaccharides, or any other organic or non-organic, synthetic or non-synthetic, at least partially water-soluble, and / or at least partially transparent polymer or substance, and / or- has a maximum thickness of 1 m m , and / or- comprises or consists of a material that has a water solubility of at least 0.1 g / L at a temperature greater than 0°C and less than 100°C.

3. The system (100) according to claim 1 or claim 2, wherein the at least one holding device comprises at least one spacer (30) configured to adj ust the vertical level of the at least one substrate (33, 43, 53) in the at least one holding device (24) .

354. A method for collecting particles ( P) contained in air (16) exhaled by a subject for analysis comprising: collecting particles (P) from the subject, sorting the particles (P) according to their mass using an inertial impactor (10) comprising: o at least one substrate (33, 43, 53) for collecting particles (P) , and o at least one holding device (24) for securing the at least one substrate (33, 43, 53) in the inertial impactor (10) during the collection of particles (P) , wherein the method comprises using at least one substrate (33, 43, 53) that comprises or consists of a material that is at least partially water-soluble at a temperature greater than 0°C and less than 100°C and / or the method comprises using at least one substrate (33, 43, 53) that comprises or consists of a material that is at least partially transparent.

5. The method according to claim 4, wherein the method comprises using at least one substrate (33, 43, 53) :- comprises or consists of at least one of the following substances: The at least one substrate may comprise or consist of at least one of the following materials: Poly(2-ethyl- 2-oxazoline) , Poly(2-hydroxyethyl methacrylate / methacrylic acid) , Poly (2- vinyl- 1 - methylpyridinium bromide) , Poly(2- viny Ipy ridine N-oxide) , Poly (4- vinylpyridine) , Poly(Acrylic Acid) PAA, Poly(allylamine) , Poly(ethylene oxide) PEO , Poly(propylene oxide) PPO, Poly(ethylene oxide-b-propylene oxide) , Poly( I- lysine hydrobrom ide) , Poly(maleic acid) , Poly(methacrylic acid) , Poly(N-isopropylacrylam ide) , Poly(N- vinylpyrrolidone) , Poly(N-vinylpyrrolidone / vinyl acetate) , Poly(oxyethylene) , Poly(styrenesulfonic acid) , Poly(vinyl acetate) , Poly(vinyl alcohol) PVA, Poly(vinyl methyl ether) , Poly(vinyl phosphoric acid) , Poly(vinylam ine) , Poly(vinylphosphonic acid) , Poly(vinylsulfonic acid) , Polyacrylamide, polyacrylic-acid-63-soln-in-water, Poly-ethylene- oxide, Poly-l-lysine-hydrobrom ide, Poly-methacrylic-acid, collagen, gelatine, salt, polysaccharides, or any other organic or non-organic, synthetic or non-synthetic, at least partially water-soluble, and / or at least partially transparent polymer or substance and / or- has a maximum thickness of 1 m m , and / or- comprises or consists of a material that has a water solubility of at least 0.1 g / L at a temperature greater than 0°C and less than 100°C.

6. The method according to claim 4 or 5 wherein the method comprises placing at least one spacer (30) in the at least one holding device (24) to adjust the vertical level of the at least one substrate (33, 43, 53) in the at least one holding device (24) in an inertial impactor.

7. The method according to any of claims 4 to 6 wherein the method comprises dissolving at least part of the at least partially water-soluble substrate with exhaled particles (33, 43, 53) in an aqueous solution or other solvent (32) :- by allowing at least one part of the at least partially water-soluble substrate with exhaled particles (33, 43, 53) to come into contact with an aqueous solution or other solvent (32) , or- by excising at least one part of the at least partially water-soluble substrate with exhaled particles (33, 43, 53) and transferring the at least one excised part to the aqueous solution or other solvent (32) .

8. The method according to any of claims 4 to 7 wherein the method comprises quantifying and / or characterising and / or analysing exhaled particles impacted on the at least one at least partially transparent substrate (33, 43, 53) using an optical method.

9. Use of at least one substrate (33, 43, 53) that comprises or consists of a material that is water-soluble at a temperature greater than 0°C and less than 100°C and / or at least partially transparent in a system or method comprising an inertial impactor (10) configured to sort and collect particles (P) contained in air (16) exhaled by a subject according to their mass.

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