Method for separating molecules and / or molecular complexes

Abstract

The invention relates to a method for in vitro separation of molecules and / or molecular complexes having a gyration radius of less than or equal to 2 pm from a complex fluid, said method comprising the steps of: a) contacting a complex fluid with a structured capture array having topographical features in the form of a plurality of planar surfaces located between cavities, wherein the structured capture array is surrounded by humid air; b) covering the deposited complex fluid with a covering device such that the complex fluid is surrounded by a meniscus ring comprising a back meniscus and a front meniscus, wherein the surface tension of the complex fluid located between the covering device and the structured capture array defines at least the front meniscus and the back meniscus; c) dragging the covering device or the structured capture array in one direction at a speed of at most 2 mm.s' ‑1 The front meniscus and the back meniscus are displaced on and along the topographical features of said structured capture array towards said direction, wherein during the displacement of the complex fluid, the front meniscus covers exposed topographical features and the back meniscus exposes covered topographical features, resulting in: the molecules and / or the molecular complexes being trapped inside the cavities.

Description

[0001] This invention relates to the separation of molecules and / or molecular complexes.

[0002] Currently, cancer diagnosis and prognosis largely rely on solid tissue biopsies, which involve surgically obtaining a small portion of the tumor. These biopsies are used for various analyses, such as cellular state or DNA mutations. However, this technique exhibits limitations for several reasons, specifically because:

[0003] This technique is highly invasive, meaning it is uncomfortable for the patient and requires punctures at intervals.

[0004] This technique is limited to visible tumors (impossible in micrometastases); some tumors cannot be sampled; puncture is limited by space and time (it may only be performed once from a portion of the tumor); and it implies certain risks to the patient (infection, release of tumor material into the bloodstream, etc.).

[0005] - The results do not reflect the genetic heterogeneity of the tumor because different parts of the tumor accumulate different genetic mutations. When sampling only one region of the tumor, it is not possible to sequence and detect all mutations.

[0006] Considering the above, liquid biopsy appears to be an effective alternative. In fact, liquid biopsy is less invasive, can be repeated more frequently, and allows for the recovery of all genetic mutations in the tumor. Furthermore, liquid biopsy would enable more regular tracking of the entire tumor and could be a potential tool for early diagnosis. Additionally, potential cancer biomarkers, such as circulating tumor cells (CTCs), miRNAs, circulating DNA (cDNA), and exosomes, are located in many biological fluids such as saliva, urine, blood, and their derivatives (plasma and serum).

[0007] While saliva is the most easily recyclable biofluid, blood appears to be a richer and safest source when the location of the tumor is unknown. In fact, although 100% of oral cancer patients have cDNA in their saliva and 80% have cDNA in their blood, only 47% to 70% of patients with other types of cancer have been reported to have cDNA in their saliva.

[0008] In blood, circulating DNA can be single-stranded or double-stranded, and it can be nuclear, mitochondrial, or viral in origin. Cell-free DNA is typically extracted from plasma or serum using centrifugation protocols. Extraction from serum yields a larger amount of DNA, but this observation may be attributed to the lysis of leukocytes or other cellular contaminants, which then disperse their DNA into solution. Extraction protocols can also vary between studies, meaning variations in the amount of DNA obtained and its conditions. Furthermore, the time and temperature at which samples are stored prior to analysis, as well as the duration of the extraction process, can affect the degradation of cell-free genetic material in the sample, or even influence the release of nucleic acids from other cellular components.

[0009] To optimize the extraction protocol, blood samples need to be collected in tubes containing EDTA, kept cool, and processed within 2 hours of collection to minimize contamination, which is highly limiting.

[0010] The objective of this invention is to eliminate these drawbacks.

[0011] The purpose of this invention is to provide a method for easily separating molecules and / or molecular complexes.

[0012] Therefore, the present invention relates to a method for the in vitro separation of molecules and / or molecular complexes with a radius of rotation less than or equal to 2 μm, particularly from complex fluids, the method comprising the following steps:

[0013] a) Contacting a complex fluid with a structured trapping array having morphological features in the form of multiple planar surfaces located between cavities, wherein the structured trapping array is surrounded by humid air.

[0014] b) Cover the deposited complex fluid with a covering device, wherein the surface tension of the complex fluid located between the covering device and the structured trapping array defines at least the forward and backward menisci.

[0015] c) up to 2mm.s' -1 The covering device or the structured trapping array is dragged in one direction at a speed to displace the complex fluid, wherein the leading and trailing menisci are displaced over and along the topographic features of the structured trapping array toward the direction described above, wherein during the displacement of the complex fluid, the leading menisci cover the exposed topographic features and the trailing menisci expose the covered topographic features, resulting in:

[0016] Molecules and / or molecular complexes are trapped inside the cavity, and

[0017] It may extend on a flat surface in the direction of dragging, wherein the humid air has a humidity of at least 40% based on the maximum humidity of the air.

[0018] The method of this invention advantageously separates low molecular weight biomarkers, such as biomarkers (DNA, exosomes, RNA, proteins), from the entire raw complex fluid in a single step. The method of this invention can advantageously separate several (biological) biomarkers simultaneously based on physical criteria such as radius of gyration. The separation can advantageously be performed on the raw complex fluid without any pretreatment. This invention can be implemented in a wide variety of applications, from marine science to healthcare.

[0019] In this invention, "molecule" refers to a group of two or more atoms bonded together by covalent bonds. This term encompasses naturally occurring molecules found in living organisms and synthetic molecules, such as those produced by the chemical industry.

[0020] In this invention, a “molecular complex” refers to several molecules linked together by non-covalent bonds.

[0021] The radius of gyration is a dimensionless measure of the spatial size of an object in rotational motion. This mechanical concept has been extended to polymer physical properties, thus allowing the specific dimensions of a polymer in solution to be described as a function of its total length, its degree of polymerization, or its molecular weight. This specific dimension depends on the molecular interactions along the polymer chains and varies depending on the properties of the monomer and the solvent. The radius of gyration of molecules and molecular complexes can be measured using physical methods based on the propagation of electromagnetic waves or neutrons. Examples of such physical methods are given in: D.G. Ballard et al., *European Polymer Journal*, 9, 9, 1973, 965-969.

[0022] In this invention, a complex fluid is defined as a liquid suspension comprising a complex mixture of various components such as molecules, macromolecules, polymers, cells, particles, and aggregates. This complex fluid is a non-Newtonian fluid and deviates from the classical linear Newtonian relationship between stress and shear rate. Due to the geometric constraints imposed by phase coexistence, they exhibit unusual mechanical responses to applied stress or strain. These mechanical responses include transitions between solid-like and fluid-like behaviors, as well as fluctuations.

[0023] In this invention, "simple fluid" refers to a Newtonian fluid whose mechanical properties are characterized as a single function of temperature, viscosity, and fluid "slipperiness." The stress applied to a simple fluid is proportional to the strain rate.

[0024] In this invention, "humidity" refers to the ratio (as a percentage) of the partial pressure of water vapor to the maximum humidity of the air. Maximum humidity corresponds to the equilibrium vapor pressure of water at a given temperature. Humidity can be measured using a hygrometer. Maximum humidity can be estimated using several empirical formulas known in the art. A commonly used formula is the Arden-Buckle equation.

[0025] Step a )

[0026] The objective of the first step of the method of the present invention is to contact a complex fluid containing molecules and / or molecular complexes to be separated with a trapping array having a cavity in which the molecules and / or molecular complexes will be separated.

[0027] Such simple contact is insufficient to trigger the separation of molecules and / or molecular complexes into the cavity of the trapping array. In fact, the probability of self-separation of molecules and / or molecular complexes is very low. Specifically, due to the inherent large amount of motion within complex fluids, which drives molecules and molecular complexes in unpredictable ways in all directions, making it difficult or even preventing their separation on the trapping array.

[0028] The method of this invention is carried out in an environment with humid ambient air. The humidity of the ambient air is a critical parameter for separating molecules and / or molecular complexes. In fact, if the ambient air is not sufficiently humid, i.e., does not have a humidity of at least 40% based on the maximum water content of the air, complex fluxes become dry and their displacement becomes very difficult or even impossible. Furthermore, dry ambient air (i.e., humidity below 40%) makes the transfer of the separated molecules and / or complexes toward the printing surface difficult, or even hinders, the transfer of the separated molecules and / or complexes toward the printing surface for subsequent analysis / detection. The latter will be disclosed in more detail later.

[0029] Specifically, the humidity is 40% to 80% based on the maximum moisture content of the surrounding air. "40% to 80%" in this invention means 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, and 80%.

[0030] Specifically, the humidity is 40% to 60% based on the maximum moisture content of the surrounding air, and more particularly 43% to 55% based on the maximum moisture content of the surrounding air.

[0031] Advantageously, the structured capture array is placed in a chamber, such as an airtight chamber.

[0032] Step b )

[0033] The complex fluid is then completely covered by the covering device. The surface of the complex fluid located between the covering device and the capture array is not planar and is curved due to the surface tension of the complex fluid. Therefore, the surface of the complex fluid on the capture array is higher than the surface of the complex fluid in contact with the covering device. The angle formed by the curved surface and the capture array then closes and affects the capture efficiency, as will be described in more detail below.

[0034] This curved surface is called a meniscus. The complex fluid is surrounded by a meniscus, which includes a rear and a front portion, namely the so-called rear and front menisci. The front and rear menisci are defined by the direction of dragging step c). As the complex fluid moves, the front meniscus is in front, and the rear meniscus is in the back.

[0035] Step c )

[0036] In this step, the complex fluid is moved by dragging the covering device or the trapping array, advantageously at a constant speed. Importantly, throughout step c), the complex fluid is held between the covering device and the trapping array by surface tension.

[0037] The motion of complex fluids leads to the efficient separation of molecules and / or molecular complexes via hydrodynamic mechanisms. More specifically, the inventors unexpectedly discovered that the displacement of the complex fluid at the surface of the morphologically structured array results in the generation of a simple fluid at the back-bend meniscus, termed the depletion region, which is analogous in nature to the generation of plasma from a blood sample. More specifically, the depletion region is located at the junction between the back-bend meniscus, the structured trapping array, and the surrounding moist air, a junction known as the triline. This depletion region contains high concentrations of low molecular weight molecules and / or molecular complexes with a gyration radius of less than 2 μm, including the molecules and / or molecular complexes of interest that must be separated.

[0038] Therefore, the molecules and / or molecular complexes of interest are contained within the simple fluid (depletion region) and separated from the other larger components of the complex fluid (i.e., components with radii of gyration greater than 2 μm). Thus, after step c), the other larger components are not separated in the structured trap array and remain in the complex fluid. Then, all components of the complex fluid are at least partially separated according to their radii of gyration. Components with radii of gyration less than 2 μm but greater than one of the molecules and / or molecular complexes of interest can also be interestingly separated on the structured trap array, thanks to the drag velocity used during this step, as described in detail below.

[0039] The separation of molecules and / or molecular complexes on the trapping array is made possible only by the generation of this simple fluid at the leading-edge meniscus. This is specifically due to the aforementioned various motions within the fluid, which drive molecules and molecular complexes in unpredictable ways in all directions.

[0040] Conversely, in simple fluids, predictable and controlled flow drives molecules and molecular complexes in a repeatable direction near the front of the meniscus. In this invention, as this simple fluid passes over the morphological feature at the tri-line, molecules and / or molecular complexes are pushed towards the bottom of the cavity by capillary forces until the front meniscus has completely passed. Once the front meniscus has completely passed, the molecules and / or molecular complexes remain trapped in the cavity along with a small amount of simple fluid. For molecules and molecular complexes in chain form, such as polymers or DNA, one end of the chain may be trapped in one of the cavities, while the remaining portion of the molecule containing the opposite end extends out of one of the cavities along the dragging direction on a planar surface.

[0041] In one embodiment of the present invention, the molecule is a biomolecule. Specifically, the biomolecule is a nucleic acid molecule. Specifically, the nucleic acid molecule is selected from the group consisting of: viral nucleic acid molecules, chromatin, circulating cell-free DNA, RNA, linear DNA, linear RNA, circular DNA, circular RNA, single-stranded DNA, double-stranded DNA, DNA containing G-quadruplex helices, triple-stranded DNA, and tumor DNA.

[0042] In one embodiment of the invention, the molecular complex is a biological complex. Specifically, the biomolecular complex is selected from the group consisting of: vacuoles, lysosomes, transport vesicles, secretory vesicles, liposomes, extranuclear particles, microvesicles, viruses, viral fractions, exosomes, and large complexes.

[0043] In one embodiment of the invention, the complex fluid is an individual biological fluid. Specifically, the biological fluid is selected from the group consisting of cerebrospinal fluid, pleural effusion, saliva, urine, blood, plasma, and serum. In particular, the biological fluid is blood.

[0044] The method of the present invention can be implemented with raw complex fluids, i.e. complex fluids that have not undergone any physical or chemical treatment or have had one or more compounds (in solid, liquid or gaseous form) added.

[0045] Alternatively, before or during step a), the complex fluid is blended with a surfactant, specifically a nonionic surfactant. The addition of the nonionic surfactant reduces the surface tension of the complex fluid. Therefore, the angle formed by the meniscus of the complex fluid and the trapping array is more closed than when the nonionic surfactant is absent. This more closed angle of the meniscus improves trapping efficiency because molecules and / or molecular complexes in the depletion region at the rear meniscus are closer to the cavity and better propelled into the cavity by hydrodynamic flow.

[0046] The capture efficiency can be defined as the ratio between the number of cavities occupied by molecular complexes and the total number of cavities exposed by the backbend of the meniscus.

[0047] In particular, before or during step a), the complex fluid is blended with 0.1% v / v to 0.5% v / v Triton X100, especially 0.3% v / v Triton X100. "0.1% v / v to 0.5% v / v" in the present invention means 0.10% v / v, 0.11% v / v, 0.12% v / v, 0.13% v / v, 0.14% v / v, 0.15% v / v, 0.16% v / v, 0.17% v / v, 0. 18% v / v, 0.19% v / v, 0.20% v / v, 0.21% v / v, 0.22% v / v, 0.23% v / v, 0.24% v / v, 0.25% v / v, 0.26% v / v, 0.27% v / v, 0.28% v / v, 0 .29% v / v, 0.30% v / v, 0.31% v / v, 0.32% v / v, 0.33% v / v, 0.34% v / v, 0.35% v / v, 0.36% v / v, 0.37% v / v, 0.38% v / v, 0.39% v / v, 0.40% v / v, 0.41% v / v, 0.42% v / v, 0.43% v / v, 0.44% v / v, 0.45% v / v, 0.46% v / v, 0.47% v / v, 0.48% v / v, 0.49% v / v and 0.50% v / v.

[0048] In one embodiment of the invention, prior to or during step a), a complex fluid is mixed with at least one labeling agent configured to attach to molecules and / or molecular complexes to be captured. The labeled molecules and / or molecular complexes can then be detected using an appropriate detection method based on the labeling agent used. Labeling agents and appropriate detection methods are well known to those skilled in the art.

[0049] Specifically, the at least one labeling agent can be an antibody or a dye. The labeling agent can carry the detection agent or be recognized by a second labeling agent carrying the detection agent. The detection agent is detected using an appropriate detection method. The detection agent can be a dye.

[0050] For example, when it is necessary to isolate nucleic acid molecules, the labeling agent can be selected from the group consisting of: YOYO of Formula 1. TM -1 fluorescent dye, T-(4,4,8,8-tetramethyl-4,8-diazaundecremethylene)bis[4-[(3-methylbenzo-1,3- [[2-yl]methylene]-1,4-dihydroquinoline Tetraiodide. Other compounds may also be used, such as DAPI, Hoechst33258, nucleic acid staining agents, and genetic / epigmoid probes that recognize genetic sequences and epigenetic markers.

[0051] In cases where exosomes must be isolated, the labeling agent can be an anti-CD63 antibody or an anti-C81 antibody conjugated to a detection agent, which can be a fluorescein-based dye or any other fluorescent dye.

[0052] The radius of gyration (Rg) of unmodified DNA molecules (i.e., naked DNA molecules) in solution can be readily estimated using a random coil model, provided that these molecules contain a sufficient number of nucleotides (the number of base pairs (nbp) is greater than 1000). In this invention, "naked DNA molecule" means a DNA molecule that does not contain any proteins (such as histones). This relationship is given by the following equation:

[0053]

[0054] For λ phage DNA, nbp equals 48502, and a radius of gyration of 1.28 μm is obtained.

[0055] For circulating DNA, which consists of both small and long fragments, the radius of gyration ranges from 500 nm to 4 μm. Therefore, the radius of gyration for proteins ranges from 1 nm to 20 nm. Tumor microRNAs are small molecules, and their radius of gyration is close to that of small proteins (1 nm).

[0056] Chromosomal DNA fragments released by cells in the form of chromatin chains or nucleosome chains are theoretically more complex to describe because of the combination of DNA chains with histones surrounding them. However, for small tumor fragments containing one or two histones (typical size of a histone octamer: 11 nm), the radius of gyration is on the order of approximately 10 nm to 20 nm. To give an idea of ​​the expected range of gyration variation, a 1000 nbp chromatin chain has a typical radius of gyration of approximately 100 nm, while a 1 Mbp chromatin chain has a typical radius of gyration of approximately 500 nm. Therefore, for chromosomal fragments, the radius of gyration ranges from 10 nm to 1 μm.

[0057] For proteins in solution, the radius of gyration depends on their three-dimensional conformation. For small proteins with a molecular weight of around 10,000 Daltons, this radius of gyration is 1 nm. For larger proteins with a molecular weight of several million Daltons, the radius of gyration is around 20 nm.

[0058] When the extracted exosomes were further observed using an electron microscope, their shape was found to be spherical. Therefore, their radius of gyration is very close to the radius of this sphere. Current literature reports radii of gyration ranging from 15 nm to 150 nm.

[0059] To define the size (width or diameter and depth) of the cavity of the capture array, and to determine the drag speed during step c), the radius of gyration of the molecules and / or molecular complexes to be captured is the most relevant parameter.

[0060] In fact, when the cavities are circular and the radius of each cavity is comparable to the radius of gyration of the molecule and / or molecular complex, more efficient trapping of molecules and / or molecular complexes is achieved on the morphological surface. In fact, the probability of trapping on the morphological surface is highest when their sizes are equal. When the cavity size is smaller than the size of the molecule and / or molecular complex, these cavities are too small for the molecule and / or molecular complex to enter. When the cavity size is larger than the size of the molecule and / or molecular complex, the probability of the molecule and / or molecular complex being released from the cavity toward the depletion region increases, and more than one molecule and / or molecular complex can be trapped in a single cavity, complicating subsequent quantification / analysis. However, as described below, the use of a combination of nanopores and micropores improves the trapping of components of interest with nanoscale radii of gyration, not to mention in micropores.

[0061] The cavities of a structured trap array can be nanopores and / or micropores.

[0062] As used herein, the terms “micropore” and “nanopore” refer to the porous structure of a structured trapping array having a depth or diameter measured in micrometers or nanometers (e.g., an open region at a surface). For example, micropores can have a diameter of 1 μm to 50 μm and a depth of 1 μm to 50 μm. Nanopores, on the other hand, can have a diameter of 10 nm to 900 nm and a depth of 10 nm to 900 nm.

[0063] In this invention, "1μm to 50μm" means 1μm, 2μm, 3μm, 4μm, 5μm, 6μm, 7μm, 8μm, 9μm, 10μm, 11μm, 12μm, 13μm, 14μm, 15μm, 16μm, 17μm, 18μm, 19μm, 20μm, 21μm, 22μm, 23μm, 24μm, 2 5μm, 26μm, 27μm, 28μm, 29μm, 30μm, 31μm, 32μm, 33μm, 34μm, 35μm, 36μm, 37μm, 38μm, 39μm, 40μm, 41μm, 42μm, 43μm, 44μm, 45μm, 46μm, 47μm, 48μm, 49μm and 50μm.

[0064] In this invention, "10nm to 900nm" means 10nm, 20nm, 30nm, 40nm, 50nm, 60nm, 70nm, 80nm, 90nm, 100nm, 110nm, 120nm, 130nm, 140nm, 150nm, 160nm, 170nm, 180nm, 190nm, 200nm, 210nm, 220nm, 230nm, 240nm, 250nm, 260nm, 270nm, 280nm, 2 90nm, 300nm, 310nm, 320nm, 330nm, 340nm, 350nm, 360nm, 370nm, 380nm, 390nm, 400nm, 410nm, 420nm, 430nm, 440nm, 450nm, 460nm, 470nm, 480nm, 490nm, 500nm, 510nm, 520nm, 530nm, 540nm, 550nm, 560nm, 570nm, 580nm,

[0065] 590nm, 600nm, 610nm, 620nm, 630nm, 640nm, 650nm, 660nm, 670nm, 680nm, 690nm, 700nm, 710nm, 720nm, 730nm, 740nm, 750nm, 760nm, 770nm, 780nm, 790nm, 800nm, 810nm, 820nm, 830nm, 840nm, 850nm, 860nm, 870nm, 880nm, 890nm and 900nm.

[0066] Regarding the dragging speed in step c), for a given assembly medium, there exists a specific capture time T, which is required to establish an interaction between the molecules and / or molecular complexes and the morphological surface of the capture array, thereby allowing the molecules and / or molecular complexes to remain trapped within the cavity. As a result, the capture time depends on the properties of the complex fluid, humidity, and the affinity of the molecules and / or molecular complexes for the surface capture array.

[0067] The interaction between the molecules and / or molecular complexes to be captured and the capture array competes with the viscosity phenomena occurring within complex solutions. In practice, molecules and / or molecular complexes need to be transferred from the complex fluid to the simple fluid at the triaxial line. Therefore, as the complex fluid becomes more “complex,” the specific capture time is longer, as defined above. Similarly, if the humidity is high, the evaporation rate at the meniscus becomes lower, and the transfer of molecules and / or molecular complexes towards the triaxial line slows down, resulting in a higher capture time. Finally, the capture time also depends on the time required for stable interactions to occur between the molecules and / or molecular complexes and the cavity surface, and therefore on their affinity. Increasing affinity through surface functionalization shortens the capture time. For blood, a typical value for τ is 50 ms for 50% humidity based on maximum moisture content and for a surface cavity formed in pristine polydimethylsiloxane.

[0068] The drag speed (v) is defined by the following formula:

[0069]

[0070] Where R g This represents the radius of gyration. It is not necessary to know the radius of gyration of the molecule and / or molecular complex to be captured with high precision; orders of magnitude accuracy are sufficient for properly adjusting the drag speed. The optimal drag speed for a fluid containing a given molecule or molecular complex varies and is lower in a blood droplet compared to a simple fluid such as PBS buffer.

[0071] When the complex fluid is blood, the relationship between the 50% humidity at maximum moisture content and the surface area, drag speed, and radius of rotation of the cavity formed in the original polydimethylsiloxane is as follows:

[0072] v 血液 (μm.s -1 ) = 20.R g (μm)

[0073] Based on this relationship and the aforementioned range of turning radii, the following drag speeds are determined under these specific conditions:

[0074] • MicroRNAs and proteins: 0.4 μm / s to 20 μm / s

[0075] • Exosomes: 1 μm / s to 100 μm / s

[0076] • Chromatin: 1 μm / s to 50 μm / s

[0077] • Circulating DNA: 1 μm / s to 1000 μm / s

[0078] As seen here, the class of molecules or molecular complexes can be defined by the range of their radii of gyration. When the molecules and / or molecular complexes to be captured belong to different classes or have very different radii of gyration, it will be necessary to adjust the size of the cavity of the capture array, as described in more detail below.

[0079] In one embodiment of the invention, the dragging in step c) is not performed continuously, but includes interruptions in the movement to improve the separation of molecules and / or molecular complexes. Specifically, the interruption lasts for 1 second and is repeated every 30 seconds. This interruption gives molecules and / or molecular complexes more time to move from the depletion region toward the cavity of the capture array. When the dragging speed is low, i.e., below 10 μm·s… 1 This is particularly interesting. Specifically, the interruption occurs when the back-curved meniscus is on the cavity.

[0080] The method of this invention enables the separation of molecules and / or molecular complexes with different radii of rotation on a structured trap array. This can be carried out in one or two steps, as described below.

[0081] In one embodiment of the invention, molecules and / or molecular complexes with different radii of rotation must be separated, and step c) is performed at least twice at different speeds for each radii of rotation.

[0082] As seen above, the separation rate depends on the radius of gyration. Therefore, if two molecules and / or molecular complexes that do not belong to the same "category" must be separated, step c) is performed twice for each type of molecule and / or molecular complex to be separated at its respective separation rate. This results in the successive separation of the two molecules and / or molecular complexes within the cavity of the structured trap array.

[0083] Alternatively or complementaryly, if it is necessary to separate molecules and / or molecular complexes with different radii of rotation, the structured trapping array includes at least a first portion and a second portion with morphological features, wherein the first portion has a cavity larger than the second portion, such that step c) results in the spatial separation of the separated molecules and / or molecular complexes with different radii of rotation on the structured trapping array. Specifically, the first portion has nanopores as morphological features, and the second portion has micropores as morphological features.

[0084] This aspect of the invention relates to a combined method for spatially separating different molecules and / or molecular complexes. Even if different molecules and / or molecular complexes belong to the same separation "category" (meaning the separation rate is the same), this aspect makes it possible to separate them differentially in space thanks to the different cavity sizes. In this case, step c) can be performed only once. It will be understood that step c) can be performed two or more times in this respect if desired, especially if different rates are required.

[0085] Specifically, the structured trapping array may present only a first part and a second part. In particular, the second part follows the first part in the direction of dragging. Therefore, molecules and / or molecular complexes with low radii of gyration are highly trapped in the first part, while being rarely trapped in the second part.

[0086] The structured trapping array may also have several first and second sections. Specifically, the structured trapping array includes alternating first and second sections in the direction of dragging. In particular, the pores of the second section may partially overlap with the pores of the first section. Specifically, 10% to 70% of the diameter of the pores in the second section overlaps with the diameter of the pores in the first section. This configuration enables the trapping of two types of molecules and / or molecular complexes at the same time and in the same location, with molecules and / or molecular complexes with larger radii of gyration located in the region of the cavity of the first section, and molecules and / or molecular complexes with smaller radii of gyration located in the region of the second section overlapping at the same cavity.

[0087] A structured capture array can include different regions with different distributions of a first part and a second part.

[0088] Specifically, the cavity in the first part is a micropore, while the cavity in the second part is a nanopore. The first part may include micropores of different sizes, and the second part may include nanopores of different sizes.

[0089] Specifically, the cavities of the first and / or second portions are distributed along a line in a direction perpendicular to the dragging direction and / or in the dragging direction. In particular, the cavities of the first and / or second portions are evenly spaced in both directions. Alternatively, the cavities of the first and / or second portions are spaced further apart in the dragging direction than in the vertical direction, particularly to provide space for the chain molecules and / or molecular complexes to be elongated during step c).

[0090] In a specific implementation, the cavity of the first part is a micron-sized hole, having a diameter and depth of 1 μm to 10 μm, especially 5 μm, distributed linearly in the dragging direction and / or vertical direction, and spaced 20 μm to 100 μm, especially 60 μm, in the dragging direction, and spaced 5 μm to 15 μm, especially 9 μm, in the vertical direction.

[0091] Alternatively, or in a complementary manner, the cavity of the second portion is a nanopore, having a diameter and depth of 400 nm to 900 nm, particularly 800 nm, in a linear distribution in the dragging direction and / or the vertical direction, and spaced 20 μm to 100 μm, particularly 60 μm, in the dragging direction, and 200 nm to 600 nm, particularly 450 nm, in the vertical direction. When combined with the above-described specific embodiments, the cavities of the first and second portions are spaced 10 μm to 50 μm, particularly 30 μm, in the dragging direction.

[0092] In a specific implementation, the cavity of the second part is a nanopore, which is linearly distributed in the dragging direction and the vertical direction, has a diameter and depth of 500 nm, and is spaced 60 μm apart in the dragging direction and 750 nm apart in the vertical direction.

[0093] In a specific implementation, the cavity of the second part is a nanopore, which is linearly distributed in the dragging direction and the vertical direction, has a diameter and depth of 800 nm, and is spaced 450 nm apart in both the dragging direction and the vertical direction.

[0094] In one embodiment of the invention, the morphological features have a hydrophobic surface. In particular, the hydrophobic surface comprises a polymer material. Preferably, the polymer material is selected from the group consisting of poly(dimethylsiloxane), poly(p-xylene), poly(methyl methacrylate), polyethylene, ethylene resin, and acrylates. This aspect of the invention is of interest for the isolation of nucleic acids and is suitable for mediating the isolated nucleic acid solvent onto a printing surface, as disclosed below.

[0095] In one embodiment of the invention, the material of the covering device is selected from the group consisting of glass and silicon oxide. Specifically, the covering device is unfunctionalized. Alternatively, the covering device may be functionalized with bovine serum albumin or other non-adhesive molecules.

[0096] The method of the present invention can be combined with immune capture to locally alter surface tension (local hydrophilicity), thereby enhancing the interaction between molecules and / or complex molecules and the structured capture array, their localization and attachment, and thus enhancing the amount of captured molecules and / or molecular complexes.

[0097] Specifically, the cavity of the structured trap array can be functionalized with connecting elements constructed for trapping molecules and / or complexes. Such connecting elements increase the efficiency of retaining molecules and / or molecular complexes within the cavity. Specifically, the connecting elements can be attached to the bottom of the cavity of the structured trap array. Alternatively, the functionalization of the cavity can be performed by using a functionalized solution containing the connecting elements, prior to steps a) to c) with a complex fluid containing the molecules and / or complexes of interest. Thus, the connecting elements are trapped in the cavity before the molecules and / or complexes of interest are trapped within it. Different embodiments of the invention described in conjunction with dragging a complex fluid are applied to the functionalized solution with necessary modifications. The dragging step using the functionalized solution can be performed at 10 μm / s. The functionalized solution may contain phosphate-buffered saline (PBS) with Triton-X, for example, at a concentration of 0.5%.

[0098] Specifically, the linker element can be an antibody targeting the molecule and / or the molecular complex. In particular, when the molecular complex to be separated is an exosome, the antibody is an antibody targeting the CD9, CD63, and / or CD81 epitopes.

[0099] Once molecules and / or molecular complexes are isolated within the cavities of the structured trap array, they can be detected directly on the trap array or after transfer to another support. In fact, the method of this invention is compatible with all known characterization methods such as sequencing, fluorescence scanning, or microscopy.

[0100] Specifically, the method of the present invention includes another step d):

[0101] d) Contact the surface of the structured trap array with the printed surface to transfer the trapped molecules and / or molecular complexes from the surface of the structured trap array to the printed surface.

[0102] Specifically, the material of the printed surface is selected from the group consisting of: glass, silicon, support for mass spectrometry analysis, gold surface for quartz crystal microbalance, and gold surface for surface plasmon resonance.

[0103] Specifically, the printed surface is functionalized with a transfer agent configured to adhere to the separated molecules and / or molecular complexes.

[0104] The transfer agent can be an antibody against the isolated molecule and / or molecular complex. For example, with respect to isolated exosomes, the transfer agent can be an antibody against epitopes CD9, CD63, and / or CD81.

[0105] The transfer agent can be 3-aminopropyltriethoxysilane (APTES). APTES is commonly used for functionalizing substrates because it can form a tightly adhered amine-reactive film on the surface. APTES transfer agents are particularly used for transferring nucleic acids. The substrate can specifically be plasma-activated glass (hydroxyl) or APTMS (trimethoxysilane).

[0106] The transfer agent can be (3-glycidoxypropyl)trimethoxysilane (GPTMS). GPTMS is particularly used for the transfer of liposomes.

[0107] When the captured molecules and / or complex molecules are dried in the cavity, or when the printing surface is hydrophobic, the printing surface can be covered with a solvent film that will accelerate the transfer of molecules and / or complex molecules through evaporation. The solvent can be ethanol, deionized water, or a saline buffer, or a mixture of both.

[0108] The present invention also relates to the use of a structured trap array for the in vitro separation of molecules and / or molecular complexes with a radius of rotation of less than 2 μm from complex fluids containing multiple components, wherein the structured trap array has morphological features in the form of multiple planar surfaces located between cavities. Attached Figure Description

[0109] Figure 1 A set of three epifluorescence images (A to C) of DNA strands isolated from blood samples and printed on functionalized coverslips on a structured capture array. Each white line represents a DNA strand or an assembly of DNA strands. Figure 1 In A, the dragging speed is 1 mm / s. -1 .exist Figure 1 In B, the dragging speed is 200 μm / s. 1 .exist Figure 1 In C, the drag speed is 20 μm / s. -1 .

[0110] Figure 2 Two epifluorescence images (A and B) of circulating DNA strands isolated from blood samples and printed on functionalized coverslips on a structured capture array. Each white line represents a DNA strand or an assembly of DNA strands.

[0111] Figure 3 Epifluorescence image of liposomes isolated from a blood sample and printed on a functionalized coverslip on a structured capture array. Each solid circle represents multiple liposomes and corresponds to the size of the circular pores where the liposomes were separated.

[0112] Figure 4 This illustrates the construction of two molds and the corresponding capture of DNA strands and fluorescent nanoparticles. Figure 4a) indicates the "discontinuous" structure of the mold as observed by scanning electron microscopy (SEM), and Figure 4 c) An epifluorescence image of DNA strands and fluorescent nanoparticles isolated from a blood sample on the mold. White circles surround the captured fluorescent nanoparticles. Figure 4 b) indicates the "discontinuous" structure of the mold as observed by scanning electron microscopy (SEM), and Figure 4 d) An epifluorescence image of DNA strands and fluorescent nanoparticles isolated from the blood sample on the mold. White circles surround the captured fluorescent nanoparticles.

[0113] Figure 5 This represents the assembly of fluorescent polystyrene (PS) nanoparticles at different speeds. Figure 5 a) An epifluorescence image of polystyrene nanoparticles captured at 2 μm / s on a discontinuous compression mold. Figure 5 b) An epifluorescence image of polystyrene nanoparticles captured at 3 μm / s on a discontinuous compression mold. Figure 5 c) An epifluorescence image of polystyrene nanoparticles captured at 5 μm / s on a discontinuous compression mold. Figure 5 d) is an epifluorescence image of polystyrene nanoparticles captured at 7 μm / s on a discontinuous compression mold. Figure 5 e) A bar chart showing the average number of nanoparticle aggregates separated within a 20-micrometer cavity as a function of assembly rate. Figure 5 f) is a bar chart representing the average number of nanoparticle aggregates separated within 10650 nanocavities as a function of assembly rate.

[0114] Figure 6 This involves a comparison of the fibers and DNA chains of polymerized plasma proteins observed in fluorescence and in bright field. Figure 6 a) An epifluorescence image of assembled fibers from unspiked blood (without spiked DNA strands) captured at 10 μm / s on a non-sequential molding die. Figure 6 b) for in Figure 6 Bright-field image of the assembled fibers observed at point a). Figure 6 c) An epifluorescence image of assembled DNA strands from spiked blood, captured at 10 μm / s on a non-sequential molding die. Figure 6 d) for in Figure 6 Bright-field image of assembled DNA strands observed at point c).

[0115] Figure 7 This study involved an analysis of the effect of Triton concentration on the occupancy rate of polymerized plasma protein fibers. Figure 7a) An epifluorescence image of assembled fibers from unspiked blood (without spiked DNA strands) containing 0.5% Triton-X, captured at 2 μm / s on a non-sequential molding surface. Figure 7 b) An epifluorescence image of assembled fibers from unspiked blood (without spiked DNA strands) containing 0.25% Triton-X, captured at 2 μm / s on a non-sequential molding surface. Figure 7 c) An epifluorescence image of assembled fibers from unspiked blood (without spiked DNA strands) containing 0.125% Triton-X, captured at 2 μm / s on a non-sequential molding die. Figure 7 d) A bar chart showing the fiber occupancy of the mold cavity as a function of Triton-X concentration (TX).

[0116] Figure 8 This involves the biofunctionalization of the bottom of a surface cavity assembled via capillary tubes. Figure 8 a) An epifluorescence image of the control experiment, in which a PBS solution containing 0.5% Triton-X was assembled on a non-sequential mold at 10 μm / s. Figure 8 b) Epifluorescence image of PBS solution containing 0.5% Triton-X and 20 μg / mL fluorescently labeled anti-CD81 antibody assembled on a non-sequential mold at 10 μm / s.

[0117] Figure 9 It involves the assembly of exosomes derived from whole blood. Figure 9 a) indicates a 5 μm cavity from the non-sequential mold, observed by SEM after assembling the control solution. Figure 9 b) shows a 5 μm cavity from the non-sequential mold as observed by SEM after assembling exosomes from spiked blood onto the non-sequential mold. Figure 9 c) An epifluorescence image of the mold after assembling the control solution. No fluorescence was observed on the mold. Figure 9 d) An epifluorescence image of a mold after assembling exosomes from spiked blood and incubating them with a fluorescent antibody against the exosomes. Exosomes (white dots) appear in the cavity of the mold.

[0118] Figure 10 This involves different methods for characterizing the combined capture of exosomes and circulating cell-free DNA (cfDNA). Figure 10 a), 10b, and 10c are images obtained by SEM at different magnifications after sample 1 was assembled onto a non-sequential mold at 10 μm / s. Exosomes can be observed as white spots inside the cavities. Figure 10 d) Epifluorescence images of DNA strands and exosomes after assembly of sample 2. The captured exosomes are highlighted by white circles. Figure 10e) An epifluorescence image of the captured DNA strands after assembly of sample 3. Figure 10 f) is an epifluorescence image of the exosomes captured after assembly of sample 3 (highlighted by white circles). Example

[0119] Example 1: Isolation of DNA strands from blood samples rich in DNA extracts

[0120] 1. Preparation of polydimethylsiloxane (PDMS) compression molding

[0121] The structured trap array is used for PDMS molding. The morphology of the PDMS molding is formed using a silicon mold. The silicon mold comprises several pillars spaced 20 μm apart to create pores within the PDMS molding. This is achieved using Sylgard... TM PDMS was prepared using kit 184. One dose of curing agent was mixed with ten doses of base material, according to the completion notes. The curing agent contained dimethylvinyl-terminated dimethylsiloxane (CAS No.: 68083-19-2), dimethylvinyl- and trimethyl-substituted silica (CAS No.: 68988-89-6), tetrakis(trimethoxysiloxy)silane (CAS No.: 3555-47-3), and ethylbenzene (CAS No.: 100-41-4). The base material contained dimethyl, methylhydrosiloxane (CAS No.: 68037-59-2), dimethylvinyl-terminated dimethylsiloxane (CAS No.: 68083-19-2), dimethylvinyl- and trimethyl-substituted silica (CAS No.: 68988-89-6), tetramethyltetravinylcyclotetrasiloxane (CAS No.: 2554-06-5), and ethylbenzene (CAS No.: 100-41-4). The PDMS mixture is then poured into a mold and baked at 80°C for 2 hours to polymerize and solidify.

[0122] 2. Preparation of Functionalized Coverslips for Printing

[0123] a. Cleaning the coverslip

[0124] First, one side of the coverslip was cleaned with acetone solution, then with ethanol solution, and finally with deionized water solution. The cleaned coverslip was then placed side up on a paper towel and dried with nitrogen using an air gun. The coverslip was then moved to a new spot on the towel and dried again with the air gun. The coverslip was then removed with tweezers, and nitrogen was blown from the side to remove any residual water. Finally, it was subjected to RF plasma treatment for 5 minutes at 0.6 mbar air and 100% power (50 W).

[0125] b. Using APTES solution (1% silane in 95% EtOH / 5% ddH2O) 2 O-functionalized cover glass

[0126] Preheat the hot plate to 140°C. Prepare the solvent by mixing 47.5 mL of ethanol and 2.5 mL of distilled water. Using a syringe, invert 0.5 mL of the APTES solution and calculate the volume of the bubbles formed inside the syringe. Mix the solvent and APTES solution in a glass dish, then cover with aluminum foil to limit evaporation for 5 minutes. Replace the air atmosphere in the culture dish with nitrogen to limit air contact during hydrolysis. Remove the aluminum foil to allow the plasma-activated coverslip to be introduced into the dish for 20 minutes. Then remove the functionalized coverslip, thoroughly clean it with ethanol and ddH2O, dry it with a nitrogen torch, and finally place it on a hot plate at 140°C for 5 minutes.

[0127] 3. Preparation of Triton-X100 and YOYO-1 solutions

[0128] A 10% Triton-X100 solution was prepared by mixing 1 ml of Triton-X with 9 ml of PBS in a 15 mL Falcon tube.

[0129] YOYO-1 is a nucleic acid staining agent. A 1:10 dilution of the YOYO-1 stock solution is prepared by diluting the 1 mM stock solution with PBS.

[0130] 4. Preparation of DNA Extracts

[0131] λ phage DNA extract obtained from New England Biolab (NEB)

[0132] 5. Preparation of assembly solution

[0133] From Etablissement Blood is obtained by du Sang (EFS) and collected in EDTA-coated tubes to prevent clotting.

[0134] The assembly solution was prepared by mixing 45.25 μL of blood with 2.5 μL of DNA extract, 0.75 μL of YOYO-1 solution, and 1.5 μL of Triton-X100 solution. The final concentrations in the assembly solution are as follows:

[0135] Triton-X100: 0.3% v / v

[0136] YOYO-1: 7.5 μM

[0137] DNA extract: 25 μg / ml.

[0138] 6. The assembly solution was sealed onto a PDMS mold.

[0139] The temperature was set to 20°C and the humidity to 40%, as measured using a digital hygrometer. The coverslip was cleaned as described in point 2.a. With the morphology feature side facing up, the PDMS mold was placed on the PDMS Petit's dish, with the long side of the mold perpendicular to the dragging motion (long side horizontal). The cleaned coverslip was placed and held approximately 2-3 mm above the PDMS mold. 40 μL of the assembly solution was placed between the PDMS mold and the coverslip. The droplet was then allowed to spread evenly along one short side of the PDMS mold.

[0140] 7. Isolation and printing of extracted DNA

[0141] The coverslip was moved along the long side of the PDMS mold at speeds of 20 μm / s, 200 μm / s, and 1 mm / s. Once the other short side of the mold was reached, the mold was removed, and any remaining droplets were absorbed using paper. All residual water was removed to avoid any diffusion upon contact with the functionalized coverslip. The morphological side of the PDMS mold was then brought into contact with the functionalized side of the functionalized coverslip for 1 minute. Afterward, the PDMS mold was removed, and the coverslip was stored in the dark.

[0142] 8. Epifluorescence microscopy

[0143] The samples were observed using an inverted microscope (Olympus, exposure: 30ms; camera gain: 100; cyan; laser power: 30; Zeiss, camera gain: 3; exposure time: 200ms; laser power: 100%; cyan) at a magnification of x100.

[0144] The results obtained for each speed are as follows: Figure 1 Shown at points A to 1C. The white lines correspond to the elongated DNA strands printed on the surface of the functionalized coverslip. In the photographs, some DNA strands appear thicker and brighter than others. The brighter strands correspond to a group of strands, and the lighter strands correspond to a single strand, which explains the brightness and thickness.

[0145] The inventors achieved better reproducibility and better coverage of the functionalized coverslips at a drag speed of 20 μm / s (due to better separation of DNA strands into the pores of the PDMS mold).

[0146] Example 2: Isolation of circulating DNA from blood samples

[0147] The objective of this embodiment is to isolate circulating DNA from blood samples from patients suffering from severe cancer.

[0148] Repeat points 1 to 3, 5 to 8 of Example 1, except that

[0149] - At point 5, no DNA was added to the assembly solution, and a 47 μl blood sample recovered from a clinical trial of a patient suffering from severe cancer was mixed with 1.5 μl of YOYO-1 solution and 1.5 μl of Triton-X100 solution, and the difference was...

[0150] - At point 7, the velocity of step a) is 20 μm / s.

[0151] The results indicate that Figure 2 A and 2B.

[0152] Example 3: Isolation of liposomes added to blood samples

[0153] Repeat points 1 to 3, 6 and 7 of Example 1, except that

[0154] In point 2, clean coverslips are functionalized using GPTMS according to the following scheme;

[0155] - At point 7, the velocity of step a) is 20 μm / s.

[0156] Replace points 4 and 5 in Example 1 with points 2 and 3 below, respectively.

[0157] 1. Functionalizing coverslips using GPTMS

[0158] Mix 1.25 ml of GPTMS solution with 48.75 ml of pure ethanol. Place the plasma-activated coverslip sides on the mixture for 30 minutes. Thoroughly clean the functionalized coverslip sides with ethanol only.

[0159] Dry the glass slides with an air spray gun.

[0160] 2. Preparation of liposomes

[0161] The lipids used in this scheme are phosphatidylcholine (POPC) and 1-palmitoyl-2-{6-[(7-nitro-2-1,3-benzo[]] from Avanti Polar Lipids. [Diazol-4-yl]amino]hexanoyl}-sn-glycero-3-phosphocholine (NBD-PC). NBD-PC is a fluorescent lipid used for observing liposomes in epifluorescence (excitation wavelength 460 nm; emission wavelength 534 nm).

[0162] a. Preparation of lipid solutions

[0163] The phospholipids were stored in chloroform (Sigma Aldrich) solution at -20°C with a POPC concentration of 10 mg / ml and an NBD-PC concentration of 1 mg / ml.

[0164] b. Preparation of phospholipids

[0165] Mix 100 μl of POPC solution with 10 μl of NBD-PC solution in a 4 mL glass flask. Then heat the flask in a drying bath at 55 °C while simultaneously using an air gun directed towards the inside of the flask to generate an airflow to evaporate the chloroform. Next, place the flask in a vacuum chamber for 2 hours to ensure all chloroform has evaporated. Cover the chamber with aluminum foil to prevent the fluorophore from being destroyed by sunlight. Finally, seal the flask with parafilm and store at 4 °C if not used immediately.

[0166] c. Suspended lipids to obtain multilayered vesicles

[0167] Add 0.5 ml of PBS to the flask and vortex until completely homogenized. Formation of 80 nm small monolayer vesicles (SUVs) by extrusion using the Avanti Polar Lipids Micro Extruder Kit.

[0168] The SUV suspension has a concentration of 2 mg / ml and can be stored at 4°C and used within 3 days.

[0169] 3. Preparation of assembly solution

[0170] The assembly solution was prepared by mixing 25 μl of blood with 22.5 μl of liposome solution and 2.5 μl of Triton-X100 solution.

[0171] 4. Epifluorescence microscopy

[0172] The samples were observed using an inverted microscope (Olympus, exposure: 30ms; camera gain: 100; cyan; laser power: 30; Zeiss, camera gain: 3; exposure time: 200ms; laser power: 100%; cyan) at a magnification of x100.

[0173] The results for a velocity of 10 μm / s are expressed in Figure 3 The figure shows that liposomes were effectively separated into the pores and transferred onto the functionalized coverslip.

[0174] Example 4: Construction of the embossed pattern

[0175] 1. The purpose of the experiment

[0176] To implement combined liquid biopsy capture, a new [system / mechanism] has been established. dieThe molds used in Examples 1 to 3 contained only micron cavities with a diameter of 5 μm. The purpose of this example is to develop a new mold structure that includes a 5 μm micron cavity suitable for DNA strand capture and a 500 nm nanon cavity suitable for nanoparticle (specifically, exosome) capture.

[0177] 2. Materials and methods

[0178] Two configurations of the embossing pattern were tested. The first configuration, termed "non-sequential," consisted of alternating micron and nano patterns along the dragging direction. The second configuration, "sequential," was divided into two parts: one half of the embossing surface was equipped with micron patterns, while the other half was equipped with nano patterns. Both configurations... Figure 4 Seen in a) and 4b).

[0179] Thanks to microfabrication processes, new molds consisting of two constructions are fabricated in a cleanroom. Each mold comprises a 4-inch silicon wafer, on which different chips are defined. One half of the chip corresponds to a non-sequential construction, and the other half corresponds to a sequential construction. PDMS (polydimethylsiloxane) replicas are produced using standard mass production processes, and after cross-linking, the molds are cut according to the chip boundaries. After demolding, the different molds consist of micron- and nano-cavities with either sequential or non-sequential constructions.

[0180] To investigate the applicability of these moldings to assembly, 100 nm fluorescent polystyrene nanoparticles (concentration 2.5 μg / mL = 5 × 10⁻⁶) were prepared. 9 (particles / mL) and a blood solution spiked with DNA strands (25 μg / mL) containing 0.5% Triton-X and 0.75 μM YOYO-1. A drop of 35 μL of this solution was assembled at 10 μm / s and then at 2 μm / s for each construct.

[0181] The captured nanoparticles and DNA strands were observed using an inverted microscope (Zeiss, camera gain: 3; exposure time: 200 ms; laser power: 100%; cyan) at a magnification of x100.

[0182] The mold structure was observed using a scanning electron microscope (SEM Helios 600i FEI, accelerating voltage 15kV, electron beam current 86pA, secondary electron signal).

[0183] 3. result

[0184] The results are presented in Figure 4c) and 4d). In both constructions, DNA strands and nanoparticles were assembled. Nanoparticles were observable in the microcavities of both constructions (indicated by circles). Inside the nanocavities, nanoparticles were not detected by fluorescence. The results obtained between the two constructions are equivalent in terms of DNA occupancy, the length and good condition of the captured DNA strands, and the ratio of occupied cavities with nanoparticles.

[0185] Example 5: Nanoparticle Assembly

[0186] 1. The purpose of the experiment

[0187] Exosomes are extracellular vesicles, typically ranging in size from 30 nm to 140 nm. They contain proteins, miRNAs, DNA, and other biomarkers that provide information about disease. They are considered novel biomarkers, and their research in the context of cancer research is increasingly developing. However, due to their small size, they are difficult to isolate. To determine the optimal parameters for capturing them, 100 nm fluorescent polystyrene nanoparticles were used to simulate exosomes in this experiment. Therefore, the aim of this experiment was to perform nanoparticle assembly in blood at concentrations close to those of real exosomes and to determine the optimal assembly rate.

[0188] 2. Materials and methods

[0189] Preparation of 100 nm fluorescent polystyrene nanoparticles (2.5 μg / mL = 5 × 10⁻⁶) 9 (particles / mL) and a 0.5% Triton-X-spikeped blood solution. A 35 μL drop of this solution was assembled at 2 μm / s, 3 μm / s, 5 μm / s, and 7 μm / s onto sequential and non-sequential molds as described in Example 4 to determine the optimal assembly speed. After assembly, each mold was placed on a clean coverslip and observed under a microscope through the coverslip.

[0190] The captured nanoparticles were observed at 100x magnification using an inverted microscope (Zeiss, camera gain: 3; exposure time: 200ms; laser power: 100%; cyan).

[0191] 3. result

[0192] The result is Figure 5 As shown in the diagram, nanoparticles are separated into micron-cavities and nanocavities. Figure 5 e) and 5f) show that as the speed increases, the number of nanoparticle aggregates separated in both the micron-cavity and the nanocavity decreases, which makes it possible to establish a correlation between scanning speed and the number of nanoparticles captured.

[0193] 4. in conclusion

[0194] Nanomaterials such as nanoparticles are assembled from whole blood in all types of cavities (micrometers and nanometers). Optimizing the drag speed optimizes the number of nanoparticles separated. Here, the optimal speed is given as 2 μm / s, although satisfactory results can be obtained at higher speeds.

[0195] Example 6: Capture of plasma protein fibrils

[0196] 1. The purpose of the experiment

[0197] In several cases, without any DNA molecule spiking, some fibers assembled in whole blood during experiments. They differed from DNA because, as observed by SEM and optical fluorescence, they appeared coarser, more dispersed, and less fluorescent. The inventors hypothesized that these fibers were formed during capillary-assisted polymerization via plasma proteins such as fibrin. The purpose of the following experiments was to confirm that these fibers were not DNA molecules and, if necessary, to determine how they were confined.

[0198] 2. Materials and methods

[0199] A blood solution containing 0.5% Triton-X was prepared, and assembly was performed at 10 μm / s using a 35 μL drop of this solution on a non-sequential mold, as described in Example 4. At the end of assembly, the mold was observed under a microscope through a glass coverslip, as described in Example 4. The results were compared with another sample assembled (at 10 μm / s, a 35 μL drop) from blood spiked with λ-phage DNA chains stained with 0.75 μM YOYO-1. Both samples were observed under a microscope in fluorescence and in bright field, as described in Example 4. Because DNA chains cannot be observed in bright field under these conditions, while protein fibers can be observed due to their coarser structure (which increases light diffusion), it was possible to distinguish DNA chains from protein fibers.

[0200] To determine how to limit the formation of these protein fibers, three blood solutions containing 0.5%, 0.25%, or 0.125% Triton-X were prepared and assembled on non-sequential molds at 2 μm / s, as described in Example 4. The three molds (one for each concentration) were observed and compared under a microscope using coverslips.

[0201] 3. result

[0202] Figure 6 a) represents assembled fibers from unspiked blood in fluorescence, and Figure 1b) indicates bright field. It can be seen that aggregated plasma protein fibers are observed at the same locations in both images. This means that these captured objects are visible in bright field.

[0203] Figure 6 c) and 6d) represent assembled spiked DNA strands stained with YOYO-1 from blood in fluorescent (6c) and bright-field (6d) conditions, respectively. Figure 6 In d), no strands were observed, confirming that DNA strands are only observable in fluorescence.

[0204] Figure 7 This represents the assembly of unspecified blood at different Triton-X concentrations. Figure 7 In a), 7b) and 7c), which are assembly of Triton-X in blood at concentrations of 0.5%, 0.25% and 0.125%, respectively, the number of fibers observed decreased with decreasing Triton-X concentration. Figure 7 The graph in d) confirms this result, as the occupancy rate decreases as the Triton-X concentration decreases.

[0205] Other experiments (not described here) have demonstrated that speed also affects the number of fibers. In fact, plasma proteins polymerized at 2 μm / s produced more fibers than those polymerized at 10 μm / s.

[0206] 4. in conclusion

[0207] It can be confirmed that the fibers assembled from blood are not DNA strands, because the latter cannot be observed in bright field.

[0208] A correlation can be established between the occupancy rate of polymerized plasma protein fibers and the Triton-X concentration, because a decrease in Triton-X concentration leads to a decrease in the number of assembled fibers. The assembly rate of these fibers is also an important factor, as an increase in rate results in a decrease in the number of fibers.

[0209] In summary, very low scan rates combined with high Triton-X concentrations favor the formation of protein fibrils in whole blood. If it is necessary to limit this phenomenon, operating parameters need to be adjusted, such as setting the rate to 10 μm / s or higher and using Triton-X concentrations below 0.125%.

[0210] Example 7: Biofunctionalization of the bottom of the surface cavity using specific antibodies

[0211] 1. The purpose of the experiment

[0212] The aim of this experiment was to functionalize the bottom of the surface lumen to facilitate the assembly of exosomes within the lumen while preventing their adsorption onto the top surface of the mold. This functionalization was attempted by incubating antibodies on the mold for one hour. The mold was then printed by contacting different coverslips one after another to gradually remove antibody molecules adsorbed on the top surface. However, the results were not excellent and were not reproducible between molds. Therefore, the inventors decided to functionalize the bottom lumen through capillary assembly. In other words, they used the same biomarker capture principle, but they manipulated the microvolume of a solution containing selected antibodies rather than the microvolume of blood. Thus, capillary action drives these molecules to the interior of the lumen rather than the top surface of the mold. The experiment demonstrated this principle.

[0213] 2. Materials and methods

[0214] A PBS (1X) solution was prepared in PBS using 0.5% Triton-X and 20 μg / mL fluorescently labeled anti-CD81 antibody. CD81 is a protein that inserts into the exosome membrane. Then, as described in Example 4, a drop of 35 μL of this solution was assembled onto a non-sequentially molded surface at a speed of 10 μm / s.

[0215] A control solution containing only PBS and 0.5% Triton-X was also prepared. Assembly was achieved at 10 μm / s on another non-sequential molding die.

[0216] The two molds (i.e., the control mold and the mold with antibody) were observed and compared using the optical microscope described in Example 4 through coverslips.

[0217] 3. result

[0218] The results are presented in Figure 8 Above. No fluorescence was observed in the left-hand image (control). In contrast, fluorescent spots are visible in the right-hand image sample, demonstrating antibody immobilization at the bottom of each micron-sized cavity on the mold. Interestingly, it can be noted that fluorescence is only visible inside the cavities and not on the top surface.

[0219] 4. in conclusion

[0220] Thanks to capillary assembly technology, antibodies can be placed only at the bottom of the lumen. Advantageously, the redistribution of antibodies throughout all lumens is uniform, and lumen functionalization can be achieved in a single step. Furthermore, this functionalization does not require a cleaning step, as the antibodies are present only within the lumen.

[0221] Example 8: Capture of exosomes from whole blood

[0222] 1. The purpose of the experiment

[0223] The aim of this experiment was to capture exosomes from whole blood via capillary assembly on a biofunctionalized mold (bottom cavity). The inventors characterized the captures using optical fluorescence microscopy and scanning electron microscopy (SEM).

[0224] 2. Materials and methods

[0225] Prepare three solutions:

[0226] The functionalized solution contains PBS with 0.5% Triton-X and 20 μg / mL anti-CD81 antibody in PBS.

[0227] ο Exosome solution, which contains a concentration close to 10 9 A blood sample containing exosomes at a concentration of / mL and 0.5% Triton-X, and

[0228] The control solution consisted of unspecified blood and 0.5% Triton-X.

[0229] Both optical fluorescence and SEM observations characterized the two samples:

[0230] 1) Control (unlabeled blood)

[0231] 2) Assemble exosomes from spiked blood on a biofunctionalized mold (bottom cavity).

[0232] For the control, a 35 μL drop of anti-CD81 solution was assembled onto a non-sequential mold at 10 μm / s. Immediately after functionalization, another assembly was performed using a 35 μL drop of the control solution at 2 μm / s.

[0233] For exosome assembly on a biofunctionalized mold, a 35 μL drop of anti-CD81 solution was assembled onto a non-sequential mold at a speed of 10 μm / s. Immediately after functionalization, another assembly was performed using a 35 μL drop of exosome solution at a speed of 2 μm / s.

[0234] When the captured cells were observed in fluorescence, at the end of assembly of the exosome solution or control solution, incubation was performed for 1 hour with a 1:100 volume diluted FITC anti-CD63 solution in PBS. This second fluorescent antibody revealed the presence of exosomes; CD63 is a protein typically found in the exosome envelope. After incubation, the mold was washed four times with PBS.

[0235] SEM observation was performed directly without any incubation with the fluorescent secondary antibody (SEM Hélios600i FEI, accelerating voltage 15kV, electron beam current 86pA, secondary electron signal).

[0236] 3. result

[0237] The results are presented in Figure 9 Above. In Figure 9 (a) In the control group, no elements compatible with the shape and size of exosomes were observed at the bottom of the lumen by SEM. Therefore, no exosomes were captured. This was achieved through... Figure 9 c) This was confirmed when the control was observed in fluorescence after incubation, and no signal from the bottom of the cavity was observed. Background fluorescence was only present at the surface of the mold.

[0238] exist Figure 9 (b) On exosomes on a functionalized mold, nanospheres are clearly observed at the bottom of the cavity. Based on their size and typical contrast, they correspond to exosomes. In fact, exosomes are vesicles with typical sizes between 30 nm and 140 nm, and they are identifiable under an electron microscope thanks to the ring-like contrast at their center. Figure 9 d) also confirms this, where the fluorescent spot is located at the bottom of the cavity, and when the focal plane is adjusted to that position, the presence of exosomes inside the cavity is shown.

[0239] 4. in conclusion

[0240] By comparing different images obtained via fluorescence or SEM, it was confirmed that exosomes can be assembled through the bottom of the functionalized cavity. Control samples showed that nothing was observed within the cavity if the exosomes were not spiked into the blood.

[0241] Example 9: Different methods for combining exosome capture with circulating cell-free DNA (cfDNA)

[0242] 1. The purpose of the experiment:

[0243] The objective of this experiment is to capture combined DNA strands and exosomes spiked into whole blood. The captured samples are then observed using fluorescence and scanning electron microscopy. However, to observe exosomes in fluorescence, staining with a fluorescent secondary antibody is required. This incubation is problematic for already assembled DNA strands, as they may resuspend in solution during secondary antibody incubation. The inventors have addressed this problem through the following experiment:

[0244] ο Combined capture in one step (without incubation with labeled secondary antibodies against exosomes),

[0245] ο Combined capture using a combination of two biomarkers in fluorescence (incubation with a labeled secondary antibody against exosomes).

[0246] 2. Materials and methods :

[0247] Prepare a PBS solution containing 0.5% Triton-X and 20 μg / mL anti-CD81 antibody in PBS. Prepare a PBS solution containing 25 μg / mL DNA strands and exosomes (approximately 10 μg / mL). 9 mL -1 (concentration) and another blood solution of 0.5% Triton-X and 0.75 μM YOYO-1.

[0248] Three samples were generated:

[0249] 1. One sample is used for SEM observation.

[0250] 2. A sample is assembled with two biomarkers (DNA strands and exosomes) on the same mold for simultaneous observation using fluorescence microscopy.

[0251] 3. Before incubating the exosomes with the fluorescent antibody, both biomarkers were first assembled onto the same mold, but the DNA strand was transferred onto an APTES-functionalized coverslip, as previously described in Example 1. Thus, the DNA strand was on the printed coverslip, while the exosomes remained inside the cavity of the mold. Finally, the two biomarkers were observed separately using fluorescence microscopy.

[0252] For Sample 1, as described in Example 4, the biofunctionalized solution containing anti-CD81 antibody was assembled on a non-sequential mold at 10 μm / s. Then, another assembly was performed using a drop of 35 μL of spiked blood containing exosomes and DNA strands at 10 μm / s. After assembly, a 5 nm Au / Pd thin metal film was deposited on the mold surface. The sample was then observed by SEM (SEM Hélios 600i FEI, accelerating voltage 15 kV, electron beam current 86 μA, secondary electron signal).

[0253] For Sample 2, as described in Example 4, the functionalized solution containing anti-CD81 antibody was assembled at 10 μm / s on a non-sequential mold. Another assembly was then performed at 2 μm / s using a drop of 35 μL of spiked blood containing exosomes and DNA strands. Incubation for one hour was performed using a second-labeled FITC anti-CD63 (Thermofisher, MA1-19602) solution diluted 1:100 in PBS. After incubation, the mold was rinsed four times with PBS. Another assembly was then performed at 10 μm / s using a drop of 35 μL of the same spiked blood containing exosomes and DNA strands to capture fresh DNA strands and compensate for those resuspended during secondary antibody incubation.

[0254] For Sample 3, as described in Example 4, the functionalized solution containing anti-CD81 antibody was assembled on a non-sequential mold at 10 μm / s. Then, another assembly was performed using a drop of 35 μL of spiked blood containing exosomes and DNA strands at 2 μm / s. As described in Example 1, the mold was pressed onto an APTES (3-aminopropyltriethoxysilane) functionalized coverslip for one minute to transfer the assembled DNA strands via electrostatic interactions. After transfer, the mold was incubated for one hour with a second-labeled FITC anti-CD63 (Thermofisher, MA1-19602) solution diluted 1:100 in PBS to label the exosomes trapped inside the cavity. After incubation, the mold was rinsed four times with PBS. The DNA strands were observed on a coverslip using the microscope described in Example 4 after transfer via nanocontact printing, while the exosomes were observed directly on the mold using the microscope through a protective coverslip.

[0255] result

[0256] The SEM observation results of sample 1 are presented in Figure 10 On a), 10b), and 10c), it can be seen that the exosomes are assembled at the bottom of the lumen (white dots). DNA strands, appearing as long white lines originating from the lumen, can also be observed on the deposited metal film used in SEM observation.

[0257] Fluorescence characterization of the two biomarkers on the same support (sample 2) is shown in Figure 10 d) Above. It can be seen that exosomes and DNA strands are well characterized by fluorescence on the same image.

[0258] like Figure 10 As shown in e) and 10f), biomarkers can also be observed independently (sample 3). Figure 10 e) On the receiving coverslip, white lines corresponding to DNA strands were observed. Furthermore, Figure 10f) Exosomes were observed directly on the mold (highlighted by white circles).

[0259] in conclusion

[0260] Thanks to SEM examination, biomarkers can be observed after only one assembly. The inventors have also made it possible to observe biomarkers in fluorescence.

[0261] - Both remain on the mold or

[0262] -Observe by removing one onto another support and observing the remaining one on the mold.

Claims

1. A method for in vitro separation of molecules and / or molecular complexes with a radius of rotation less than or equal to 2 µm from complex fluids, the method comprising the following steps: a) Contacting a complex fluid with a structured trapping array having morphological features in the form of multiple planar surfaces located between cavities, wherein the structured trapping array is surrounded by humid air, and wherein the complex fluid is a non-Newtonian fluid. b) Cover the deposited complex fluid with a covering device such that the complex fluid is surrounded by a meniscus including a back meniscus and a front meniscus, wherein the surface tension of the complex fluid located between the covering device and the structured trapping array at least defines the front meniscus and the back meniscus. c) up to 2mm.s' 1 The covering device or the structured trapping array is dragged in one direction at a speed to displace the complex fluid, wherein the forward and backward menisci are displaced on and along the topographic features of the structured trapping array toward the direction, wherein during the displacement of the complex fluid, the forward menisci cover the exposed topographic features and the backward menisci expose the covered topographic features, resulting in: The molecules and / or the molecular complexes are trapped inside the cavity and can elongate on the planar surface in the direction of the dragging. The humid air described therein has a humidity of at least 40% based on the maximum humidity of the air.

2. The method of claim 1, wherein the humidity is 40% to 80% based on the maximum moisture content of the surrounding air.

3. The method according to claim 1, wherein the molecule is a biomolecule.

4. The method according to claim 1, wherein the molecular complex is chromatin.

5. The method according to claim 3, wherein the biomolecule is a nucleic acid molecule.

6. The method of claim 5, wherein the nucleic acid molecule is selected from the group consisting of linear DNA, linear RNA, circular DNA, and circular RNA.

7. The method of claim 5, wherein the nucleic acid molecule is selected from the group consisting of single-stranded DNA and double-stranded DNA.

8. The method according to claim 5, wherein the nucleic acid molecule is RNA.

9. The method according to claim 5, wherein the nucleic acid molecule is a viral nucleic acid molecule.

10. The method according to claim 5, wherein the nucleic acid molecule is circulating free DNA.

11. The method according to claim 5, wherein the nucleic acid molecule is tumor DNA.

12. The method according to claim 1, wherein the molecular complex is a biological complex.

13. The method of claim 12, wherein the biological complex is selected from the group consisting of vacuoles and lysosomes.

14. The method of claim 12, wherein the biological complex is selected from the group consisting of transport vesicles and secretory vesicles.

15. The method of claim 12, wherein the biological complex is selected from the group consisting of extranuclear particles, microvesicles, and exosomes.

16. The method of claim 12, wherein the biocomplex is a liposome.

17. The method of claim 12, wherein the biological complex is a virus or a part of a virus.

18. The method of claim 12, wherein the biological complex is a large complex.

19. The method of claim 1, wherein the complex fluid is an individual biological fluid.

20. The method of claim 19, wherein the biological fluid is selected from the group consisting of cerebrospinal fluid, pleural effusion, saliva, urine, and blood.

21. The method of claim 19, wherein the biofluid is selected from plasma and serum.

22. The method of claim 1, wherein the complex fluid is blended with a surfactant before or during step a).

23. The method of claim 22, wherein the surfactant is a nonionic surfactant.

24. The method of claim 22, wherein the complex fluid is blended with 0.1% to 0.5% v / v Triton X100 before or during step a).

25. The method of claim 22, wherein the complex fluid is blended with 0.3% v / v Triton X100 before or during step a).

26. The method according to any one of claims 1 to 25, wherein molecules and / or molecular complexes with different radii of rotation must be separated, and wherein step c) is performed at least twice at different speeds for each radii of rotation.

27. The method according to any one of claims 1 to 25, wherein molecules and / or molecular complexes with different radii of rotation must be separated, wherein the structured trap array comprises at least a first portion and a second portion of morphological features, wherein the first portion has a cavity larger than the second portion, such that step c) results in spatial separation of the separated molecules and / or molecular complexes with different radii of rotation on the structured trap array.

28. The method according to any one of claims 1 to 25, wherein the cavity of the structured trapping array is functionalized with connecting elements configured for trapping the molecules and / or complexes.

29. The method according to any one of claims 1 to 25, wherein the method comprises an additional step d): d) Contact the surface of the structured trap array with the printed surface to transfer the trapped molecules and / or molecular complexes from the surface of the structured trap array to the printed surface.

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