EP1222031B1 - Method and device for moving and placing liquid drops in a controlled manner - Google Patents

Abstract

The invention relates to a method and a device which serves for moving and dosing amounts of liquid on a microscopic scale with a volume of especially 10<SUP>-12 </SUP>to 10<SUP>-6 </SUP>liters by means of an inhomogeneous electric field using a support having an ultraphobic surface.

Description

The present invention relates to a method and apparatus for moving and metering liquid quantities on a microscopic scale with a volume of in particular 10 ^-12 to 10 ^-6 liters with an electric field using a carrier with an ultraphobic surface optionally in combination with an ultraphobic dosing tip ,

The manipulation and in particular the metering of minute liquid drops, which have a volume in the order of 10 ^-12 - 10 ^-6 liters or a diameter in the order of about 0.01 - 1 mm, is still a problem today, because in this process, also referred to as micro-dosing, even the smallest losses of liquid already lead to considerable deviations from the desired dosing quantity. Such liquid losses occur, for example, when the liquid drop is displaced along a conventional surface, because even with very smooth surfaces, part of the liquid drop adheres to the surface.

It is therefore an object to provide a method for moving and dosing of liquid droplets with a volume of, in particular smaller than 10 ^-6 liters without significant loss of liquid available.

The object is achieved by providing a method for Microdosing liquid droplets solved in which the liquid droplets with an inhomogeneous electric field on a support with an ultraphobic surface be moved lossless.

The invention relates to a substrate according to claim 1 and a method according to claim 10 and a method according to Claim 16 and uses of the substrate according to claims 14 and 15.

Preferably, the manipulator is an electrically charged tip or a wire, in particular a tip or a wire with an ultraphobic surface used.

In a preferred embodiment, to generate the electric field between the manipulator and the carrier a voltage of 100 to 1000 volts, preferably from 400 to 600 volts applied. The tension can vary greatly depending on Geometry of the arrangement.

A liquid drop according to the invention consists of any liquid and preferably has a volume of 10 ^-12 to 10 ^-6 liters, more preferably from 10 ^-9 to 10 ^-6 liters. According to the invention, such a drop is moved without loss with a displaceable electric field on an ultraphobic surface.

Further preferred is a liquid drop by means of the electric field divided off a liquid reservoir. Several drops of liquid can by means of of the electric field united on an ultraphobic surface and to be mixed. All of these process steps can also be in any Combined with each other.

The closest prior art is described by the document US5674592, this is a substrate with the properties of a partially extremely hydrophobic surface describes their wetting properties through a coating and a nanostructure can be modified controlled. Drops can be made on this surface with the help of a electrostatically charged object, which brought without contact in the vicinity of the drops will be moved, directed and controlled. However, this document does not describe any in the substrate embedded electrodes, to the controlled movement or Association of drops varies according to the destination and direction of movement Voltages can be applied.

In a preferred embodiment, the electric field is between a Tip, which preferably has a diameter of 0.01 to 1 mm, any Length has an ultraphobic surface, and one preferably metallic carrier. With this tip drops of liquid on the ultraphoben Surface moved. This makes the tip an ultraphobic surface have no liquid components adhere to the tip.

As the liquid drops both at the top and on the ultraphobic surface assume almost the shape of a sphere, their volumes can be very simple from the, e.g. calculated under a microscope diameter.

In a further preferred embodiment, the liquid reservoir of the Device on an arrangement for electrostatic charging.

Ultraphobic surfaces according to the invention are characterized in that the Contact angle of a water drop lying on the surface, more than 150 ° is and the roll angle does not exceed 10 °.

As a rolling angle here is the inclination angle of a basically planar but textured surface against the horizontal understood, where a standing Drops of water of volume 10μl due to gravity is moved when the Surface is tilted.

Such ultraphobic surfaces are e.g. in the publications WO 98/23549, WO 96/04123, WO 96/21523 and WO 96/34697, which are hereby incorporated by reference Reference, and thus are considered part of the disclosure.

In a preferred embodiment, the ultraphobic surface has a surface topography in which the spatial frequency f of the individual Fourier components and their amplitudes a (f) expressed by the integral of the function S (log f) = a (f) · f calculated between the integration limits log (f ₁ / μm ^-1 ) = -3 and log (f ₁ / μm ^-1 ) = 3, is at least 0.5 and consists of a hydrophobic or especially oleophobic material or a durable hydrophobicized or especially durable oleophobated material. Such an ultraphobic surface is described in international patent application WO 99/10322.

A hydrophobic material in the context of the invention is a material that is on a even, non-textured surface a contact angle, relative to water, of greater than 90 °.

An oleophobic material in the context of the invention is a material which is based on a even, non-textured surface a contact angle, based on long-chain n-alkanes, such as n-decane, of greater than 90 °.

Preferably, the ultraphobic surface is an aluminum surface that has microstructures provided, anodized, optionally gesealt, calcined, optionally with coated with a primer layer and then with a hydrophobic and / or oleophobic coating is provided, as it is in the international Patent application WO 99/10323 is described.

The manipulator and / or the carrier can be made entirely of aluminum or preferably has an aluminum coating, wherein the aluminum, such as treated above.

Also preferably, the ultraphobic surface is an aluminum surface that optionally anodically oxidized, with hot water or steam gesealt, optionally coated with a primer layer and then with a hydrophobic and / or oleophobic coating is provided, as in the international patent application WO 99/10324. The dosing tip can be made entirely of aluminum or preferably has one Aluminum coating, wherein the aluminum treated as stated above becomes.

Furthermore, the ultraphobic surface is preferably a surface which is coated with Ni (OH) ₂ particles, optionally coated with an adhesion promoter and subsequently provided with a hydrophobic and / or oleophobic coating, as described in international patent application WO 99/10111 is. Preferably, the Ni (OH) ₂ particles have a diameter d ₅₀ of 0.5 to 20 microns.

In a further advantageous embodiment, the ultraphobic surface is made Tungsten carbide structured with a laser, optionally with a coupling agent coated and then with a hydrophobic and / or oleophobic Cover is provided, as in the international patent application WO 99/10113 is described. Preferably, the metering tip is only with Tungsten carbide, which is then treated as indicated above. Preferably The tungsten carbide has a layer thickness of 10 to 500 microns.

In addition, preferably, the surface is sandblasted with a blasting agent, optionally coated with a primer layer and then with a provided hydrophobic and / or oleophobic coating, as in the international Patent application WO 99/10112 is described.

Suitable as a hydrophobic and / or oleophobic coating of said surfaces all interface-active repellents with any molecular weights. at these compounds are cationic, anionic, amphoteric and / or non-ionic surface-active compounds, such as e.g. in the register "Surfactants Europe, A Dictionary of Surface Active Agents available in Europe, Edited by Gordon L. Hollis, Royal Socity of Chemistry, Cambridge, 1995 become.

Examples of anionic repellents which may be mentioned are: alkyl sulfates, Ether sulfates, ether carboxylates, phosphate esters, sulfosuccinates, sulfosuccinamides, Paraffin sulfonates, olefin sulfonates, sarcosinates, isothionates, taurates and lingnins Links.

As cationic repellent auxiliary agents are, for example, quaternary alkylammonium compounds and to call imidazoles.

Amphoteric repellents are, for example, betaines, glycinates, propionates and imidazoles.

Nonionic repellents are, for example: alkoxylates, alkylamides, Esters, amine oxides and alkyl polyglycosides. Also suitable: reaction products of alkylene oxides with alkylatable compounds, such as. B. fatty alcohols, Fatty amines, fatty acids, phenols, alkylphenols, arylalkylphenols, such as Styrene-phenol condensates, carboxylic acid amides and resin acids.

Particularly preferred are repellents in which 1 to 100%, especially preferably 60 to 95% of the hydrogen atoms are substituted by fluorine atoms. exemplary be perfluorinated alkyl sulfate, perfluorinated alkyl sulfonates, perfluorinated Alkyl phosphonates, perfluorinated alkyl phosphinates and perfluorinated carboxylic acids called.

Preference is given to using compounds having a molecular weight M _w > 500 to 1,000,000, preferably 1,000 to 500,000 and particularly preferably 1,500 to 20,000, as the polymeric repellent auxiliary agents for the hydrophobic coating or as the polymeric hydrophobic material for the surface. These polymeric repellents may be nonionic, anionic, cationic or amphoteric compounds. Furthermore, these polymeric repellents may be homo- and copolymers, graft and graft copolymers and random block polymers.

Particularly preferred polymeric repellents are those of the type AB-, BAB and ABC block polymers. In the AB or BAB block polymers is the A segment a hydrophilic homopolymer or copolymer, and the B block is a hydrophobic one Homopolymer or copolymer or a salt thereof.

Also particularly preferred are anionic, polymeric repellents, in particular Condensation products of aromatic sulfonic acids with formaldehyde and alkylnaphthalenesulfonic acids or from formaldehyde, naphthalenesulfonic acids and / or benzenesulfonic acids, condensation products of optionally substituted Phenol with formaldehyde and sodium bisulfite.

Also preferred are condensation products obtained by reaction of Naphthols with alkanols, additions of alkylene oxide and at least partial Conversion of the terminal hydroxy groups in sulfo groups or half esters of Maleic acid and phthalic acid or succinic acid are available.

In another preferred embodiment, the repellent auxiliary is selected from the group of sulfosuccinic acid esters and alkylbenzenesulfonates. Also preferred are sulfated, alkoxylated fatty acids or salts thereof. As alkoxylated fatty acid alcohols are in particular those having from 5 to 120, with 6 to 60, most preferably provided with 7 to 30 ethylene oxide C ₆ -C ₂₂ -Fettsäurealkohole, which are saturated or unsaturated, in particular stearyl, understood. The sulfated alkoxylated fatty acid alcohols are preferably present as salt, in particular as alkali or amine salts, preferably as diethylamine salt.

Die Polymerasekettenreaktion PCR (polymerase chain reaction), ELISA (enzyme linked immunosorbent assay) oder die Bestimmung von Enzymaktivitäten.

Preferred fields of application for the method according to the invention and the device according to the invention are biochemical or chemical methods in which microscopic liquid volumes must be moved, mixed or metered. As examples may be mentioned here:

The polymerase chain reaction (PCR), enzyme linked immunosorbent assay (ELISA) or the determination of enzyme activities.

The inventive method is easier to perform than the conventional Microdosing with the help of pressing. Due to the minimal adhesion of the liquid drops on the ultraphobic surfaces, the manupulation is of the smallest Liquid quantities without losses possible. As a result, dosing errors can be avoided become.

Another object of the invention is the use of the device according to the invention for metering liquids on a microscopic scale, in particular in the range of 10 ^-6 to 10 ^-12 liters.

The device according to the invention will now be described with reference to FIG. 1 and Example 1 exemplified in more detail.

Fig. 1

zeigt eine Kunststoffplatte 2 zum Verschieben von Flüssigkeitstropfen 4,5 mit einer Vielzahl von Elektroden 3

Fig. 2

zeigt eine Aluminiumplatte 7 mit einer elektrisch geladenen Spitze 10 als Manipulator

Fig. 3

zeigt eine runde Spitze 12 mit Ringelektrode 13 zur Entnahme kleiner Flüssigkeitsvolumina 15 aus einem Vorrat 14
(Querschnittszeichnung).

Fig. 4

zeigt eine Anordnung von drei Spitzen 16 zur Bildung eines nahezu dreieckförmigen Spaltes M, der anstelle der Ringelektrode 13 in Fig. 3 zur Entnahme kleiner Flüssigkeitsmengen aus einem Vorrat verwendet werden kann.

Figures 2-4 and Examples 2-5 relate to similar devices and methods, which devices and methods are not the subject of this invention.

Fig. 1

shows a plastic plate 2 for moving liquid droplets 4, 5 with a multiplicity of electrodes 3

Fig. 2

shows an aluminum plate 7 with an electrically charged tip 10 as a manipulator

Fig. 3

shows a round tip 12 with ring electrode 13 for removing small volumes of liquid 15 from a supply 14th
(Cross-section drawing).

Fig. 4

shows an arrangement of three tips 16 to form a nearly triangular gap M, which can be used instead of the ring electrode 13 in Fig. 3 for taking small amounts of liquid from a supply.

Examples example 1

FIG. 1 shows a device 1 according to the invention for residue-free movement of liquid drops (here aqueous solutions) on solid surfaces.

The device consists of a substrate 2 (here Plexiglas), on its surface round electrically conductive electrodes 3 (diameter 1 mm, spacing 5 mm) introduced are flush with the surface of the substrate. To the individual Electrodes 3 different voltages can be applied against each other.

The surface of the substrate 2 is coated with an approximately 5 microns thick electrically insulating provided with ultraphobic coating. This is on the substrate about 5 microns thick Layer of aluminum evaporated. The Al layer is anodized, with treated with hot steam and provided with a hydrophobic coating. to Preparation of the hydrophobic coating, the substrate is 5 hours at pH 7 in a 1 wt .-% solution of Fluowet PL80 Clariant immersed, with water rinsed and dried at 60 ° C.

a. Metallisierung:

Auf das Substrat wird eine ca. 5µm dicke Aluminiumschicht thermisch aufgedampft. Die Oberfläche wurde anschließend in destilliertem Chlorform (CHCl₃) 3 min. entfettet.

b. Anodische Oxidation:

Die anodische Oxidation der Aluminiumoberfläche wurde in In Schwefelsäure unter kontinuierlicher Elektrolytbewegung bei laminaren Strömungsbedingungen durchgeführt. Die Elektrolyttemperatur von 20°C wurde durch einen Thermostat geregelt. Der Abstand des Substratmaterials zur Gegenelektrode aus AlMg3, halbhart betrug 5 cm. Die Stromdichte während der anodischen Oxidation wurde konstant auf 10 mA/cm² geregelt. Die Oxidation wurde solange fortgeführt bis eine etwa 2-3 µm dicke Oxidschicht entstanden war.

c. Wasserbehandlung:

Nach der anodischen Oxidation wurde die Probe 5 min. in destilliertem Wasser und anschließend 1 min. in Methanol gespült. Nach dem Trocknen (Luft, Raumtemperatur) wurde die Probe in einem Becherglas, das zuvor mehrfach mit destilliertem Wasser gekocht wurde, in destilliertem Wasser bei 100°C 15min behandelt. Nach dieser Behandlung.wurde in Methanol gespült (1 min) bei 80°C im Trockenschrank 1 Stunde getrocknet.

Preparation of the ultrahydrophobic coating:

a. metallization:

On the substrate, an approximately 5 .mu.m thick aluminum layer is thermally evaporated. The surface was then in distilled chlorine (CHCl ₃ ) 3 min. degreased.

b. Anodic oxidation:

The anodic oxidation of the aluminum surface was carried out in sulfuric acid under continuous electrolyte movement under laminar flow conditions. The electrolyte temperature of 20 ° C was controlled by a thermostat. The distance of the substrate material to the counter electrode made of AlMg3, semi-hard was 5 cm. The current density during the anodic oxidation was constantly controlled to 10 mA / cm ² . The oxidation was continued until an approximately 2-3 μm thick oxide layer was formed.

c. Water Treatment:

After anodic oxidation, the sample was allowed to stand for 5 min. in distilled water and then 1 min. rinsed in methanol. After drying (air, room temperature), the sample was treated in distilled water at 100 ° C for 15 min in a beaker previously boiled several times with distilled water. After this treatment, it was rinsed in methanol (1 min) at 80 ° C in a drying oven for 1 hour.

The Al layer is completely in an aluminum oxide layer by this treatment been converted.

Handling of the device:

First, all electrodes 3 are at the same electrical potential. One Drop 5 may be on the surface in the direction of a directly adjacent electrode be moved by facing this electrode to a potential of 800V the other electrodes is switched. Then the drop is above the relevant one Electrode.

By multiple executed switching of the electrodes 3, the movement can be of the droplet 5 on the surface arbitrarily control within the electrode grid. In this way, also different drops 4, 5 moved to the same place and united with each other.

The movement of the drops 4, 5 takes place without residue on the ultraphobic surface, ie without adhesion of liquid residues along the path of movement. This can be seen as follows: A drop 4 (diameter about 1 mm) of a solution of 4- (6-diethylamino-3-diethylimino-3H-xanthe-9-yl) -1,3-benzodisulfonic acid (Kiton Red, concentration 1 x 10 ^-2 mol / l in water) is located on the ultraphobic surface. The drop 4 is displaced along a closed path over 8 electrodes (length of the path 40 mm). This process is repeated 10 times, so that the total distance is 400 mm. Subsequently, the drop is removed and a drop of pure water along the previously used closed path also moved 10 times.

This water droplet is examined spectrophotometrically. Up to the detection limit of 10 ^-10 mol / l (based on the drop volume) no dye can be detected. The losses due to the displacement of the droplet are thus less than 10 ppb.

The example shown here can also be used for liquid drops used, which are surrounded by solid walls from all sides, e.g. in Columns or tubes. These designs thus allow lossless promotion of liquids solely by the change of electric fields, i. without mechanically moving parts.

Example 2

FIG. 2 shows a device 6 for complete transmission of liquid drops (here aqueous solutions) by means of a mobile Tip 10.

The device has a support plate 7 made of aluminum with an ultraphoben Plating and a top 10 on. The tip also has an ultraphobic surface on. The preparation of the ultraphobic coating is carried out according to Example 1.

Handling of the device:

A drop 8 of a solution of 4- (6-diethylamino-3-diethylimino-2H-xanthe-9-yl) -1,3-benzodisulfonic acid (Kiton Red, concentration 1 × 10 ^-2 mol / l in water) is on the ultraphobic surface. The volume is V = (3.00 ± 0.05) x 10 ^-9 liters. The volume determination was carried out on the basis of the diameter of the spherical drop with a measuring microscope.

With the help of the tip 10, the drop 8 can be recorded. This is approached the tip to a distance of about 5 mm, with between 10 and the top Substrate plate 7, a voltage of 800 V is applied. The radius of the tip is approx. 0.5 mm. The drop hanging on top is placed in a jar containing 65μl of water by switching off the voltage.

The dye concentration in the water was then determined spectrophotometrically at 4.54 × 10 ^-7 mol / l. This corresponds to a volume transmitted by the tip of V = 2.95 nl. The transfer was carried out 5 times in the same way, resulting in no loss of the transferred volume within the relative metering error of 1.5%.

Example 3

Another example shows the metering and complete transfer of liquid drops with the aid of the device in FIG. 2.

A drop 8 of a solution of 4- (6-diethylamino-3-diethylimino-3H-xanthe-9-yl) -1,3-benzodisulfonic acid (Kiton Red, concentration 1 x 10 ^-2 mol / l in water) is on the ultraphobic surface. The volume is V ₃ = (3.00 ± 0.05) x 10 ^-9 liters.

Another drop 9 of a solution of 1,1'-diethyl-4,4'-dicarbocyanine iodide (concentration 1 × 10 ^-2 mol / l in water) is located on the ultraphobic surface. The volume is V ₄ = (3.00 ± 0.05) x 10 ^-9 liters.

With the help of the tip 10 of the drop 8 is recorded as in Example 2. The on the tip hanging drops is in a recess 11 of the device by switching off the voltage is stored. The other drop 9 is taken up with the tip and united with the drop 8 in the well. Then be pick up both drops with the tip and place in a container with 65 μl of water according to Example 2 transferred.

The dye concentrations in the water were then determined spectrophotometrically. The transfer was carried out 5 times in the same way, resulting in no loss of the transferred volumes V ₃ and V ₄ within the relative dosing error of 1.5%.

Example 4

Fig. 3 shows an arrangement for the controlled removal of small known liquid volumes from a supply (cross-sectional drawing). The arrangement consists of an electrode 12 with a round tip (diameter 1 mm) and an annular electrode 13 (inner diameter 0.5 mm). Both electrodes are provided with an ultrahydrophobic coating, the preparation of which is described in Example 1. The assembly is immersed in an aqueous solution of 4- (6-diethylamino-3-diethylimino-3Hxanthe-9-yl) -1,3-benzodisulfonic acid (Kiton Red, concentration 10 ^-2 mol / L in water) (as shown in FIG. 3). When a voltage of 900 V is applied between the ring 13 and the electrode 12, a drop of liquid 15 is removed from the reservoir 14 and remains adhered to the electrode 12. By lateral tilting and dropping the electric field, the drop can be transferred to another vessel. By measuring the fluorescence intensity of the dye in a known volume of water, the volume of the drop 15 was determined. After 30 repetitions of the withdrawal, a volume of (65.0 ± 0.2) × 10 ^-9 liters is obtained.

Example 5

Instead of the annular electrode 13 of the device in FIG. 3, an arrangement as in FIG. 4 may also be used. Here, three round electrodes 16 (diameter 1 mm) are provided with an ultrahydrophobic coating, whose preparation is described in Example 1. The electrodes 16 are arranged as described in Fig. 4 for forming a nearly triangular-shaped spade M, which has the same function of the ring electrode 13 in FIG. With this arrangement, as in Example 15, a drop of liquid is removed from a supply. Reproducing the dose 30 times gives a volume of (50.0 ± 0.3) × 10 ^-12 liters.

Similarly, other structures (in cross-section and plan view, respectively) may be used round, square or arbitrarily shaped column) instead of the ring 13 in Fig. 3rd be used for dosing. Structures that are particularly suitable for this purpose are known microstructure techniques (e.g., light, X-ray, or electron lithographic Techniques) can be generated because small volumes to be dosed accordingly need small structures.

Claims (18)

1. Substrate (2) into which two or more electrodes (3) are introduced, to which, individually, various voltages can be fed against one another, and which is provided with an ultraphobic coating.
2. Substrate according to Claim 1, characterized in that the electrodes (3) are flush with the surface of the substrate (2).
3. Substrate according to Claim 2, characterized in that the electrodes are arranged in a uniform grid.
4. Substrate according to one of the preceding claims, characterized in that the ultraphobic surface has a surface topography in which the spatial frequency f of the individual Fourier components and their amplitudes a(f) expressed by the integral S(log (f)) = a · (f)-f calculated between the integration limits log (f₁/µm^-1) = -3 and log (f₁/µm^-1) = 3 is at least 0.3, and consists of ultraphobic polymers or durably ultraphobic materials.
5. Substrate according to one of the preceding claims, characterized in that the ultraphobic surface is an aluminium surface which is structured and coated with a hydrophobic and/or oleophobic material.
6. Substrate according to one of Claims 1-4, characterized in that the ultraphobic surface is an aluminium surface treated with steam and coated with a hydrophobic and/or oleophobic material.
7. Substrate according to one of Claims 1-4, characterized in that the ultraphobic surface is a surface coated with Ni(OH)₂ particles and coated with a hydrophobic and/or oleophobic material.
8. Substrate according to one of Claims 1-4, characterized in that the ultraphobic surface is sandblasted and a surface coated with a hydrophobic and/or oleophobic material.
9. Substrate and device according to one of Claims 1-4, characterized in that the ultraphobic surface is a tungsten carbide surface which has been laser-structured and coated with a hydrophobic and/or oleophobic material.
10. Method of moving or dosing a liquid drop on a microscopic scale using a substrate according to one of Claims 1-9, characterized in that the liquid drop (8, 9) is moved using the electrodes (3).
11. Method according to Claim 10, characterized in that various voltages against one another are fed individually to the electrodes.
12. Method according to Claim 10 or 11, characterized in that a liquid drop is moved as desired within an electrode grid.
13. Method according to Claim 12, characterized in that two or more drops are moved within the electrode grid and thereby combined with one another.
14. Use of the substrate according to one of Claims 1-9 and of the method according to one of Claims 10-13 for dosing liquids on a microscopic scale, preferably in the range from 10^-6-10^-12 litres, particularly preferably in the range from 10^-6-10^-9 litres.
15. Use of the substrate according to one of Claims 1-9 and of the method according to one of Claims 10-13 for carrying out chemical or biochemical processes, preferably for PCR, ELISA and/or determination of enzyme activities.
16. Method for the preparation of a substrate according to one of Claims 1-9, characterized in that the electrodes are introduced into the substrate and the surface of the substrate is provided with an ultraphobic coating.
17. Method according to Claim 16, characterized in that the electrodes are introduced such that they are flush with the surface of the substrate.
18. Method according to one of Claims 16 or 17, characterized in that the electrodes are introduced into the substrate in accordance with a uniform grid.

Images