WO2006005881A1

WO2006005881A1 - Method and device for analysing small volumes of liquid

Info

Publication number: WO2006005881A1
Application number: PCT/FR2005/050516
Authority: WO
Inventors: Jérôme BOUTET; François PERRAUT; Yves Fouillet; Philippe Rizo
Original assignee: Commissariat A L'energie Atomique
Priority date: 2004-07-01
Filing date: 2005-06-29
Publication date: 2006-01-19
Also published as: FR2872575A1

Abstract

The invention relates to a method for the optical analysis of a drop (2) of a liquid medium. The inventive method comprises the following steps consisting in: bringing a drop of liquid into contact with a hydrophobic surface (8); and deforming the drop on said surface by means of electrowetting, in order to position and deform same on the path of an optical beam (50).

Description

METHOD AND DEVICE FOR ANALYZING SMALL VOLUMES OF

LIQUID

DESCRIPTION

TECHNICAL FIELD AND PRIOR ART

The invention relates to the field of optical detection, in particular in very small volumes of liquid such as drops.

Fluorescence detection in a liquid medium is generally done using one or more lasers passing through the liquid contained in a bowl. Detection is often done by photomultiplier.

Another possibility is to use a fluorescence microscope (epi-illumination) to illuminate one or more cuvettes containing one or more liquids. The reading is then done using a camera, then by average gray levels of the image on regions of interest corresponding to the interior of the cuvettes. The separation between the excitation and emission wavelengths is carried out using a dichroic filter.

Detection can also be done in transmission. In this case, the excitation and detection chains are diametrically opposed. Such a device has the advantage of not requiring a dichroic filter, but has the disadvantage of making the filtering of the emission more difficult.

It is also possible to find bent systems for which the excitation and emission axes form an angle equal to or close to 90 °. The Light sources can be lasers, LEDs, fluorescent discharge, arc lamps. The detectors may be photomultipliers, CCD or CMOS cameras, photodiodes. This kind of system is not easy to implement for very small volumes of liquid. It is indeed necessary to have a bowl of a volume as small as the volume of liquid to be measured.

In addition, there is a problem of optimization of the signal to be measured.

A very small volume of liquid, of the order of a few microliters, or even sometimes of the order of a nanoliter, implies signals which are also very weak. Traditional methods are therefore very difficult to implement.

The document by J-C Roulet et al. entitled

"Performance of an Integrated Microoptical System for

Fluorescence Detection in Microfluidic System ", Analytical Chemistry, Vol.74, No.14, p.3400-3407, 2002 describes a chip for fluorescence measurements.

However, there is the problem of finding a less complex method of implementation.

Preferably, such a method and such a device must make it possible to carry out various types of measurements, such as measurements of fluorescence or spectrometry (absorption in particular) or of colorimetry. I / INVENTION STATEMENT

The invention firstly relates to a method for optical analysis of a drop of a liquid medium comprising: - bringing a drop of liquid into contact with a hydrophobic surface,

the deformation of the drop on this surface by electrowetting, in order to deform it on the path of an optical beam. According to the invention, an electrowetting method is used to deform or stretch or lengthen, or possibly position or move, a drop of liquid in one direction, so as to increase the optical path of the light in this drop. For example, in the context of a lab on a chip, when the drop, possibly after a displacement, is on a detection zone, one or more electrodes, under the hydrophobic layer, can be activated to give the drop an optimal geometry for detection.

The shape of the drop may be given by a matrix or by a row of electrodes of identical sizes.

In this case, it is the activation of some electrodes and the deactivation of others that gives the drop the shape that optimizes the conditions for detection.

According to one variant, its shape can be given to the drop by a single activated electrode whose shape is optimal for detection. In the case of a collimated beam excitation (laser or lentil fiber for example), the optimal shape of the drop could be as close as possible to that of a cylinder. The elongation or deformation of the drop can be applied to any form of optical detection, colorimetry, or spectrometry, or fluorescence.

The latter can be observed by any means, such as a photomultiplier or a photodiode or a camera or a microscope.

The drop may be confined, at least during its deformation, between said hydrophobic surface and an upper substrate. The drop may, before deformation, not be confined by the upper substrate. It is then a mixed system, in which the drop is initially unconfined and then confined.

It can also be before deformation, already confined by the upper substrate.

The invention also relates to a device for optical analysis of a drop of a liquid medium comprising:

a first substrate comprising a hydrophobic surface,

means for generating a radiation for generating a light beam parallel to the hydrophobic surface,

means for deforming a drop, on this surface, by electrowetting, in order to deform in the path of a light beam generated by the radiation generating means.

Such a device may further comprise a second substrate disposed facing the hydrophobic surface.

Such a second substrate allows a confinement of the drop.

This second substrate may itself further comprise a superficial hydrophobic layer, and / or an electrode.

The means for deforming a drop on the hydrophobic surface of the first substrate may comprise a plurality of electrodes under the hydrophobic surface. These electrodes may all be of identical size, or at least one of the electrodes may be of elongate shape.

A device according to the invention may further comprise means for reflecting the light beam after passing through a drop of liquid positioned on the hydrophobic surface, which further increases the path of the beam in the drop.

The drop may also be moved on the hydrophobic surface, the device may comprise means for moving the drop, it is for example the means for deforming the drop that can be used to move it. BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1C represent an embodiment of a device according to the invention, in an open system; FIG. 2 represents a deformation of a drop, in the path of an optical beam, in an open system;

FIGS. 3A and 3B show top views of a device and a drop, before and during a measurement according to the invention,

FIG. 4 represents a device implemented in the context of the invention, in a closed system,

FIGS. 5A and 5B show an implementation of the invention, in an open and closed mixed system,

- Figure 6 shows a device implemented in the context of the invention in closed system with conductive cover. FIGS. 7A and 7B show a device implemented in the context of the invention, in a mixed system with a conductive cover.

DETAILED PRESENTATION OF PARTICULAR EMBODIMENTS

A first embodiment of the invention, in open system, is illustrated in Figures IA - IC.

This embodiment implements a device for moving or handling drops of liquid based on the principle of 1 electrowetting on a dielectric. Examples of such devices are described in the article by MG Pollack, AD Shendorov,

R.B. Fair, entitled "Electro-wetting-based actuation of droplets for integrated microfluidics", Lab Chip 2 (1) (2002) 96-101.

The forces used for the displacement of liquid drops are then electrostatic forces.

The document FR 2 841 063 describes a device implementing, in addition, a catenary facing the electrodes activated for displacement.

The principle of this type of displacement is synthesized in Figures IA - IC.

A drop 2 rests on a network 4 of electrodes, from which it is isolated by a dielectric layer 6 and a hydrophobic layer 8 (Figure IA).

The hydrophobic nature of this layer means that the drop has a contact angle, on this layer, greater than 90 °. The electrodes 4 are themselves formed on the surface of a substrate 1.

When the electrode 4-1 located near the drop 2 is activated, using switching means 14, the closure of which makes contact between this electrode and a voltage source 13 via a common conductor 16, the layer dielectric 6 and the hydrophobic layer 8 between this activated electrode and the drop under voltage act as a capacitance.

The counter-electrode 10 allows a possible displacement by electrowetting on the surface of the hydrophobic surface; she maintains a contact electric with the drop during such a displacement. This counter electrode can be either a catenary as in FR - 2 841 063, or a buried wire or a planar electrode in the hood of a confined system (such a confined system is described below).

In open system, if there is no displacement, it is possible to spread the drop on the hydrophobic surface, without counter-electrode. This is for example the case if the drop can be brought to the hydrophobic surface by a conventional dispensing system, the electrodes 4-1, 4-2 serving only to spread or deform the drop where it has been deposited. .

The drop may thus be optionally displaced step by step (FIG. 1C) on the hydrophobic surface 8 by successive activation of the electrodes 4-1, 4-2, etc., along the catenary 10.

It is therefore possible to move liquids, but also to mix them (by bringing drops of different liquids near), and to perform complex protocols.

The documents cited above give examples of implementations of adjacent electrode series for the manipulation of a drop in a plane, the electrodes can indeed be arranged in a linear manner, but also in two dimensions, thus defining a plan of displacement of the drops.

As illustrated in FIG. 2, the simultaneous activation of several electrodes, indicated in this figure by the references 4-1, 4-2, 4-3, will allow, that there has been a displacement of the drop or not, to lengthen the drop 2 in the desired direction, this direction being in fact determined by the electrodes 4-1, 4-2, 4-3 selected. FIGS. 3A and 3B show a view from above of the system, in which the electrodes 4 are schematized by squares, the drop always being designated by the reference 2.

Before activation of the electrodes, it rests on one of them; after activation of the electrodes 4-1, 4-2, 4-3, 4-4, the drop is stretched and positioned above these same four electrodes. Figure 2 shows the case where only three electrodes are selected, thus stretching the drop on these 3 electrodes.

The drop is then in a state in which its length L (see FIG. 2), measured parallel to the hydrophobic surface 8, is greater than its height h, for example in a ratio L / h of the order of 2 to 4 or more.

A light beam 50 of wavelength adapted to the desired measurement (absorption or fluorescence or colorimetry or spectrometry) thus sees its path in the drop significantly increased due to the elongation thereof.

The measurement signal is amplified. This is particularly advantageous when the volume of liquid examined is of the order, at most, of a few microliters, and can even reach very low values, for example of the order of one nanolitre. Conveniently, light emitting means 52 will be positioned to direct a beam 50 along the substrate or parallel to the substrate. These means may be for example a microlaser or a diode.

A detector will detect the resulting optical signal.

The activation of the electrodes 4 will allow, possibly first, possibly, to move or position, then to extend and spread or deform the drop 2 along the beam 50, as explained above.

FIG. 4 represents another embodiment of the invention, in a closed or confined system.

In this figure, reference numerals identical to those of FIGS. 1A-2 designate the same elements.

This device further comprises an upper substrate 100, preferably also covered with a hydrophobic layer 108. This set may be optionally transparent, allowing observation from above.

Again, a light beam can be directed between the two substrates, parallel to them.

Conveniently, light emitting means similar to the means 52 of FIG. 2 will be positioned to direct a beam 50 along the substrate. The selection of electrodes 4 of the lower substrate first makes it possible to move or position, then to lengthen and spread or deform the drop 2 along the beam 50. FIGS. 5A and 5B, on which numerals identical to those of FIG. 4 denote identical or similar elements, represent a mixed system, in which a drop 2 is initially in an open medium (FIG. 5A), the activation of electrodes 4-1, 4-2, 4 3 allowing an alignment and a flattening of the drop (FIG. 5B), in a closed system, in an area where the system is provided with a hood, as illustrated above with reference to FIG. 4. FIG. variant of the closed system, with a conductive cover 100, comprising an electrode or an array of electrodes 112, as well as an insulating layer 106 and a hydrophobic layer 108. The catenary 10 of the preceding figures is replaced, in this embodiment, by the electrode 112. The activation of this electrode 112 and the electrodes 4 makes it possible to move the droplet into the desired position and then to stretch it or to deform it, to bring it into the path of a light beam 50.

FIGS. 7A and 7B, on which reference numerals identical to those of FIG. 6 denote identical or similar elements, represent a mixed system, in which a drop 2 is initially in an open medium (FIG. 7A), activation of FIG. electrodes 4-1, 4-2, 4-3 allowing a alignment and flattening of the drop (FIG. 7B), in a closed system, in an area where the system is provided with a hood, as illustrated above in connection with FIG. 6. In the embodiments described above several electrodes 4 are activated to deform the drop. It is also possible to provide an elongated electrode whose activation would flatten the drop, again by electrowetting effect.

A device according to the invention may further comprise means which will make it possible to control or activate the electrodes 4, for example a PC-type computer and a relay system connected to the device or the chip, such as the relays 14 of the FIG. 1A, these relays being controlled by the PC type means.

Typically, the distance between a possible conductor 10 (Figures IA - 5B) on the one hand and the hydrophobic surface 8 on the other hand is for example between 1 micron and 100 microns or 500 microns.

This conductor 10 may be for example in the form of a wire diameter between 10 microns and a few hundred microns, for example 200 microns. This wire may be a gold or aluminum wire or tungsten or other conductive materials.

When two substrates 1, 100 are used (FIGS. 6 to 7B), they are separated by a distance of between, for example, 10 μm and 100 μm or 500 μm. Whatever the embodiment considered, a drop of liquid 2 will have a volume between, for example, 1 nanolitre and a few microliters, for example between 1 ni and 5 μl or 10 μl.

In addition, each of the electrodes 4 will for example have a surface of the order of a few tens of μm ² (for example 10 μm ² ) up to 1 mm ² , depending on the size of the drops to be transported, the spacing between adjacent electrodes being for example between 1 .mu.m and 10 .mu.m.

The structuring of the electrodes 4 can be obtained by conventional methods of micro ^¬ technologies, for example by photolithography.

Methods for producing chips incorporating a device according to the invention can be directly derived from the processes described in document FR-2 841 063.

Conductors, and in particular conductors 110 may be made by depositing a conductive layer and etching of this layer in the appropriate pattern of conductors, before deposition of the hydrophobic layer 108.

The electrodes may be made by deposition of a metal layer (for example a metal selected from Au, Al, ITO, Pt, Cr, Cu) by photolithography. The substrate is then covered with a dielectric layer of Si3N4 or SiO2. Finally a deposit of a hydrophobic layer is performed, such as a teflon deposit made by spinning.

A system according to the invention has many advantages: • this system increases the optical path of the excitation light 50. It can be further improved adding a reflective device, for example a micro-mirror, to the end of the optical path, this device returning the beam into the drop; a separating cube may allow, in some cases, and especially absorption spectroscopy, to separate the reflected beam;

• the excitation light is also better preserved in the drop. When the drop is stretched, the rays strike the interface at the periphery of the drop at a lower angle of incidence than in the case of a spherical drop (this is very clear in FIG. 2). The rays reflected inside the drop are of stronger intensity. There is therefore an optical guidance of the excitation light in the drop.

• the rays resulting from the excitement transmitted

(those who come out of gout) are weaker, since more light is absorbed into the drop. Optical filtering is thus made easier.

• In the case of a deformed drop shaped cylinder or half-cylinder (which is particularly the case for an open or uncontained system), the excitation light is distributed more evenly. This makes the detection less noisy by possible local variations in marker concentration in the liquid of the drop.

• The excitation light thus guided does not come into contact with the support or the cover, thus preventing any parasitic fluorescence. She does not Also comes not in contact with the detector (all the light can be directed through the drop), thus simplifying the filtering.

The possibility, in a confined system, of forming square or rectangle-shaped droplets makes it possible to improve the optical path by producing almost a liquid tank.

Claims

1. A method for optical analysis of a drop (2) of a liquid medium comprising: - the positioning of light emitting means, so as to direct an optical beam along the substrate,

contacting a drop of liquid with a hydrophobic surface (8), deformation of the drop on this surface by electrowetting, in order to deform it in the path of said optical beam (50).

2. Method according to claim 1, the drop being confined, at least during its deformation, between said hydrophobic surface (8) and an upper substrate (100).

3. Method according to claim 2, the drop being, before deformation, not confined by the upper substrate.

4. Method according to claim 2, the drop being, before deformation, confined by the upper substrate.

5. Method according to one of claims 1 to 4, further comprising a displacement of the drop on the hydrophobic surface.

6. Method according to one of claims 1 to 5, the displacement and / or the deformation being obtained by activation of a plurality of electrodes (4), located under the hydrophobic layer (8).

7. Method according to one of claims 1 to 5, the displacement and / or the deformation being obtained by activation of a single electrode, located under the hydrophobic layer.

8. Method according to one of claims 1 to 7, the optical analysis being a colorimetric analysis, or by spectrometry or fluorescence.

9. Method according to one of the claims

1 to 8, the optical beam being reflected after passing through the drop and the reflected beam back through the drop after reflection.

10. Method according to one of the claims

1 to 9, comprising the implementation of an electrowetting device, comprising a first substrate covered with said hydrophobic layer (8), and a plurality of electrodes (4) disposed under this hydrophobic layer.

11. Apparatus for optical analysis of a droplet (2) of a liquid medium comprising:

a first substrate comprising a hydrophobic surface (8), means (52) for generating a radiation for generating an optical beam parallel to the hydrophobic surface,

- Means for deforming a drop, on this surface, by electrowetting, in order to deform it in the path of an optical beam (50) generated by the means (52) for generating radiation.

12. Device according to claim 11, further comprising a second substrate (100) arranged opposite the hydrophobic surface.

13. The device of claim 12, the second substrate further comprising a hydrophobic surface layer (108).

14. Device according to claim 12 or 13, the second substrate further comprising an electrode (112).

15. Device according to one of claims 11 to 14, the means for deforming a drop, on this surface, by electrowetting, comprising a plurality of electrodes (4) under the hydrophobic surface.

16. Device according to claim 15 the electrodes under the hydrophobic surface being all of identical size.

17. Device according to claim 15 at least one of the electrodes under the hydrophobic surface being of elongate shape.

18. Device according to one of claims 15 to 17, further comprising means for controlling said plurality of electrodes so as to deform or stretch or lengthen a drop and to increase the optical path of the light in this drop.

19. Device according to one of claims 11 to 18, further comprising means for reflecting said optical beam after passing through a drop of liquid positioned on the hydrophobic surface.