A METHOD FOR ISOLATING MOLECULES AND/OR MOLECULAR COMPLEXES

Abstract

A method for isolating molecules and/or molecular complexes having a radius of gyration smaller or equal to 2 m from a complex fluid, including the steps of: a) contacting a complex fluid with a structured capture array having topographical features, wherein the structured capture array is placed in an environment with surrounding humid air humidity of at least 40% based on the maximal moisture content, b) covering the deposited complex fluid with a covering element, wherein the surface tension of the complex fluid between the covering element and the structured capture array defines at least a front and a rear meniscus; and c) dragging either the covering element or the structured capture array in one direction at a speed of at most 2 mm.Math.s.sup.1 for displacing the complex fluid, resulting in that the molecules and/or the molecular complexes are trapped inside the cavities.

Claims

1-12. (canceled)

13. A method for in vitro isolating molecules and/or molecular complexes having a radius of gyration smaller or equal to 2 m from a complex fluid, said method comprising the following steps: a) contacting a complex fluid with a structured capture array having topographical features in the form of a plurality of plane surfaces in-between cavities, wherein the structured capture array is surrounded by humid air and wherein said complex fluid is a non-Newtonian fluid, b) covering the deposited complex fluid with a covering means such that the complex fluid is surrounded by a meniscus which comprises a rear meniscus and a front meniscus, wherein the surface tension of the complex fluid between the covering means and the structured capture array defines at least the front and the rear meniscus; and c) dragging either the covering means or the structured capture array in one direction at a speed of at most 2 mm.Math.s.sup.1 for displacing the complex fluid, wherein the front and the rear menisci are displaced on and along the topographical features of said structured capture array toward said direction, wherein the front meniscus covers uncovered topographical features and the rear meniscus uncovers covered topographical features during displacement of the complex fluid, resulting in that: the molecules and/or the molecular complexes are trapped inside the cavities, and possibly elongated on the plane surfaces toward the direction of the dragging, wherein the humid air has a humidity of at least 40% based on the air maximal humidity.

14. The method for in vitro isolating molecules and/or molecular complexes according to claim 13, wherein the humidity in step c) is from 40 to 80% based on the maximal moisture content of the surrounding air.

15. The method for in vitro isolating molecules and/or molecular complexes according to claim 13, wherein the molecules are biological molecules, in particular the biological molecules are nucleic acid molecules, particularly the nucleic acid molecules are selected from the group comprising viral nucleic acid molecules, chromatin, circulating free DNA, RNA, linear DNA, linear RNA, circular DNA, circular RNA, single-stranded DNA, double-stranded DNA, and tumoral DNA.

16. The method for in vitro isolating molecules and/or molecular complexes according to claim 13, wherein the molecular complexes are biological complexes, in particular the biological molecular complexes are selected in the group comprising vacuoles, lysosomes, transport vesicles, secretory vesicles, liposomes, ectosomes, microvesicles, virus, part of virus, exosomes and macro complex.

17. The method for in vitro isolating molecules and/or molecular complexes according to claim 13, wherein the complex fluid is a biological fluid of an individual, in particular said biological fluid is selected in the group consisting of cerebrospinal fluid, pleural effusion, saliva, urine, blood, plasma and serum, especially the biological fluid is blood.

18. The method for in vitro isolating molecules and/or molecular complexes according to claim 13, wherein before or at step a), the complex fluid is blended with a surfactant, in particular a non-ionic surfactant.

19. The method for in vitro isolating molecules and/or molecular complexes according to claim 18, wherein before or at step a), the complex fluid is blended with 0.1 to % v/v Triton X100, particularly 0.3% v/v TritonX100.

20. The method for in vitro isolating molecules and/or molecular complexes according to claim 13, wherein molecules and/or molecular complexes of different radius of gyration have to be isolated, and wherein step c) is carried out at least two times at different speeds for each radius of gyration.

21. The method for in vitro isolating molecules and/or molecular complexes according to claim 13, wherein molecules and/or molecular complexes of different radius of gyration have to be isolated, wherein the structured capture array comprises at least a first and a second portions of topographical features wherein the first portion has larger cavities than the second one, such that step c) results in the spatial separation onto the structured capture array of the isolated molecules and/or molecular complexes of different radii of gyration.

22. The method for in vitro isolating molecules and/or molecular complexes according to claim 13, wherein the cavities of the structured capture array are functionalised with a linking element configured for trapping the molecules and/or complexes.

23. The method for in vitro isolating molecules and/or molecular complexes according to claim 13, wherein the process of the invention comprises a further step d): d) contacting the surface of the structured capture array with a printing surface for transferring the trapped molecules and/or molecular complexes from the surface of the structured capture array to the printing surface.

24. A structured capture array for in vitro isolating molecules and/or molecular complexes having a radius of gyration smaller than 2 m from a complex fluid comprising numerous components, wherein the structured capture array having topographical features in the form of a plurality of plane surfaces in-between cavities.

Description

LEGEND TO THE FIGURES

[0104] FIG. 1 is a set of three epifluorescence images (A to C) of DNA strands isolated from a blood sample on a structured capture array and printed on a functionalised coverslip. Each white line represents one or an assembly of DNA strands. In FIG. 1A, the speed of dragging was 1 mm.Math.s.sup.1. In FIG. 1B, the speed of dragging was 200 m.Math.s.sup.1. In FIG. 1C, the speed of dragging was 20 m.Math.s.sup.1.

[0105] FIG. 2 is a set of two epifluorescence images (A and B) of circulating DNA strands isolated from a blood sample on a structured capture array and printed on a functionalised coverslip. Each white line represents one or an assembly of DNA strands.

[0106] FIG. 3 is an epifluorescence image of liposomes isolated from a blood sample on a structured capture array and printed on a functionalised coverslip. Each solid circle represents a plurality of liposomes and fits the dimension of the round well where the liposomes were isolated.

[0107] FIG. 4 represents two stamps' configurations and the respective capture of DNA strands and fluorescent nanoparticles. FIG. 4a) represents a non-sequential configuration of a stamp observed with a Scanning Electron Microscopes (SEM) and FIG. 4c) is an epifluorescence image of DNA strands and fluorescent nanoparticles isolated from a blood sample on the said stamp. White circles surround the captured fluorescent nanoparticles. FIG. 4b) represents a non-sequential configuration of a stamp observed with a Scanning Electron Microscopes (SEM) and FIG. 4d) is an epifluorescence image of DNA strands and fluorescent nanoparticles isolated from a blood sample on the said stamp. White circles surround the captured fluorescent nanoparticles.

[0108] FIG. 5 represents assemblies of fluorescent polystyrene (PS) nanoparticles at different speeds. FIG. 5a) is an epifluorescence image of polystyrene nanoparticles captured on a non-sequential stamp at 2 m/s. FIG. 5b) is an epifluorescence image of polystyrene nanoparticles captured on a non-sequential stamp at 3 m/s. FIG. 5c) is an epifluorescence image of polystyrene nanoparticles captured on a non-sequential stamp at 5 m/s. FIG. 5d) is an epifluorescence image of polystyrene nanoparticles captured on a non-sequential stamp at 7 m/s. FIG. 5e) is an histogram representing the average number of isolated nanoparticles aggregates within 20 micro cavities as function of the assembly speed. FIG. 5f) is a histogram representing the average number of isolated nanoparticles aggregates within 10650 nanocavities as function of the assembly speed.

[0109] FIG. 6 relates to comparison of fibers of polymerized plasma proteins and DNA strands observed in fluorescence and in bright field. FIG. 6a) is an epifluorescence image of assembled fibers from unspiked blood (without spiked DNA strands) captured on a non-sequential stamp at 10 m/s. FIG. 6b) is a bright field image of the assembled fibers observed at FIG. 6a). FIG. 6c) is an epifluorescence image of assembled DNA strands from spiked blood captured on a non-sequential stamp at 10 m/s. FIG. 6d) is a bright field image of the assembled DNA strands observed at FIG. 6c).

[0110] FIG. 7 relates to an analysis of the influence of the Triton concentration on the occupation rate of polymerized plasma proteins fibers. FIG. 7a) is an epifluorescence image of assembled fibers from unspiked blood (without spiked DNA strands) with 0.5% Triton-X captured on a non-sequential stamp at 2 m/s. FIG. 7b) is an epifluorescence image of assembled fibers from unspiked blood (without spiked DNA strands) with 0,25% Triton-X captured on a non-sequential stamp at 2 m/s. FIG. 7c) is an epifluorescence image of assembled fibers from unspiked blood (without spiked DNA strands) with 0,125% Triton-X captured on a non-sequential stamp at 2 m/s. FIG. 7d) is a histogram representing the occupation rate of the cavities of the stamp by the fibers as function of the Triton-X concentration (TX).

[0111] FIG. 8 relates to Biofunctionalization of the bottom of surface cavities through capillary assembly. FIG. 8a) is an epifluorescence image of a control experiment wherein an assembly of a PBS solution with 0.5% of Triton-X at 10 m/s on a non-sequential stamp. FIG. 8b) is epifluorescence image of an assembly of a PBS solution with 0.5% of Triton-X and a fluorescent labelled anti-CD81 antibody at 20 g/mL at 10 m/s on a non-sequential stamp.

[0112] FIG. 9 relates to assembly of exosomes from whole blood. FIG. 9a) represents a 5 m cavity from a non-sequential stamp observed by SEM after assembling a control solution. FIG. 9b) represents a 5 m cavity from a non-sequential stamp observed by SEM after assembling of exosomes from spiked blood on a non-sequential stamp. FIG. 9c) is an epifluorescence image of the stamp after assembling the control solution. No fluorescence is observed on the stamp. FIG. 9d) is an epifluorescence image of the stamp after assembling the exosomes from spiked blood and incubated with a florescent antibody direct to the exosomes. Exosomes are revealed (white spots) on the cavities of the stamp.

[0113] FIG. 10 relates to Different methods for characterizing the combined capture of exosomes and circulating free DNA (cfDNA). FIGS. 10a), 10b and 10c are images obtained by SEM at different magnifications, after assembly of sample 1 on a non-sequential stamp at 10 m/s. Exosomes are observable as white dots inside the cavities. FIG. 10d) is an epifluorescence image of DNA strands and exosomes after assembling of sample 2. Captured exosomes are highlighted by white circles. FIG. 10e) is an epifluorescence image of captured DNA strands after assembling of sample 3. FIG. 10f) is an epifluorescence image of captured exosomes (highlighted by white circles) after assembling of sample 3.

EXAMPLES

Example 1: Isolation of DNA Strands from a Blood Sample Enriched in DNA Extract

[0114] 1. Preparation of a Polydimethylsiloxane (PDMS) Stamp

[0115] The structured capture array is a PDMS stamp. The topographical features of the PDMS stamp are formed using a silicon mould. The silicon mould comprises several pillars with a 20 m space between them in order to form wells in the PDMS stamp. The PDMS is prepared by using Sylgard 184 Kit. In accordance with the notes on completion, 1 dose of curing reagent is mixed with 10 doses of base. The curing agent contains Dimethyl siloxane, dimethylivinyl terminated (CAS Number: 68083-19-2), Dimethylvinylated and trimethylated silica (CAS Number: 68988-89-6), Tetra (trimethoxysiloxy) silane (CAS Number: 3555-47-3) and Ethyl benzene (CAS Number: 100-41-4). The base contains Dimethyl, methylhydrogen siloxane (CAS Number: 68037-59-2), Dimethyl siloxane, dimethylvinyl terminated (CAS Number: 68083-19-2), Dimethylvinylated and trimethylated silica (CAS Number: 68988-89-6), Tetramethyl tetravinyl cyclotetra siloxane (CAS Number: 2554-06-5, Ethyl benzene (CAS Number: 100-41-4). This PDMS mixture is then poured onto the mould, baked at 80 C. during 2 hours to polymerize and become solid.

[0116] 2. Preparation of a Functionalized Coverslip for Printing

[0117] a. Cleaning of the Coverslip

[0118] First, one side of a glass coverslip is cleaned up with a solution of acetone, then with a solution of ethanol and finally with a solution of deionised water. Then, the washed coverslip side is placed up on a paper towel and dried with nitrogen by means of an air gun. Thereafter, the coverslip is moved to a new spot on the towel and dried again with the gun. Afterwards, the coverslip is taken with tweezers and nitrogen is blown from the side of the coverslip to get rid of any water residual. Finally, a radio frequency plasma treatment is performed during 5 min at 0.6 mbar air and 100% power (50 W).

[0119] b. Functionalization of the Coverslip with an APTES Solution (1% Silane in 95% EtOH/5% ddH.sub.2O)

[0120] A hot plate is preheated to 140 C. A solvent is prepared by mixing 47.5 ml of ethanol and 2.5 ml of distilled water. 0.5 mL of APTES solution is taken using a syringe by inverting it and accounting for the volume of the air bubble forming inside the syringe. The solvent and the APTES solution are mixed in the glass dish and then covered with aluminium foil to limit evaporation for 5 minutes. The air atmosphere within the dish is replaced by nitrogen to limit air contact during hydrolysis. The aluminium foil is removed to allow the introduction of the plasma activated coverslip inside the dish for 20 minutes. The functionalised coverslip is then removed, cleaned thoroughly with ethanol and ddH.sub.2O, dried with a nitrogen gun and finally put on a hot plate at 140 C. for 5 minutes.

[0121] 3. Preparation of Triton-X100 and YOYO-1 Solutions

[0122] A 10% Triton-X100 solution is prepared by mixing 1 ml of Triton-X with 9 ml of PBS in a 15 mL Falcon tube.

[0123] YOYO-1 is a nucleic acid stain. A 1:10 dilution of the stock YOYO-1 solution is prepared by diluting with PBS a 1 mM stock solution.

[0124] 4. Preparation of the DNA Extract

[0125] Lambda phage DNA extracts were acquired from New England Biolabs (NEB)

[0126] 5. Preparation of the Assembly Solution

[0127] Blood was obtained from the Etablissement Franais du Sang (EFS), collected in EDTA coated tubes to prevent clotting.

[0128] The assembly solution was prepared by mixing 45.25 l of blood with 2.5 l of the DNA extract, 0.75 l of the solution of YOYO-1 and 1.5 l of the solution of Triton-X100. The final concentration in the assembly solution are the following: [0129] Triton-X100: 0.3% v/v, [0130] YOYO-1: 7.5 M, [0131] DNA extract: 25 g/ml.

[0132] 6. Mounting the Assembly Solution on the PDMS Stamp

[0133] The temperature is set at 20 C. and the humidity at 40% as measured with a numerical hygrometer. A coverslip is cleaned as described at point 2.a. With the topographical features facing up, the PDMS stamps is placed on a PDMS Petri dish with its long side perpendicular to the movement of dragging (long side horizontal). The cleaned coverslip is placed and held at approximately 2-3 mm above the PDMS stamp. 40 L of the assembly solution is placed between the PDMS stamp and the coverslip. Then the droplet is spread evenly all along the topographical features of one the short side of the PDMS stamp.

[0134] 7. Isolation and Printing of the Extract DNA

[0135] The coverslip is moved in the direction of the long side of the PDMS stamp at speeds of 20 m/s, 200 m/s and 1 mm/s. Once the other short side of the stamp is reached, the stamp is removed and the remaining droplet is wicked away using a paper. All of the water residuals are removed to avoid any spread upon contact with the functionalised coverslip. The topographical side of the PDMS stamp is then placed into contact with the functionalised side of the functionalised coverslip for 1 min. Thereafter, the PDMS stamp is removed and stored in dark conditions.

[0136] 8. Epifluorescence Microscopy

[0137] Samples are observed at x100 magnification using an inverted microscope (Olympus, exposure: 30 ms; camera gain: 100; cyan light; laser power 30; Zeiss, Camera gain: 3; exposure time: 200 ms; laser power: 100%; cyan light).

[0138] The results obtained for each speed are represented at FIGS. 1A to 1C. The white lines correspond to the elongated DNA strands printed at the surface of the functionalised coverslip. On the pictures, one can see some DNA strands that seem thicker and brighter than the others. The brighter strands correspond to a set of strands and the lighter strands to single strands, which would explain the brightness and thickness.

[0139] The inventors obtained a better reproducibility and a better coverage of the functionalised coverslip (resulting from a better isolation of the DNA strands into the wells of the PDMS stamp) at a 20 m/s speed of dragging.

Example 2: Isolation of Circulating DNA from a Blood Sample

[0140] The aim of this example is to isolate circulating DNA from a blood sample pertaining to a patient afflicted by cancer.

[0141] Points 1. to 3. and 5. to 8. of Example 1 were reproduced, except that [0142] in point 5, no DNA was added to the assembly solution and that 47 l of a blood sample recovered from a clinical trial with patients afflicted by cancer is mixed with 1.5 l of the solution of YOYO-1 and 1.5 l of the solution of Triton-X100, and that [0143] in point 7, the speed at step a) is 20 m/s.

[0144] The results are represented on FIGS. 2A and 2B.

Example 3: Isolation of liposomes added to a blood sample

[0145] Points 1. to 3., 6. and 7. of Example 1 were reproduced, except that [0146] in point 2., the cleaned coverslip is functionalized with GPTMS according to the below protocol; [0147] in point 7, the speed at step a) is 20 m/s.

[0148] Points 4. and 5. of Example 1 are replaced by the below points 2. and 3. respectively.

[0149] 1. Functionalisation of the Coverslip with GPTMS

[0150] 1.25 ml of a solution of GPTMS is mixed with 48.75 ml of pure ethanol. The plasma activated coverslip's side is laid on the mixture for 30 min. The functionalised coverslip's side is cleaned thoroughly with ethanol only and dry slides with an air gun.

[0151] 2. Preparation of Liposomes

[0152] The lipids used for this protocol are phosphatidylcholines (POPC) and 1-palmitoyl-2-{6-[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]hexanoyl}-sn-glycero-3-phosphocholine (NBD-PC) from Avanti Polar Lipids. NBD-PC is a fluorescent lipid used to see the liposomes in epifluorescence (excitation wavelength 460 nm; emission wavelength 534 nm).

[0153] a. Preparation of Lipids Solutions

[0154] The phospholipids are stored at 20 C. in solution in chloroform (Sigma Aldrich), at a concentration of 10 mg/ml for POPC and 1 mg/ml for NBD-PC.

[0155] b. Preparation of Phospholipids

[0156] 100 l of POPC solution is mixed with 10 l of NBD-PC solution in a 4 ml glass flask. Then the flask is heated at 55 C. in a dry bath while an air flow is creating using an air gun pointing inside the flask in order to make the chloroform evaporate. The flask is thereafter placed in a vacuum chamber for 2 hours to make sure all the chloroform is evaporated. The chamber is covered with aluminium foil to avoid fluorophore destruction by daylight. Finally, the flask is sealed with a parafilm and stored at 4 C. if not used right away.

[0157] c. Suspension of Lipids to Obtain Multilamellar Vesicles

[0158] Add 0.5 ml of PBS is added in the flask and vortexed until complete homogenisation. Formation of 80 nm diameter small unilamellar vesicles (SUV) is performed by extrusion using Avanti Polar Lipids mini extruder kit.

[0159] The SUV suspension is at 2 mg/ml concentration and can be stored at 4 C. and used within 3 days.

[0160] 3. Preparation of the Assembly Solution

[0161] The assembly solution was prepared by mixing 25 l of blood with 22.5 l of the liposome solution and 2.5 l of the solution of Triton-X100.

[0162] 4. Epifluorescence Microscopy

[0163] Samples are observed at x100 magnification using an inverted microscope (Olympus, exposure: 30 ms; camera gain: 100; cyan light; laser power 30; Zeiss, Camera gain: 3; exposure time: 200 ms; laser power: 100%; cyan light).

[0164] The results for a speed of 10 m/s are represented in FIG. 3. This figure shows that the liposomes were efficiently isolated into the wells and transferred onto the functionalised coverslip.

Example 4: Configurations of Stamp Patterns

[0165] 1. Objectives of the Experiment

[0166] New stamps have been built in order to implement a combinatorial liquid biopsy capture. The stamp used in Examples 1 to 3 contained only micro cavities with diameter of 5 m. The objective of this example was to develop new configurations of stamps which contains both micro cavities of 5 m, adapted for the DNA strands capture, and nano cavities of 500 nm, adapted for nanoparticles capture, in particular exosome.

[0167] 2. Materials and Methods

[0168] Two configurations of stamps patterns were tested. The first one is called non-sequential and consists in alternating micro patterns with nano patterns along the dragging direction. The second one, sequential, is split in two: one half of the stamp surface is equipped with micro patterns and the second half with nano patterns. Both configurations are visible in FIGS. 4a) and 4b).

[0169] Thanks to microfabrication's processes, new moulds composed with both configurations were fabricated in a clean room. Each mould consisted in a 4 inches wafer of silicon and on this wafer, different chips were defined. Half of the chips corresponded to non-sequential configuration and the other half to sequential configuration. PDMS (Polydimethylsiloxane) replica were produce by conventional molding process, after cross-linking the stamps were cut according to chip borders. After unmolding, the different stamps were composed of micro cavities and nano cavities either in sequential or in non-sequential configurations.

[0170] In order to investigate the suitability of these stamps for combinatorial assembly, a solution of blood spiked with 100 nm fluorescent polystyrene nanoparticles (concentration of 2,5 g/mL=510.sup.9 particles/mL) and DNA strands (25 g/mL) with 0.5% of Triton-X and 0,75 M of YOYO-1 was prepared. A drop of 35 L of this solution was assembled at 10 m/s then at 2 m/s on each configuration.

[0171] Captured nanoparticles and DNA strands are observed at x100 magnification using an inverted microscope (Zeiss, Camera gain: 3; exposure time: 200 ms; laser power: 100%; cyan light).

[0172] Stamps configurations are observed with a Scanning Electron Microscopes (SEM Helios 600i FEI, Acceleration Voltage 15 kV, e-beam current 86 pA, secondary electron signal).

[0173] 3. Results

[0174] The results are presented on FIGS. 4c) and 4d). In both configurations, DNA strands and nanoparticles are assembled. The nanoparticles are observable in micro cavities in both configurations (they are indicated by the circles). Inside nano-cavities the nanoparticles are not detected by fluorescence. The results obtained between the two configurations are equivalent: the DNA occupation rate, the length and the good state of the captured DNA strands, as well as the ratio of occupied cavity with nanoparticles.

Example 5: Nanoparticles Assembly

[0175] 1. Objectives of the Experiment

[0176] Exosomes are extracellular vesicles exhibiting typical sizes between 30 and 140 nm. They contain proteins, miRNA, DNA and other biomarkers bringing information about the disease. They are considered as a novel biomarker and their study in a cancer research context is more and more developed. However, due to their small size, they are difficult to isolate. In order to determine the optimal parameters for their capture, exosomes were micmicked with 100 nm fluorescent polystyrene nanoparticles in this experiment. The objective of this experiment is thus to implement the assembly of nanoparticles in blood at a concentration closed to realistic exosomes concentration and to determine the optimum speed of assembly.

[0177] 2. Materials and Methods

[0178] A solution of blood spiked with 100 nm fluorescent polystyrene nanoparticles (2,5 g/mL=10.sup.9 particles/mL) with 0.5% of Triton-X is prepared. A drop of 35 L of this solution was assembled at 2, 3, 5 and 7 m/s on sequential and non-sequential stamps as described in example 4, in order to determine the optimal assembly speed. After assembly, each stamp was put on a clean coverslip and observed on the microscope through this coverslip.

[0179] Captured nanoparticles are observed at x100 magnification using an inverted microscope (Zeiss, Camera gain: 3; exposure time: 200 ms; laser power: 100%; cyan light).

[0180] 3. Results

[0181] The results are shown in FIG. 5. Nanoparticles were isolated in micro cavities as well as in nano cavities. The graphs 5e) and 5f) show a decrease of the number of isolated nanoparticles aggregates in both micro and nano cavities with the increasing of the speed, which enables to establish a link between scanning speed and quantity of captured nanoparticles.

[0182] 4. Conclusion

[0183] Nano species such as nanoparticles were assembled from whole blood in all type of cavities (micro and nano). Optimizing the speed of dragging optimizes the quantity of isolated nanoparticles. Here, the optimum speed was given at 2 m/s, despite that satisfying result can be obtained at higher speeds.

Example 6: Capture of Plasma Proteins Fibers

[0184] 1. Objectives of the Experiment

[0185] At multiple occasions, some fibers were assembled during experiments in whole blood without any spiking of DNA molecules. They are different from DNA because through SEM and optical fluorescence observation they appear to be thicker, more diffuse and less fluorescent. The inventors hypothesized these fibers were formed during capture by capillary assisted polymerization of plasma proteins such as fibrine. The objectives of the following experiments are to confirm these fibers are not DNA molecules and to determine a way to limit them, if desired.

[0186] 2. Materials and Methods

[0187] A solution of blood with 0.5% Triton-X is prepared and an assembly on a non-sequential stamp, as described in example 4, at 10 m/s with a drop of 35 L of this solution is carried out. At the end of the assembly, the stamp is observed through a glass coverslip on the microscope described in example 4. The result is compared to another sample (at 10 m/s with a drop of 35 l) corresponding to the assembly in blood spiked with lambda-phage DNA strands dyed with YOYO-1 at 0,75 M. Both samples are visualized in fluorescence and in bright field on the microscope described in example 4. As DNA strands cannot be observed in bright field under these conditions while protein fibers do because of their thicker structure which increases light diffusion, that makes it possible to distinguish DNA strands from protein fibers. In order to determine a way to limit the apparition of these protein fibers, three solutions of blood with 0.5%, 0,25% or 0,125% Triton-X were prepared and assemblies at 2 m/s are performed on a non-sequential stamp, as described in example 4. The three stamps (one per concentration) are observed on a microscope through a coverslip and compared.

[0188] 3. Results

[0189] FIG. 6a) represents the assembled fibers from unspiked blood in fluorescence and FIG. 1b) in bright field. One can see that fibers of polymerized plasma proteins are observable at the same locations in both images. It means that these captured objects are visible in bright field.

[0190] FIGS. 6c) and 6d) represent the assembled spiked DNA strands dyed with YOYO-1 from blood in fluorescence (6c)) and in bright field (6d)). In FIG. 6d), no strand appeared, confirming DNA strands are only observable in fluorescence.

[0191] FIG. 7 represents assemblies of unspiked blood at different Triton-X concentrations. On FIGS. 7a), 7b) and 7c), which are respectively assemblies at 0.5%, 0,25% and 0,125% of Triton-X in blood, one can see that the number of observed fibers decreases with the decrease of Triton-X concentration. The graph on FIG. 7d) confirms this result since the occupation rate decreases when the Triton-X concentrations decreases.

[0192] Other experiments (not described here) proved that the speed also has an influence on the quantity of fibers. Indeed, there are more fibers of polymerized plasma proteins at 2 m/s than at 10 m/s.

[0193] 4. Conclusion

[0194] It can be affirmed that the fibers assembled from blood are not DNA strands, since the latter cannot be observed in bright field.

[0195] A link between the occupation rate of polymerized plasma proteins fibers and Triton-X concentration can be established, since the number of assembled fibers decreases if the Triton-X concentration decreases. The speed is also an important factor for the assembly of those fibers since their number decreases when the speed increases.

[0196] In conclusion, very low scanning velocity combined with high Triton-X concentration favors the formation of protein fibers in whole blood. If such phenomenon needs to be limited, the operation parameters need to be tuned such as speed is set at 10 m/s or more and Triton-X concentration is used at a concentration below 0,125%.

Example 7: Biofunctionalization of the Bottom of the Surface Cavities with a Specific Antibody

[0197] 1. Objectives of the Experiment

[0198] The objective of this experiment was to functionalize the bottom of the surface cavities in order to facilitate the exosomes assembly inside the cavities while preventing their adsorption on the top surface of the stamp. This functionalization has been already attempted by incubation of an antibody on the stamp for one hour. A printing of the stamp was then realized by putting the stamp in contact with different coverslips, one after another, in order to progressively remove the antibody molecules adsorbed on the top surface. However, the results were not excellent and reproducible from stamp to stamp. The inventors thus decided to functionalize the bottom cavities by capillary assembly. In other words, they used the same principle of biomarker capture but instead of manipulating a blood microvolume, they manipulate a microvolume of a solution containing the selected antibody. Capillary effects thus drive these molecules inside the cavities and not on the top surface of the stamp. The described experiment demonstrates this principle.

[0199] 2. Materials and Methods

[0200] A solution of PBS (1X) is prepared with 0.5% of Triton-X and with a fluorescent labelled anti-CD81 antibody at 20 g/mL in PBS. CD81 is a protein inserted inside the envelope of exosomes. A drop of 35 L of this solution is then assembled on the surface of a non-sequential stamp, as described in Example 4, at a speed of 10 m/s.

[0201] A control solution with only PBS and 0.5% of Triton-X was also prepared. An assembly is realized at 10 m/s on another non-sequential stamp.

[0202] Both stamps, the control one and the one with the antibody are observed with the optical microscope described in Example 4 through a coverslip and compared.

[0203] 3. Results

[0204] The results are presented on FIG. 8. On the left picture (control), no fluorescence is observed. On the contrary, fluorescents dots are visible on the right picture sample attesting for the immobilization of the anti-body at the bottom of each micro-cavities present on the stamp. Interestingly, one can note that the fluorescence is only visible inside the cavities, not on the top surface.

[0205] 4. Conclusion

[0206] Thanks to the capillary assembly technique, the antibody can be placed only in the bottom of the cavities. Advantageously, the repartition of the antibody is homogeneous in all the cavities and solely one step is to be performed to functionalize the cavities. Moreover, this functionalization does not require cleaning step(s) since the antibody is only present inside the cavities.

Example 8: Exosome Capture from Whole Blood

[0207] 1. Objectives of the Experiment

[0208] The objective of this experiment is to capture exosomes from whole blood by capillary assembly on a bio-functionalized stamp (bottom cavities). The inventors characterized the capture by optical fluorescence microscopy and by Scanning Electron Microscopy (SEM).

[0209] 2. Materials and Methods

[0210] Three solutions were prepared: [0211] a functionalization solution comprising PBS with 0.5% of Triton-X and anti-CD81 antibody at 20 g/mL in PBS, [0212] an exosomes solution comprising a blood sample enriched with exosomes at a concentration close to 10.sup.9/mL and 0,5%, of Triton-X and [0213] a control solution which is composed of unspiked blood and 0.5% of Triton-X.

[0214] For both optical fluorescence and SEM observation, two samples were characterized: [0215] 1) A control (unspiked blood) [0216] 2) An assembly of exosomes from spiked blood on a biofunctionalized stamp (bottom cavities)

[0217] For the control, a drop of 35 L of the anti-CD81 solution is assembled on a non-sequential stamp at 10 m/s. Immediately after the functionalization, another assembly is performed at 2 m/s with a drop of 35 L of the control solution.

[0218] For the exosomes assembly on a biofunctionalized stamp, a drop of 35 L of the anti-CD81 solution is assembled on a non-sequential stamp at 10 m/s. Immediately after the functionalization, another assembly is performed at 2 m/s with a drop of 35 L of the exosomes solution.

[0219] When the resulting capture is observed in fluorescence, an incubation is carried out for one hour with a solution of fluorescein isothiocyanate (FITC) anti-CD63 (Thermofisher, MA1-19602) diluted at 1:100 in volume in PBS at the end of the assembly of the exosomes solution or the control solution. This secondary fluorescent antibody reveals the presence of exosomes, the CD63 being a protein commonly found in the envelope of exosomes. After the incubation, the stamp is rinsed four times with PBS.

[0220] SEM observations were performed directly without performing any incubation with the fluorescent secondary antibody (SEM Helios 600i FEI, Acceleration Voltage 15 kV, e-beam current 86 pA, secondary electron signal).

[0221] 3. Results

[0222] The results are presented on FIG. 9. On FIG. 9a) (the control), by SEM no element compatible with exosome shape and dimension can be observed at the bottom of the cavities. Accordingly, no exosome was captured. This is confirmed by FIG. 9c) where the control is observed in fluorescence after incubation and no signal coming from the bottom of the cavities can be observed. The background fluorescence is only at the surface of the stamp.

[0223] On FIG. 9b) (exosomes on functionalized stamp), nano-spheres are clearly observed at the bottom of the cavities. They correspond to exosomes according to their size and typical contrast. Indeed, exosomes are vesicles with typical sizes between 30 and 140 nm and they are recognizable in electron microscopy thanks to a donut like contrast in their center. This is also confirmed by FIG. 9d) where fluorescence dots are located at the bottom of the cavities, when the focus plane is adjusted at that location, revealing the presence of exosomes inside the cavities.

[0224] 4. Conclusion

[0225] By comparing the different images, obtained by fluorescence or SEM, it can be affirmed that, by functionalizing the bottom of the cavities, exosomes can be assembled. The control samples demonstrates that nothing is observed inside the cavities if exosomes are not spiked into blood.

Example 9: Different Methods for Combining the Capture of Exosomes and Circulating Free DNA (cfDNA)

[0226] 1. Objectives of the Experiment:

[0227] The objective of this experiment is now to perform the combinatory capture with DNA strands and exosomes both spiked inside whole blood. Captured samples were observed in fluorescence and Scanning Electron Microscopy. However, in order to observe exosomes in fluorescence, they need to be dyed with a secondary fluorescent antibody by incubation. This incubation is problematic for the already assembled DNA strands since they can be resuspended in the solution during secondary antibody incubation. Here the inventors solved this problem with the following experiments: [0228] a combinatory capture in one step (without incubation of a secondary labelling antibody for exosomes), [0229] a combinatory capture using combination of both biomarkers in fluorescence (with the incubation of the secondary labelling antibody for exosomes)

[0230] 2. Materials and Methods:

[0231] A solution of PBS with 0.5% of Triton-X and an anti-CD81 antibody at 20 g/mL in PBS was prepared. Another solution of blood with DNA strands at 25 g/mL and exosomes (approximate concentration of 10.sup.9 mL.sup.1) with 0.5% Triton-X and 0,75 OA of YOYO-1 was prepared.

[0232] Three samples were realized: [0233] 1. one for SEM observation, [0234] 2. one with both biomarkers (DNA strands and exosomes) assembled on a same stamp in order to be observed together by fluorescence microscopy, [0235] 3. one with both biomarkers first assembled on a same stamp but the DNA strands are transferred on an APTES functionalized coverslip, as previously described in Example 1, before the exosomes incubation with a fluorescent antibody. Hence, the DNA strands are on the printed coverslip while the exosomes still remain inside the cavities of the stamp. The two biomarkers are finally observed separately by fluorescence microscopy.

[0236] For sample 1, an assembly of the bio-functionalization solution with the anti-CD81 antibody is performed at 10 m/s on a non-sequential stamp as described in Example 4. Another assembly is then realized with a drop of 35 L of the spiked blood with exosomes and DNA strands at 10 m/s. After the assemblies, a thin metallic film of 5 nm of Au/Pd is deposited at the stamp surface. The sample is then observed by SEM (SEM Hlios 600i FEI, Acceleration Voltage 15 kV, e-beam current 86 pA, secondary electron signal).

[0237] For sample 2, an assembly of the functionalization solution with the anti-CD81 antibody is performed at 10 m/s on a non-sequential stamp as described in Example 4. Another assembly is then realized with a drop of 35 L of the spiked blood with exosomes and DNA strands at 2 m/s. An incubation is carried out for one hour with a solution of a secondary labelled FITC anti-CD63 (Thermofisher, MA1-19602) diluted at 1:100 in volume in PBS. After the incubation, the stamp is rinsed four times with PBS. Another assembly is then performed with a drop of 35 L of the same spiked blood with exosomes and DNA strands at 10 m/s, in order to capture fresh DNA strands and compensate those re-suspended during the incubation of the secondary antibody.

[0238] For sample 3, an assembly of the functionalization solution with the anti-CD81 antibody was performed at 10 m/s on a non-sequential stamp as described in Example 4. Another assembly is then realized with a drop of 35 L of spiked blood with exosomes and DNA strands at 2 m/s. The stamp is pressed on a APTES (3-Aminopropyl-triethoxysilane) functionalized coverslip as described in Example 1 for one minute in order to transfer the assembled DNA strands by electrostatic interactions. After the transfer, an incubation is carried out on the stamp for one hour with a solution of a secondary labelled FITC anti-CD63 (Thermofisher, MA1-19602) diluted at 1:100 in volume in PBS in order to label the exosomes captured inside the cavities. After the incubation, the stamp is rinsed four times with PBS. DNA strands were observed, with the microscope described on example 4, the coverslip after their transfer by nano-contact printing while the exosomes were observed directly on the stamp with the said microscope through a protecting coverslip.

[0239] Results

[0240] SEM observations of sample 1 are presented on FIGS. 10a), 10b) and 10c). As one can see, exosomes were assembled at the bottom of the cavities (white dots). DNA strands were also observable as long white line starting from the cavities on the deposited thin metallic film used in SEM observation.

[0241] The fluorescence characterization of both biomarkers on the same support (sample 2) is represented on FIG. 10d). As one can see, exosomes and DNA strands were well characterized by fluorescence on the same image.

[0242] The biomarkers can also be observed separately (sample 3), as shown on FIGS. 10e) and 10f). On FIG. 10e), white lines corresponding to DNA strands were observed on the receiving coverslip, and besides, on FIG. 10f) exosomes were observed directly on the stamp (highlining by white circle).

[0243] Conclusion

[0244] The biomarkers can be observed after only one assembly thanks to SEM inspection. The inventors made it also possible to observe them in fluorescence [0245] with both remaining on the stamp or [0246] by removing one on another support for observation and observing the remaining one on the stamp.