EP1251946A1

EP1251946A1 - Capillary force mixer

Info

Publication number: EP1251946A1
Application number: EP00974506A
Authority: EP
Inventors: Robert Seidel; Andreas Wolf
Original assignee: Albert Ludwigs Universitaet Freiburg
Current assignee: Micro Biolytics GmbH
Priority date: 2000-01-21
Filing date: 2000-11-03
Publication date: 2002-10-30
Anticipated expiration: 2020-11-03
Also published as: DE10002500A1; WO2001052975A1; DE50012871D1; EP1251946B1; ATE327820T1; AU2001212785A1

Abstract

The invention relates to an analytical microfluid channel array with a high chamber density, on the base of a microtiter plate. Said array is used for analysing chemical, biochemical, biological, physical or other measurable values in separately accommodated liquid samples which are mixed in the analysis chamber. The individual microfluid channel system acts as a capillary force mixer and comprises an inert matrix (1, 2), a detection area (5) and channels (3, 6) which are able to transport liquid in a capillary manner and which interconnect the sample feed openings (4, 7). The capillary liquid transport channel is at least partially formed by the matrix and the detection area. The detection area is suitable e.g., for carrying out analyses using spectrophotometric (e.g., IR) or electrochemical measuring procedures. The invention also relates to the use of said analytical capillary force mixer or test element arrays for determining a measurement in one or more liquids and to a method for determining chemical, biochemical, biological, physical or other measurable values in one or more liquid samples using said capillary force mixer or said microfluid channel array.

Description

capillary force

SUMMARY OF THE INVENTION The invention relates to an analytical capillary force mixer for determining one or more analytes in an aqueous or other usable solvent, preferably in separately applied sample liquids that mix in the analysis chamber, consisting of a carrier, a detection zone and a channel capable of capillary liquid transport , which combines two or more liquid application openings, the use of the aforementioned capillary mixer to determine one or more measured quantities in one or more liquids, and a method or a method for determining chemical, biochemical, biological, physical or other measurable quantities in one or more liquid samples Using said analytical capillary force meter, and / or method of making and using it

Background of the Invention

Carrier-bound or free tests are often used for the guarative or guarantative analytical determination of constituents of body fluids, in particular blood. In these tests, reagents are either embedded in appropriate layers of a solid carrier which is brought into contact with the sample, or the reagents are mixed with the sample in a chamber The reaction of liquid sample and reagents leads to a detectable signal in the presence of a target analyte, in particular to a color change, which can be evaluated visually or with the aid of a device, usually reflection-photometric

The simultaneous analysis of a large number of samples is often desirable and possible, for example in the case of flow cytometry, DNA segregation, different types of

Chromatography and electrophoresis and in ligand-receptor studies Rapid DNA analysis plays a major role in the Human Genome Project, for example, and research in the pharmaceutical or agricultural industry is without the simultaneous screening of a large number of substances for their biological Impact no longer conceivable

So-called microtiter plates, mostly with a standardized size of 12 7 x 8.5 cm, are used here. These are plates that contain a large number of small vessels, for example 96 or 192, but also fewer or considerably more, are customary, which are filled with different reagents. The amount of reagents in these analysis plates generally ranges from milliliters (10 ³ L) to microliters (10 ⁶ L) are the reagents valuable because they are only available in small quantities or are expensive, or both, therefore, miniaturization of the tests is sought, especially if many tests are to be or have to be carried out. The filling of the analysis trough (wells) is usually carried out by machines, as well further analysis is automated with preferred forms of use. For high-throughput screenmg (HTS), microtiter plates, for example from Greiner, 64943 Hirschberg, (micro-assay plate) with 1536 wells are known. Here, the working volume with 4-8 μl is relative high The sequential (sequential) introduction of the analytes is associated with the risk of cross-contamination. Appropriate caution is advised Use or the drop size limits the attempt to miniaturize the test. The working volume of a microtiter plate from Corning Costar (55924 Bodenheim, Germany) is only 1-2 μl, but here too the independent mixture of analytes introduced into separate wells is not A device for mixing two analytes by spontaneous mixing or vibration is described in US Pat. No. 4,088,448, but there is only one sample application point and detection reagents are preferably in solid form, which necessitates pre-loading with reagent and thus the flexible use of the device limited US Pat. No. 5,300,779 also describes an apparatus for mixing two analytes, but there is only one sample application point and reagents are already integrated. US Pat. No. 5,627,041 presents a single-use cuvette for the analysis of biological samples, this being a series of channels, Kapill aren, reservoirs and stop points that allow the controlled flow of sample, reagent and solvent. The complex design of this cuvette is space-intensive and not suitable for the design on microtiter plates. In addition, the flow of the analytes through the device does not take place only under centrifugal force spontaneous The US Pat. No. 5,222,808 describes a capillary mixing apparatus in which the mixing is not carried out spontaneously but by means of magnetic particles

General Description of the Invention The object of the present invention is then to eliminate the disadvantages of the prior art. In particular, an easy-to-use, independently volume-dosing test element is to be made available, with which, using minimal sample volumes, not only a spatial separation of the detection zone and samples - is possible, but additionally a mixture of two or more separately applied liquids takes place.Maximum possible flexibility for use for a wide variety of samples and analytes should be ensured.In addition, the mixing of the liquids and the transport of the liquids to the detection zone should be so fast that the analysis of a Sample is not limited in time by this. Another aspect in the elaboration of the described invention is the need to enable the inclusion of newer measurement methods, such as, without being restricted to this example, for example IR spectroscopy, in the analysis methods also those for these methods the absorption properties of conventional biological solvents, the maximum diameter of the capillary channel must be taken into account. Furthermore, a simple construction of the test element is intended to result in an inexpensive fer that is easy to produce in terms of production technology This enables the use of the microfluidic channel array for mass testing (high throughput screening, HTS) using standardized robots and machines

This is achieved by the subject matter of the invention, as described in the patent claims and below and explained in more detail. The advantages achieved with the invention are, in particular, that an inexpensive to produce capillary force mixer is presented, which is also more compact when used in parallel in multiple versions Arrangement can be used in particular on the base of a conventional microtiter plate for automated HTS, for example in pharmaceutical or agricultural research and development. It allows reactions with a minimum of sample and reagent, both of which can only be applied shortly before the analysis In addition, the design of the novel capillary mixer enables the use of new spectroscopic methods, such as modern variants of UV / VIS or fluorescence spectroscopy or TIRF, in particular IR spectroscopy, for analysis b iological and other substances in water or other water or biological media The structure of the new test system allows the analysis of substances in the low nanohter (10 ⁹ L) range. Very many systems can be used on a small one, for example, as specified by the standard Mirotiter plate Combine space, and the filling of the sample feed openings can be carried out automatically. Due to the chosen structure and the small size of the system, an active mixture is not necessary, but mixing takes place automatically by diffusion within a short time. For example, all variants can be used to detect the analyte the spectrometer can be used, provided the measuring beam focusing is appropriately adapted to the width of the detection zone. Due to the special structure, it is possible to carry out infrared (IR) spectroscopic investigations in particular in water solutions which are used in conventional fluid channel systems due to the high absorption in this spectral range of the water used as a solvent is not possible

List of pictures

Figure 1 (Fig. 1) shows a single capillary force mixer according to the invention. Figure 2 (Fig. 2) shows the function of a capillary force mixer according to the invention. Figure 3 (Fig. 3) shows such a capillary force mixer with a hydrophobic separating layer. Figure 4 (Fig. 4) shows an example of the liquid task in a capillary force mixer according to the invention

Detailed description of the invention

The invention relates to an analytical microfluidic channel array, hereinafter referred to as a capillary force mixer, for determining one or more analytes or the state of one or more analytes in a water-based or furthermore another usable (in particular hydrophilic) solvent or solvent mixture (analyte plus solvent (mixture) = subsequently "Liquid"), consisting of an inert carrier, a detection zone and a channel capable of capillary liquid transport, which connects two or more sample application openings. The structure of the new test system allows the analysis of very small quantities, ie those in the low nanohter (10 ⁹ L) - Range, in particular in the range from 0.05 to 800 nl, preferably 0.1 to 100, in particular 0.1 to 50 nl. Many systems can be combined in the small space specified by the standard microtiter plates, and the Filling of the test opening can be done automatically Due to the chosen structure and the small size of the system, an active mixture is not necessary, but a mixture takes place independently by diffusion within a few minutes.The capillary mixer also has the advantages of easy filling and good dosing. The separate liquid feed points reduce the risk of cross -Contamination reduced, and the capillary force mixer can be flexibly adapted to a given sample to be analyzed by selecting a suitable reagent solution shortly before the measurement In particular, it is a capillary force mixer for the analysis of (in particular two or more) liquids which comprise (in particular two or more) reagents (reactants) which are brought together and mixed for the reaction (their bringing together in particular to obtain closer ( quantitative or qualitative) information about at least one of the reagents (analyte) or forms a new component through a physical or chemical reaction), characterized in that the capillary force mixer includes at least two liquid application points (= sample application openings) and capillary areas (especially supply capillaries) ) which enable the liquids to be taken up by capillary force without the addition of external forces into the capillary force mixer and to have a capillary-active mixing zone into which the liquids enter by capillary force and in which a mixture of the liquids in the essentially by diffusion

The invention relates in particular to a capillary force mixer according to the preceding paragraph, characterized in that there are two liquid feed points, each of which flows into a capillary, which can be filled separately or in succession and mouth in a common, also capillary-active mixing zone, the capillaries preferably parallel to the edges the mixing zone

In another embodiment, the invention relates to a capillary force mixer according to one of the last two paragraphs, the capillary-active mixing zone itself being connected directly to the liquid feed points, so that there are no additional capillaries

A capillary force mixer according to one of the last three paragraphs is more preferably characterized in that there are no moving or active parts for the function

A capillary force mixer is particularly preferred in accordance with the last paragraph, in which the layer thickness of the liquids in the mixing zone is fixed

A capillary force mixer according to the last paragraph is preferred, characterized in that the layer thickness of the liquids in the mixing zone is preset in a range from 0.1 to 10,000 μm, preferably in a range from 1 to 100 μm

A capillary force mixer is more preferred after the penultimate or last paragraph, the mixing zone of which is designed in such a way that the layer thickness of the liquids in the mixing zone is set such that IR spectroscopic measurements are carried out in aqueous solvents. can be led; the layer thickness of the liquids in the mixing zone is preferably preset in a range from below 30 μm, in particular from 3 to 30, especially from 5 to 10 μm.

A capillary force mixer according to one of the preceding seven paragraphs is particularly preferred, characterized in that one or more functional areas are equipped with temperature control means, or a capillary force mixer according to one of these paragraphs, in which functional layers are introduced in the area of the mixing zone.

A capillary force mixer according to the last paragraph is even more preferred, characterized in that one or more functional parts are equipped with sensors, in particular electrochemical sensors.

A capillary force mixer according to one of the last nine paragraphs is very particularly preferred, characterized in that one or, in the case of more than two liquid feed points, one or more hydrophobic or differently designed separating layers are present (= applied to the inner surface of the capillary area, in particular the mixing zone) which prevent premature mixing of the liquids brought into the mixing zone by capillary force, in particular further characterized in that there is a ventilation duct (ventilation shaft) for completely filling the mixing zone; preferably the volume calibration (amount of liquid) of the liquids (to be applied) is predetermined by the dimensions of the mixing zone up to the hydrophobic zone.

A capillary force mixer according to one of the last 10 paragraphs is even more preferred, characterized in that the volume of the supply capillaries and the mixing zone (in one variant of the invention without supply capillaries only the volume of the mixing zone) is preset in the range from 0.05 to 800 nl, preferably 0 , 1 to 100, in particular 0.1 to 50 nl.

A capillary force mixer according to one of the last eleven paragraphs, which is composed of two individual parts (cover and substrate layer), is very preferred.

In particular, a capillary force mixer is preferred according to one of the last twelve paragraphs, which consists of silicon, glass or both (enables high-precision construction). Even more preferred is a capillary force mixer according to one of the last thirteen paragraphs, in which there are means for detection by optical, electrochemical or other suitable means of reactions which take place within the mixing zone integrated into the capillary system after the liquids have been mixed

A capillary force mixer according to one of the preceding fourteen paragraphs, which has a lens or a Fresnel lens, in particular in the area of the mixing zone, is preferably integrated on the outside of the capillary force mixer, especially in the case of plastic designs (primarily an optically transparent area). , includes

A further preferred embodiment of the invention is based on the use of the capillary force mixer according to one of the last fifteen paragraphs for the analysis of at least two liquids which are brought together and mixed for the reaction and the combination of which leads to obtaining more detailed information about at least one of the liquids, or the reaction thereof forms a new component, characterized in that all liquids are filled into the capillary force mixer by capillary force without the addition of external forces, and their mixing takes place essentially by diffusion

In particular, the use of the capillary mixer according to the preceding paragraph is preferred, the measuring method being a spectroscopic method, in particular a spectroscopic measuring method, which detects electromagnetic waves with a wavelength of about 160 to about 20,000 nm, preferably of about 180 to about 15,000 nm, especially IR. Light with a wavelength of about 700 to about 15000 nm, especially about 2500 to 15000 nm, is used

The use of a capillary mixer as described above is also preferred for analysis by electrochemical or enzymatic methods

The use according to one of the last three paragraphs of a capillary force mixer is particularly preferred, characterized in that, in order to measure a reference value, a detection is carried out directly in situ before filling with the second or later liquid (reagent solution or sample solution)

The use of a capillary mixer according to one of the last four paragraphs is very preferred, one of the liquids being a biological sample, in particular whole blood Very preferred embodiments of the methods and devices according to the invention can be found in the examples

The liquids are on the one hand samples, in particular biological samples such as blood (whole blood), blood plasma, blood serum, saliva, sweat, urine, pus, gastric juice, bile, other digestive juices, cerebrospinal fluid, tear fluid, milk, glandular secretions , Synovial fluid, but also hamolymph from insects, liquid foods or food components in nutritional analysis, culture media, cell plasma, medication, or water samples, for example from lakes, flowing water or clear systems, extracts or other liquids to be analyzed, each according to Refurbishment, such as centrifugation, filtration, chromatography or the like, or in particular without refurbishment (in particular in the case of blood, which may also contain cellular constituents which can be excluded from the mixing area, in particular in the case of a thin layer of the mixing zone, or in the case of larger layers) due to their slow diffusion in the ge In contrast to the surrounding liquid, diffuse very slowly into an area of the mixing zone with a reagent solution, so that there

Properties of the surrounding liquid can be measured directly) can be applied to the liquid application zones, these samples being able to detect qualitatively or quantitatively reactants (analytes), in particular enzymes, antibodies, nucleic acids, other biomacromolecules, sugars, lipids, liposaccharides, organic acids, amino acids , Drugs or other xenobiotics, metabohts, enzyme substrates, other organic compounds, heavy metals, ions, gases or the like. On the other hand, there are solutions of detection reagents ("Reagenzιen0" reagent solutions ") with complementary (ie to some kind of detectable interaction , eg chemical (essential biochemical or biological) or physical type, with one or more analytes from the samples, capable) reactants (in particular possibly fluorescent, enzyme or other denatured antibodies, low molecular weight reagents (for example substrates un d / or cosubstrates for enzymatic reactions with components in the samples or inter-near (e.g. fluorescent) dyes), enzymes or other catalysts, mixtures of enzymes or other catalysts with substrates and / or cosubstrates, complexing agents, etc which are preferably mixed after mixing cause a specific change in a physical or chemical property ("signal") of a sample, which can be detected and evaluated for analytical purposes, especially computer-based supports Preferred applications are also PCR and hybridization of nucleic acids or derivatives (eg fluorescence-labeled, or with modified bases and / or backbones) thereof

The fact that the mixing takes place essentially by diffusion means that no additional auxiliaries for mixing (such as vibration, stirring, centrifugation or the like) are necessary and in particular no such means are used to produce the mixture of applied liquids (at most to overcome hydrophobic separating layers, to bring the applied liquids into contact) In particular, it is the internal diffusion of 2 or more liquids, in contrast to the diffusion of solid substances into a liquid

The detection of signals resulting from the mixing (or previous ones, in order to enable calibration in particular) is possible with conventional spectrometers, only the measuring beam focusing should be adapted to the width of the detection zone. Due to the special design, it is possible, especially infrared (IR) spectros - carry out copying investigations in watery solutions, which are not possible in conventional fluid channel systems due to the high absorption in this spectral range of the water used as solvent. IR spectroscopy can only with sufficient reliability and reproducibility up to a water layer thickness of about 10 μm (in particular Traps can also be operated up to 30 μm) The thickness variation of the measuring chamber must be kept as low as possible for reliable reproducibility

IR spectroscopy is a routine method and is used by organic chemists, biochemists, and others for qualitative and quantitative molecular detection. When infrared light is passed through a sample of an organic substance, some of the frequencies are absorbed, while other frequencies pass through the sample unimpeded Applications in the analysis of organic substances use almost exclusively IR-radiated with wave numbers in the range of 650-4000 cm ¹ (mid-IR) Wave numbers below 650 cm ¹ are referred to as far infrared (FIR), those above 4000 cm ¹ as near infrared (near infrared = NIR)

IR spectroscopy also includes laser Raman spectroscopy, including Raman confocal laser spectroscopy, Fouπer transform IR (FTIR) spectroscopy or Hadamard transform IR, or any other IR spectroscopic method using the loading particularly preferred founer transform (FT-IR) spectroscopes are possible without further qualitative and quantitative determinations of analytes in the picogram (pg) range. With other optical methods, such as UV / VIS or in particular fluorescence or chemistry or fluorescence spectroscopy, can still much smaller amounts can also be detected. The "Total infernal reflection fluorescence technique (TIRF, see Garlamd PB et al Optical evanescent methods for study of biomolecular mteractions, Quart Rev Biophys 29 91-117 (1996), which is based on detection from outside the main beam based on rays carried out of the material can be used

As with all methods of spectroscopy, the signal obtained in IR spectroscopy is dependent on the layer thickness of the sample under investigation, so that the IR signal obtained from a <10 μm cell is very weak. This disadvantage can be caused by an immediate previous calibration is largely absorbed, as this largely eliminates the measurement inaccuracies. The control and software required for this are fundamentally already state of the art and can often be found in spectrometers. The possibility of calibrating the samples directly before mixing with the exact same optical path length as in the mixed state is a decisive advantage of the new microfluid channel system. This also enables differential measurements (comparison before / after)

Suitable analysis devices, in particular FT-IR devices, are easily accessible, in particular commercially available, for example from Bruker (Karlsruhe, Germany), for example Bruker Spectrum GX FT-IR Spectrometer Bruker Equmox 55, examples of UV / VIS devices are lambda 40 or the Lambda 800 UV / VIS Spectrometer System from Perkm Eimer

An inert or multi-part substrate is used as the inert carrier (matrix) for a capillary force mixer according to the invention, which substrate is not attacked by the liquids to be appended, or is not attacked, or not attacked significantly, within the time period required for application and measurement, in particular from one or more of the materials mentioned below

In a preferred embodiment of the invention, the new microfluidic channel system has a very simple structure. Due to the lack of any movable components, the (preferred) structure of the carrier is made up of two microtechnologically processed in a batch process Slabs (top and substrate layers) possible. This method is very inexpensive and is based on the latest state of the art. In a particular embodiment of the invention, a matrix-shaped array, for example consisting of a cover and substrate layer, which contains several of the capillary force mixers according to the invention (optionally with ventilation channels, hydrophobic separating layers or other features of preferred embodiments of these capillary force mixers, as described here) in a matrix-like arrangement , for example in total with the dimensions of conventional microtiter plates, which in particular can be produced directly from two parts (cover and substrate layer).

A capillary mixer according to the invention is produced in particular by joining the components of the capillary mixer according to the invention (preferably directed) and connecting them to one another by holding devices such as clamps or the like. The components, e.g. Plates (top and substrate layer) of the microfluidic channel array can also be glued (for example by means of polymerization, polyaddition or polycondensation materials, which can also be filled with fillers, eg to increase the electrical conductivity with silver particles), welding, glass Fusion bonding or other microtechnical processes such as anodic or eutectic bonding. This must be done in a directed manner, i.e. the components must be exactly aligned ("alignment") in order to ensure congruence, for example existing channels, and the assembly must not create any additional volume in the capillary or mixing zone area.

A number of materials can be used. Preferably, since the devices are manufactured using microtechnology, the materials are selected so that they are compatible with known microfabrication techniques. These include, for example, methods for removing material from the surface, such as wet etching, electrochemical etching, anisotropic etching or, in particular, chemical wet etching, for example in the case of Si0 ₂ using hydrogen fluoride / ammonium fluoride in the presence or absence of acetic acid, or dry etching, such as plasma etching processes, such as Sputtering, plasma chemical etching, reactive ion etching, ion beam etching or anisotropic deep etching using RIE, ion beam methods such as ion beam etching, laser ablation (laser ablation), photolithography, in particular laser ablation; Application of material, for example using PVD, be it without plasma support (e.g. thermal evaporation, flash evaporation, MBE, VPE or electronic jet blasting), CVD (thermal or plasma-based (PECVD)), laser-based material deposition or wet-chemical metal deposition (galvanic or chemical); or direct molding processes, such as vacuum molding, injection molding or other primary molding methods, pressing, embossing (for example hot embossing), and the like, depending on the suitability for the respective material, or combinations thereof. The materials are also selected for their suitability for the full range of conditions to which the microfluidic systems are to be exposed, including extreme temperature, pH or salt concentration conditions or the application of electrical or magnetic fields. In a preferred embodiment of the invention, the materials are those which are also used in the semiconductor industry, where microfabrication techniques are used regularly, in particular based on silicon, such as silicon, or other semiconductor materials, such as gallium arsenide. Other preferred materials are silicon dioxide, glass, such as borosilicate glasses, quartz, quartz glass, silicone or polysilicon. In the case of semiconductor materials, it can often be expedient to layer an insulating layer, for example made of silicon dioxide, over the material, in particular where electrical fields are to be used on the microfluidic element. Other preferred materials are plastics, including polymers such as polymethyl methacrylate (PMMA), polycarbonate, polyesters such as polybutylene terephthalate or polyethylene terephthalate, polyamides, polytetrafluoroethylene (TEFLON "), polyvinyl chloride (PVC), polydimethylsiloxane (PDMS), polysulfone, polystyrene, Polyolefins, such as polymethylpentene, polypropylene or polyethylene, polyvinylidine fluoride, acrylonitrile-butadiene copolymer (ABS), cycloolefin polymers such as TOPAS "(thermoplastic olefin polymer with amorphous structure from Hoechst AG, Frankfurt, Germany), block copolymers or mixtures of two or more thereof usable. Parts consisting of such polymers, in particular substrate and cover layers, can be produced by known microfabrication processes, for example those mentioned above, or by means of microtechnologically produced mother molds using known molding processes, such as injection molding, embossing or stamping, or by polymerizing the monomeric precursor material within the mold (primary shaping). Such polymeric materials are preferred for many purposes because they can be easily manufactured, inexpensive and also disposable. Ceramic materials are also possible, particularly in the case of hybrid microsystems. Microfluidic elements based on glass or silicon are particularly preferred. All of the materials mentioned can contain specifically treated or coated surfaces or surface sections within the capillary or further the sample application areas, for example hydrophobized areas (in particular separating layers with linear Expansion), for example by silanation (coating with a silane to which a connecting molecule carrying a hydroxyl group is coupled) more functional, such as hydroxyl, groups or metal production, in particular with gold, for example by vapor deposition, or (above all to increase it) the capillary forces) hydrophilization, for example in the case of glass with a medium approach, so that the surface has many hydroxyl groups, for example with Caroic acid, or other introduction of polar groups (for example silanization with silanes containing amino or hydroxyl groups), or in the case of plastics by treatment with argon plasma, plasma etching, corona discharge (eg 10-25 W at 13.56 MHz, 1 Torr chamber pressure, 5-10 min), by covalent bonding of photoreactive hydrophilic polymers, by absorbing wetting agent-like layers or coating with Nanocomposites using SolJGel technology, by thin Al ₂ 0 ₃ coating (vapor deposition with Al, subsequent oxidation by electrochemical means, by Erhi etching in water or by means of steam), in the case of PMMA in particular by etching with KOH, or by other thermal, physical or chemical methods, depending on the suitability for the respective material

The capillary-active mixing zone is either connected to the liquid supply points via capillaries or directly connected to them, so that there are no separate supply capillaries.The mixing zone comprises the detection zone (detection zone) for the physical or chemical signals resulting from the mixture, for example optical windows, sensors or like

In order to enable optical evaluation, it is possible to use transparent materials for the intended optical measuring range, for example, without wanting to derive a restriction, clear plastics or glass for visible light, preferably special optical glass or for short-wave (UV) light Quartz glass and, for long-wave light (IR), preferably silicon. For the filling of the channel by capillary force, the wettability of the matrix is imperative, which can be achieved by appropriate treatment of the surfaces (see above)

Functional areas (layers) can be introduced in the mixing zone, for example electrical or biochemical in nature, which are based on the determination of the desired measurement variable.These are preferably sensors (transducers), such as, in particular, chemical sensors (e.g. chemical resistors, chemical capacitors, metal oxide - gas sensors chemically sensitive field effect transistors, such as ISFETS or CHEMFETS, Microelectrodes (potentiometric or amperometric), SAW sensors, e.g. vacuum resonators, or chemosensor arrays), or biological sensors (especially CMOS-compatible), or radiation sensors (e.g. photodiodes or image sensors, especially for fluorescence or chemiluminescence measurements), and also thermal converters , Transducers for ionizing radiation, magnetic or mechanical transducers, and any combination of such sensors. Means for tempering can also be integrated, such as heating elements, e.g. polysilicon resistors or thermogenerators, or for cooling channels for coolants. However, such functional layers are necessary for the production of the invention the subject matter is not absolutely necessary - in preferred embodiments of the invention they are not present. Temporary agents may alternatively or additionally also be present externally

The area of the mixing zone does not require any grooves or grooves or other means for aligning the capillary flow of introduced liquids along predetermined paths, so that preferably completely smooth surfaces are present

The liquid application points can contain means for improved connection of the application point to the capillary area, for example one or more small notches in the transition area between the liquid application point and the capillary area or areas hydrophobicized at the edge, hydrophilic or hydrophilized areas at the transition to the capillary area.However, such means are not absolutely necessary, but on the other hand in preferred embodiments of the inventive capillary mixer are available

Venting channels are preferably designed so that they only allow gas to be transported, not liquid (for example, by hydrophobizing the surface, at least in the border area to the mixing zone of the capillary mixer, due to the large cross-section, so that the capillary force is no longer sufficient at the transition from the mixing zone to the venting channel for the further transport of the liquid (stop junction), through formation as a microporous hydrophobic stopper which allows air to pass through but not hydrophilic liquids, or the like

In a preferred embodiment of the invention, there is a matrix of several of the capillary force mixers according to the invention, the ventilation channels of which are all connected or coupled to one another in groups, which, for example, in the case of hydrophobic separation tunnels in the mixing chamber, by pressure or, in particular, vacuum shock to overcome the separation of applied samples in all or several mixing zones at the same time and thus to initiate the contact between the samples and their subsequent mixture in a synchronized manner. Even in the case of separate ventilation ducts, a pressure or vacuum surge in several capillary force mixers can simultaneously initiate the transfer of hydrophobic separating layers , for example if it is in a pressure chamber

Examples The following explanations serve to illustrate the invention without wishing to restrict its scope

Example 1 Structure of a Single Capillary Force Mixer Fig. 1 shows the exemplary structure of a single capillary force mixer, which is for example in a multi-system array (several such capillary force mixers are arranged in a matrix next to each other) or the like, held together The contact surfaces are previously processed by hot forming or casting, advantageously by means of micro-technical processes such as photohthography, dry or wet etching, such as "synchrotron radiation ablation" or also by application processes, for example spinning, CVD or the like that the finished product per microfluidic channel system has a supply capillary (3 and 6) for each of the liquids and a zone (5) for mixing and detection. The plates (cover and substrate layer) of the microfluidic channel array can also be glued, Weld or on whose microtechnical processes, such as anodic or eutectic bonding, are to be merged. The process must be directed and the merging must not create any additional volume

Example 2 Capillary Force Mixer for Mixing Two Liquids The capillary force mixer shown schematically as an example in Fig. 2a for the mixing of two liquids with reactants has the following functions, which also apply analogously to capillary force mixers for mixing more than two liquids. The two capillaries (3 and 6 ) are connected in the mixing and detection zone (5). When filling the first capillary (3) through the full opening (= liquid application point) (4) with sample liquid (8), the introduced sample is transferred into the mixing and detection zone by capillary force ( 5) pulled, while the second capillary (6) remains free of liquid (9) This is shown in Fig 2b. Now the calibration can be carried out by appropriate methods, preferably but not exclusively, by spectroscopic methods, particularly preferably IR spectroscopic methods. Subsequently, the second capillary (6) is filled with reagent liquid (10) through the full opening (= liquid application point) (7) at the contact surface with the sample liquid ( 8) in the mixing and detection zone (5), a mixture (11) takes place immediately (Fig. 2c). The mixing front proceeds in the direction of the first capillary (3). This can be time-resolved by suitable methods, for example methods of spectroscopy (especially IR ), the entire length of the mixing and detection zone (5) is available for this purpose. The measurement can be distributed simultaneously at several points over the mixing and detection zone (5), or in chronological order at the same point in the mixing - And detection zone (5) are carried out

Example 3 Capillary force mixer with a hydrophobic separating layer

In a particularly preferred embodiment (Fig. 3) of the capillary mixer, a layer (13) is introduced in the mixing and detection zone, which gives the site a hydrophobic character. Such layers can be implemented by special surface properties such as roughness, but preferably by a hydrophobic coating , for example made of hydrophobic metal layers such as gold, in an even more preferred embodiment of hydrophobic plastics. As an example of such a hydrophobic plastic, without deriving from it, Teflon is also called. Silanization, in particular with alkylsilanes, is also possible. This layer (13) forms one Separation zone and causes that during the introduction (Fig. 3a) of the aqueous liquid (eg sample) (14) the capillary force only penetrates the liquid up to the hydrophobic interface (13) (15). This is shown schematically in Fig. 3b Likewise, the second liquid (e.g. reagent) (16, Fig 3c) only drawn up to the opposite side of the boundary surface (13) (17, Fig. 3d) In this way, calibration can be carried out on both liquids (14, 16) in the capillary areas 15 and 17 before the start of the reaction by mixing Separation by the hydrophobic forces of the separation zone using suitable methods, for example slight vibration, pressure change (in particular by applying a vacuum surge across the ventilation duct 19) or hydrophilization of the separation zone (eg by chemicals present in at least one of the liquids) or catalysts, such as enzymes, which break down the hydrophobic layer), a contact can be made between the two liquids (14, 16) and the mixture (18, Fig. 3e) takes place spontaneously. In this embodiment, a ventilation duct (19) as in Fig 1, be incorporated so that a complete Ful- The mixing zone is guaranteed. This preferred embodiment of the invention not only permits the spectroscopic evaluation of both liquids before the mixing, but also enables a very precise determination of the amounts required, in particular of reagent liquid. The mixing is also completed more quickly than in the first embodiment. The steps shown are analogously also transferable for capillary force mixers with more than two, in particular three or four, sample application openings and sample channels opening into a common mixing and detection zone, the separation zone having a corresponding shape, for example in the case of three application openings the shape of a Y, the legs of which each separate the separation zones of the chamber sections means, in the case of four task openings, the shape of a plus sign (+), etc

If several such microfluidic elements (for example in the form of a matrix) are present, their ventilation channels (19) can be coupled, which enables the separation zone to be overcome by coordinated (under) pressure surges

Example 4 Filling a KapiUarkraftmischer

The capillaries can be filled through the sample application opening, as shown schematically in Fig. 4, by a capillary pipette (21), at the end of which the reagent forms a microdrop (22) under gravity or light pressure (26) .The capillary pipette is inserted into the full opening ( 4 or 7) until the drop reaches the bottom of the opening (27) and makes contact with the capillary (3 or 6). The amount of liquid (eg sample) (28) that is absorbed into the microfluidic channel system is included determined by the dimensions of the capillary and a possibly applied back pressure (29) This makes it possible to use very small amounts of liquid, in particular of reagent liquid, which otherwise could not simply be pipetted due to the surface tension of the liquid

Example 5 Production of a capillary mixer a) Production of the cover layer (1) (FIG. 1) A glass wafer 10 cm long and wide and 500 μm thick is coated on both sides by means of chrome sputtering (vacuum dusting) 50 nm thick using a photoresist mask , which covers the underside and the area around the planned supply capillaries (3, 6) and the planned mixing zone (5) (together capillary area) (e.g. 0.5 x 0.5 cm), the remaining chrome layer is covered with a light-sensitive polymer ( Photoesist, e.g. Microposit 1818S Photo Resist, Shipley Europe Ltd, Coventry, England) and exposed the exposed area to UV light and developed (eg using Microposit 351 developer 5 1 in water) (photo thography) and then using HCI (36% -ιg) the unprotected area of The chrome layer is removed (patterning). Then the area of the future supply capillaries and the mixing zone is etched 10 μm deep with 50% HF and then the photoresist (using a photo resist remover such as Microposit Remover 1112A) and the chrome layer are removed Thickness of the mixing zone (10 μm) specified New vacuum deposition of chrome (50 nm) on both sides of the surface of the glass wafer, renewed photohthography with a mask that only leaves the areas of the future sample task opening open on the underside, and renewed Cr-Pattemmg with subsequent HF etching (50%, 60 μm) with subsequent photoresist and chrome removal as well as laser drilling to achieve openings that are accessible from outside as a liquid feed point len (full openings) (4, 7) to the side opposite to the etched out mixing zone area (the future upper side of the capillary power mixer) results in the cover plate (1) of the capillary power mixer to be manufactured

b) Production of the substrate layer A glass wafer as described under a) is first vacuum-coated on the top and bottom with a 50 nm chrome layer, then the indentation for the liquid task is etched in by photohthography and Cr-patterning on the top (36% HCI), analogous to that described under a). After BHF etching (50 μm), the substrate plate (2) is finished

c) Joining the top and substrate layers The top and substrate layers are joined by glass fusion bonding (joining under pressure at 250 to 600 ° C after introducing HF between the layers to be joined using steam or a drop of hydrofluoric acid, which gently attaches the glass surfaces so that they bond firmly through pressure and diffusion) and together form the finished capillary force mixer

Claims

Patent claims

1 capillary force mixer for the analysis of liquids which comprise reagents which are brought together and mixed for the reaction, characterized in that the capillary force mixer has at least two liquid supply points and it comprises capillary areas which allow the liquids to act by capillary force without adding external forces to the capillary force mixer are recorded and that there is a capillary-active mixing zone into which the liquids enter by capillary force and in which the liquids are mixed essentially by diffusion

2 capillary force mixer according to claim 1, wherein two each end in a capillary

Liquid feed points are available, which can be filled separately or one after the other and mouth in a common, also capillary-active mixing zone

3 capillary force mixer according to one of claims 1 or 2, characterized in that the capillary-active mixing zone itself is directly connected to the liquid feed points, so that no additional capillaries are present

4 capillary force mixer according to one of claims 1 to 3, characterized in that there are no moving or active parts to function

5 capillary force mixer according to claim 4, characterized in that the layer thickness of the liquids in the mixing zone is fixed

6 capillary force mixer according to claim 5, wherein the layer thickness of the liquids in the mixing zone is fixed in a range from 0.1 to 10,000 microns

7 capillary force mixer according to claim 6, wherein the layer thickness of the liquids in the mixing zone is preset in a range of 1 to 100 microns

8 capillary force mixer according to claim 7, characterized in that the layer thickness of the liquids in the mixing zone is set so that IR spectroscopic measurements can be carried out in water-based solvents 9 capillary force mixer according to claim 8, characterized in that the layer thickness of the liquids in the mixing zone is fixed in a range from 3 to 30, in particular from 5 to 10 microns, preset

10 capillary force mixer according to one of claims 1 to 9, characterized in that one or more functional parts are equipped with means for temperature control

11 capillary force mixer according to one of claims 9 or 10, characterized in that functional layers are introduced into the mixing zone

12 capillary force mixer according to claim 11, characterized in that one or more functional parts are equipped with sensors

13 capillary force mixer according to claim 12, characterized in that one or more functional parts are equipped with electrochemical sensors

14 capillary force mixer according to claims 1 to 13, wherein in the mixing zone there is a hydrophobic or differently designed separating layer which prevents premature mixing of the liquids brought into the mixing zone by capillary force

15 capillary force mixer according to claim 14, characterized in that for the complete filling of the mixing zone there is a venting passage

16 capillary force mixer according to one of claims 14 or 15, characterized in that the volume calibration of the liquids is predetermined by the dimensions of the supply capillaries and the mixing zone up to the hydrophobic zone

17 capillary force mixer according to one of claims 1 to 16, characterized in that the volume of the supply capillaries and the mixing zone is in the range from 0.05 to 800 nl, preferably 0.1 to 100, in particular 0.1 to 50 nl

18 capillary force mixer according to one of claims 1 to 17, characterized in that it is assembled from two individual parts (cover and substrate layer)

19 capillary force mixer according to one of claims 1 to 18, characterized in that it consists of silicon, glass or both Capillary force mixer according to one of claims 1 to 19, characterized in that there are furthermore means for detection by optical, electrochemical or other suitable means of reactions which take place within the mixing zone integrated into the capillary system after the liquids have been mixed

Capillary force mixer according to one of Claims 1 to 19, which comprises a lens or a fresnel lens, in particular in the region of the mixing zone, preferably integrated on the outside of the capillary force mixer

Use of the KapiUarkraftmischer according to any one of claims 1 to 21 for the analysis of at least two liquids which are brought together for reaction and mixed and the combination of which leads to obtaining more detailed information about at least one of the liquids, or the reaction of which forms a new component, wherein all liquids Capillary force can be filled in the capillary force mixer without the addition of external forces and the mixing thereof is essentially carried out by diffusion

Use according to claim 22 of the KapiUarkraftmischer, wherein the measurement method is a spectroscopic method

Use according to claim 23 of the KapiUarkraftmischer, wherein the spectroscopic measurement method uses electromagnetic waves with a wavelength of about 160 to about 20,000 nm, in particular from about 180 to about 15,000 nm

Use according to claim 23 of the KapiUarkraftmischer, wherein the spectroscopic measurement method uses IR light with a wavelength of about 700 to about 15000 nm

Use according to claim 23 of the capillary mixer for analysis by electrochemical or enzymatic methods

Use according to one of claims 22 to 26 of the KapiUarkraft mixer, characterized in that for the measurement of a reference value a detection is carried out directly in situ before filling with the second or later reagent solution

Use according to one of claims 22 to 27 of the capillary mixer, wherein one of the liquids is a biological sample, in particular whole blood