METHOD FOR FORMING A LIQUID FILM ON A SUBSTRATE

This method for forming a liquid film on a substrate comprises the steps of placing the substrate in a chamber, and depositing a composition on the substrate, the composition including water, introducing a volatile liquid into the chamber, and closing the chamber for a predetermined period, the volatile liquid evaporating in the chamber during this closing step. The method further comprises at least partial extraction, out of the chamber, vapor formed by the evaporation of the volatile liquid, this extraction producing spreading out of the composition on the substrate, said spread composition then forming the liquid film on the substrate.

BACKGROUND OF THE INVENTION

The present invention relates to a method for forming a liquid film on a substrate. The formation method comprises the following steps:

placing the substrate in a chamber, and depositing a composition on the substrate, the composition including water,

introducing a volatile liquid into the chamber, and

closing the chamber for a predetermined duration, the volatile liquid evaporating in the chamber during this closing step.

The invention in particular applies to the detection of particles of micrometric and sub-micrometric size, notably biological particles, such as cells, bacteria or further viruses. The invention also applies to the detection of particles in the form of microbeads. The composition then includes said particles to be detected, and the substrate is a transparent slide, the transparent slide being intended to be illuminated by a light source, for acquiring by means of an optical detection system at least one image of the composition comprising the particles.

In order to acquire images of these particles with a large size image sensor, i.e. a sensor having an area of a few cm2, or even a few tens of cm2, the composition should be spread out at best on the transparent slide, in order to form a film of small thickness and with an area equivalent to that of the image sensor. By small thickness, is meant a thickness with a value of less than 500 μm, preferably comprised between 50 μm and 200 μm.

A first technique, so-called spin coating, consists of depositing the composition on the substrate and of causing rotation of the substrate, in order to spread the composition over the substrate by the action of the centrifugal force. However, this first technique is complex to apply, the spreading of the composition being relatively difficult to control. Further, when the composition includes bacteria, the introduction of the composition into a device able to apply this technique may contaminate the composition.

A second technique, so-called dip coating, consists of dipping the substrate in a reservoir including the composition, and then of gently withdrawing the substrate from this reservoir. A film of this composition is then formed at the surface of the substrate. However, this requires a large volume of said composition, typically of a few tens of and such a volume is not necessarily available.

A third technique is described in the article entitled “Overcoming the ‘coffee-stain’ effect by compositional Marangoni-flow-assisted drop-drying” of Majumder et al., published in “The Journal of Physical Chemistry” in 2012. This third technique consists of depositing a drop of water on a Teflon substrate and of placing this substrate in an atmosphere saturated with ethanol, for example inside a Petri dish. The drop then spreads over the substrate during the gradual presence of the substrate in the atmosphere saturated with ethanol. The substrate should be placed in this atmosphere saturated with ethanol for a period of the order of 1,000 seconds, in order to observe proper spreading of the drop.

However, this spreading of the drop is not optimum, and requires a relatively large amount of the composition in order to obtain a film having an area of a few cm2, or even of a few tens of cm2.

SUMMARY OF THE INVENTION

The object of the invention is therefore to propose a method for forming a liquid film on a substrate, the film being formed by spreading out a composition, the method allowing improvement in the spreading of the composition over the substrate.

For this purpose, the object of the invention is a method of the aforementioned type, wherein the method further comprises at least partial extraction, outside of the chamber, of the vapor formed by the evaporation of the volatile liquid, this extraction producing a spreading of the composition over the substrate, said spread composition then forming the liquid film on the substrate.

Unlike the method of the state of the art corresponding to the third technique, the spreading of the composition according to the invention is not observed during the step for hermetically closing the chamber, i.e. during the evaporation of the volatile liquid inside the chamber, but only from the moment when the vapor formed by evaporation of the volatile liquid is at least partly extracted out of the chamber.

According to other advantageous aspects of the invention, the method comprises one or more of the following features, taken individually or according to all the technically possible combinations:

the predetermined period has a value comprised between 1 minute and 30 minutes, preferably comprised between 5 minutes and 20 minutes, still preferably substantially equal to 10 minutes;

during introduction of the volatile liquid, the volatile liquid is positioned away from the composition, in the absence of contact between the volatile liquid and the composition;

the volatile liquid introduced into the chamber includes an alcohol, such as ethanol;

the chamber is a box, such as a Petri dish, the box including a lid and a receptacle having an aperture, the lid being movable between an open position in which the lid is away from the aperture of the receptacle and a closed position in which the lid is able to obturate the aperture of the receptacle, said closed position corresponding to hermetic closure of the box, and during the closing step, the lid is moved from its open position to its closed position, and then maintained in the closed position for the predetermined period, and during the extraction step, the lid is moved from its closed position to its open position, and then maintained in the open position;

the receptacle includes a bottom, the volatile liquid is positioned against the bottom during the introduction step, and the box further includes at least one shim for maintaining the substrate away from the bottom, in order to position the volatile liquid away from the composition during the introduction step;

the composition further comprises particles, a surfactant and a hydrophilic polymer, the particles having a diameter preferably of less than 10 μm, still preferably less than 1 μm, the surfactant having a concentration preferably at least equal to the critical micellar concentration, and the hydrophilic polymer has a boiling temperature above that of water, the polymer preferably being a polyethylene glycol defined by the following formula:

H(—OCH2CH2—)nOH, wherein n represents the number of oxyethylene unit(s) of the polymer;

the composition has a volume with a value comprised between 0.5 μL and 5 μL;

the substrate is made in a hydrophilic material, such as glass or still further a plastic material;

the substrate is a transparent slide, the transparent slide being intended to be illuminated by a light source, for acquiring, by means of an optical detection system, at least one image of the composition comprising particles; and

the composition further comprises particles, the particles being biological species, such as live biological species, for example bacteria.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

InFIG. 1, a substrate10is placed in the chamber12, and a composition14including water as well as particles24, is deposited on the substrate10. A volatile liquid16is positioned in the chamber12, and the chamber12is hermetically closed, the volatile liquid16evaporating inside the chamber12when the latter is hermetically closed.

The substrate10is preferably made in a hydrophilic material, such as glass or further such as a plastic material which has been made hydrophilic.

The substrate10for example, is in the form of a transparent slide18intended to be illuminated by a light source20, for acquiring by means of an optical detection system22, at least one image of the composition14comprising the particles24, as illustrated inFIG. 4.

The chamber12for example is a box26, such as a Petri dish, the box26including a lid28and a receptacle30having a bottom31and an aperture32. The lid28is movable between an open position in which the lid28is away from the aperture32of the receptacle and a closed position in which the lid28is able to obturate the aperture32of the receptacle, said closed position corresponding to hermetic closure of the box26.

In the exemplary embodiment ofFIG. 1, the chamber12is of a cylindrical form. The chamber12for example has a diameter of about 10 cm and a height of about 1.5 cm.

Additionally, the chamber12includes at least one shim33for maintaining the substrate10away from the bottom31of the receptacle, in order to position the volatile liquid16away from the composition14when it is introduced into the chamber12. In the exemplary embodiment ofFIG. 1, the chamber12includes two maintaining shims33.

The composition14for example comprises the particles to be detected24and a solution, the solution including water, a surfactant and preferably a hydrophilic polymer. In other words, the composition14is a dispersion of particles to be detected24in an aqueous solution comprising water, the surfactant and the hydrophilic polymer.

In the described exemplary embodiment, the solution of the composition14consists of water, of the surfactant and of the hydrophilic polymer. The composition14then consists of the particles to be detected24, of water, of the surfactant and of the hydrophilic polymer.

The composition14has a volume with a value comprised between 0.5 μL and 5 μL.

The hydrophilic polymer has a boiling temperature above that of water. The hydrophilic polymer for example is a polyethylene glycol defined by the following formula:

wherein n represents the number of oxyethylene unit(s) of the polymer.

The number n of oxyethylene unit(s) is an integer, preferably comprised between 1 and 180, still preferably comprised between 4 and 16, still preferably equal to 13.

The number n of units is for example equal to 2, and the polymer is then called diethylene glycol, also noted as DiEG.

Alternatively, the number n of oxyethylene units is equal to 13, and the hydrophilic polymer is called polyethylene glycol 600, also noted as PEG 600, PEG 600 having a molecular weight of the order of 600 g/mol.

When the number n of oxyethylene units is equal to 13, the hydrophilic polymer, i.e. PEG 600, has a mass concentration preferably comprised between 0.2% and 0.8%, still preferably comprised between 0.4% and 0.6%.

Still alternatively, the number n of oxyethylene units is equal to 180, and the hydrophilic polymer is called polyethylene glycol 8000, also noted as PEG 8000, PEG 8000 having a molecular weight substantially equal to 8,000 g/mol.

The surfactant, also called a tenside, has a concentration preferably equal to the critical micellar concentration (CMC). The surfactant includes for example polyoxyethylene 20 sorbitan monolaurate, also known under the commercial name of TWEEN20.

Alternatively, the surfactant includes a copolymer of polyoxyethylene and of polyoxypropylene, such as a poloxamer, for example one known under the commercial name of PLURONIC F-68. Still alternatively, the surfactant includes sodium dodecylsulfate, also called SDS.

The surfactant, optionally completed with the hydrophilic polymer, is intended to form a film covering the particles24upon evaporation of the water of the solution14. The addition of a hydrophilic polymer gives the possibility of extending the period, or duration, during which this film covers the particles24.

The volatile liquid16positioned in the chamber12includes an alcohol, such as ethanol. In the described exemplary embodiment, the volatile liquid16consists of this alcohol, such as ethanol.

Alternatively, the volatile liquid16is isopropanol or any other volatile solvent.

The preferred volatile liquid16is however ethanol, since it is adapted to the case when the particles24are biological species, and more particularly live biological species, notably bacteria or any other microorganism. Under these particular conditions, it is actually preferable to have a not very aggressive volatile liquid16towards biological species24.

The transparent slide18has a thickness E along a longitudinal direction X corresponding to the illumination direction of the composition14by the light source20, as illustrated inFIG. 4. The thickness E for example has a value comprised between 10 μm and 100 μm, preferably comprised between 20 μm and 50 μm.

The slide18is preferably hydrophilic, so that a contact angle α, visible inFIG. 1, between the composition14and the slide18has a small value. The value of the contact angle α obtained between the hydrophilic slide18and the composition14is less than 20°, preferably less than 10°, still preferably of the order of a degree.

The hydrophilicity of the slide18is for example obtained by preparing the slide18according to the following steps. The first preparation step is sonication in soapy water for a period, or duration, of the order of 10 minutes. The soapy water for example includes water and washing up liquid, the water preferably being a pure water of type 1 according to the ISO 3696 standard, also known under the commercial name of Milli-Q. The second step is a rinsing step with water, such as Milli-Q water, with acetone and with isopropanol. The third step is a drying step, for example drying with nitrogen, and the fourth step is having an oxygen plasma pass for a period, or duration, of more than 15 seconds, preferably of the order of 30 seconds.

The method for forming a liquid film on the substrate10, the film being formed by spreading of the composition14, will now be described by means ofFIG. 2.

During the initial step50, the substrate10is placed in the chamber12and the composition14is deposited on the substrate10, the composition14notably including water.

The substrate10, for example in the form of a transparent slide18, is preferably laid out on the maintaining shims33, in order to be positioned away from the bottom31of the receptacle of the chamber12.

The volatile liquid16is then introduced into the chamber12, during step55. The volatile liquid16is for example positioned against the bottom31.

Upon introducing the volatile liquid16, the latter is positioned away from the composition14. During this introduction step, the volatile liquid16and the composition14are then not in contact with each other.

During the next step60, the chamber12is hermetically closed for a predetermined period, or predetermined duration, the volatile liquid16then evaporating in the chamber12during this closing step. In other words, the substrate10during this closing step is placed under an atmosphere of the volatile liquid16, for example under an ethanol atmosphere.

The predetermined period has a value comprised between 1 minute and 30 minutes, preferably comprised between 5 minutes and 20 minutes, still preferably equal to 10 minutes plus or minus 1 minute.

When the chamber12has the shape of the box26including the lid28and the receptacle30, the lid28during this closing step60is moved from its open position to its closed position, as illustrated by the arrow F inFIG. 1, and the lid28is then maintained in a closed position for the predetermined period.

Finally, during step65, after expiry of said predetermined period, the vapor formed by the evaporation of the liquid16is at least partly extracted out of the chamber12. This at least partial extraction of the vapor formed earlier then produces spreading of the composition14on the substrate10, this spread composition14forming the liquid film on the substrate10.

When the chamber12has the shape of the box26including the lid28and the receptacle30, the lid28during the extraction step65is moved from its closed position to its open position, as illustrated by the arrow O inFIG. 1, and the lid28is then maintained in the open position.

As the vapor of the liquid16is confined inside the chamber12during the closing step60, the opening of the box26, by displacement of the lid28from its closed position to its open position (arrow O), causes at least partial extraction of this vapor out of the chamber12.

InFIG. 3, the curve68illustrates the time-dependent change of the radius of the composition14in a plane perpendicular to the longitudinal direction X, from the moment when the vapor formed by evaporation of the liquid is extracted from the chamber, i.e. from the beginning of step65.

The curve68was obtained with a glass slide18which was made hydrophilic as described earlier, for a composition14having a volume of about 10 μl, and with the introduction of about 1 ml of ethanol into the chamber12and a predetermined period of10minutes for the closing step60. The composition14was prepared with the following proportions: for a total volume total of 1 ml of the composition14, 45 μl of 0.2% TWEEN20 surfactant and 600 μl of PEG 600 with a mass concentration equal to 0.5%.

The curve68then shows a very rapid change in the radius of the composition14versus time, i.e. a very rapid spreading of the composition14over the substrate10from the moment when the vapor formed by the evaporation of the liquid is extracted from the chamber. In the example ofFIG. 3, the composition14initially has a radius of about 4 mm, and the value of the radius is doubled in a little more than 4 seconds from the beginning of step65, the value of the radius being trebled at about 18 seconds after the beginning of step65.

Thus, when the predetermined period of step60is equal to 10 minutes, the value of the radius of the composition14is trebled in a little more than 10 minutes with the formation method according to the invention, while with the formation method of the state of the art, trebling of the value of the radius is only obtained at best after about 1,500 seconds, i.e. 25 minutes.

The method according to the invention therefore allows a significant improvement in the spreading of the composition14over the substrate10.

After forming the liquid film on the substrate10in accordance with the method according to the invention, the liquid film is illuminated by the light source20so that the particles24are detected by means of an optical detection system22, visible inFIG. 4.

The light source20is able to emit a light beam70along the longitudinal direction X, in order to illuminate the composition14positioned on the transparent slide18.

The light source20is positioned at a first distance D1from the transparent slide18along the longitudinal direction X. The first distance D1preferably has a value comprised between 1 cm and 30 cm, for example equal to 10 cm.

In the exemplary embodiment ofFIG. 4, the light source20is a point-like source. The spatial coherence of the light source20is additionally improved, for example by coupling it with a pinhole, with a diameter comprised between 50 μm and 500 μm, placed in contact with the source20.

The light source20is for example a light emitting diode, also called a LED, which is monochromatic and has sufficiently reduced dimensions so as to be considered as spatially coherent, the diameter of the light emitting diode being less than one tenth of the first distance D1separating this light emitting diode from the slide18. The light emitting diode of the light source20for example has an emission wavelength equal to 555 nm and a power equal to 1.7 W.

Alternatively, the light source20is a spatially or temporally coherent source, such as a laser diode (DL) or further a laser diode of the VCSEL (Vertical Cavity Surface Emitting Laser) type.

The optical detection system22comprises the light source20, an image acquisition device72, the transparent slide18and the composition14spread over the slide18, the composition14including the particles to be detected24.

The detection system22is intended for detecting the particles24, during evaporation of the composition14, this evaporation being de-correlated from the spreading, described earlier, of the composition14over the transparent slide18.

The particles to be detected24for example are biological particles, i.e. cells (for example red corpuscles, white corpuscles or platelets), cell components (for example mitochondria), bacteria, viruses or any other molecule or molecule aggregates, notably protein aggregates.

Alternatively, the particles to be detected24are microbeads.

The particles to be detected24have a diameter preferably less than 1 μm, the diameter of the particles24being for example comprised between 50 nm and 1 μm, still preferably comprised between 10 nm and 1 μm.

The light beam70is able to directly illuminate the composition14positioned on the transparent slide18.

The image acquisition device72comprises a photodetector array74including a plurality of pixels, not shown. Each pixel of the photodetector74has dimensions of less than or equal to 10 μm, or even 4 μm. Each pixel for example has the shape of a square, the side of which has a value of less than or equal to 10 μm, or even 4 μm. Alternatively, each pixel has the shape of a square with a side of 2.2 μm.

The acquisition device72is positioned at a second distance D2from the transparent slide18along the longitudinal direction X. The second distance D2has a value of less than 1 cm, and preferably comprised between 100 μm and 2 mm.

In the exemplary embodiment ofFIG. 4, the second distance D2is equal to 500 μm. By giving preference to a short distance between the acquisition device72and the transparent slide18, it is possible to limit phenomena of interferences between different diffraction patterns when the composition14is illuminated.

The image acquisition device72is able to acquire images of the radiation transmitted by the slide18over which is laid out the composition14illuminated by the light beam70. By transmitted radiation, is meant the radiation passing through the composition14and the slide18, so that the acquisition device72and the light source20are located on either side of the transparent slide18and of the composition14.

The photodetector array74is a two-dimensional image sensor, i.e. in a plane perpendicular to the longitudinal axis X. The photodetector array74is a pixelized image sensor, and for example a CMOS (Complementary Metal Oxide Semi-conductor) sensor.

The images acquired by the photodetector array74are formed by the radiation directly transmitted by the illuminated composition14, in the absence of any magnification optics positioned between the transparent slide18and the photodetector array74. However, this does not exclude the presence of microlenses, each being coupled with a corresponding pixel of the sensor, these microlenses allow better collection of the signal. The photodetector array74is also called a lens-less imaging device, and is able to form an image of the composition14, while being placed at a small distance from the latter. By small distance is meant a distance of less than 1 cm, the second distance D2for example being of the order of 500 μm.

For optical detection of the particles24, successive images of the composition14spread as a film and containing the particles24are then acquired by means of the optical detection system22. Upon evaporation of the film, the formation of a skin covering the particles24is observed, the skin having a very small thickness, such as a thickness with a value comprised between 10 nm and 5 μm.

When the composition14is illuminated by the light source20under these conditions, the skin then plays the role of one or several microlenses formed above the particles24, which allows improvement in the detection of these particles.

The addition of the hydrophilic polymer in the composition14allows extension of the period during which the skin remains in contact with the particles24upon evaporation of the composition14. The hydrophilic polymer gives the possibility of extending the period of occurrence of the skin, while retaining proper spreading of the composition14over the slide18. The contact angle a obtained between the slide18and the composition14actually has a value of less than 20°, preferably less than 1° and 10°, still preferably of the order of a degree.

The hydrophilic polymer also causes a reduction in the thickness of the obtained skin upon evaporation of the composition14, which allows detection of particles24with a more reduced size, while retaining a good signal-to-noise ratio. Indeed, if the thickness of the skin is too large relatively to the particles24which one wishes to detect, then the signal-to-noise ratio decreases.

When the surfactant has a concentration at least equal to the critical micellar concentration, the composition14is even better spread, while having a sufficient evaporation rate for allowing the formation of the skin.

The detection system22then allows detection of the particles24having a diameter of very small value, such as for example particles having a diameter of 200 nm.