EP1397483A1

EP1397483A1 - Micro fluid system

Info

Publication number: EP1397483A1
Application number: EP03718772A
Authority: EP
Inventors: Johan-Valentin Kahl
Original assignee: Ibidi GmbH
Current assignee: Ibidi GmbH
Priority date: 2002-04-22
Filing date: 2003-04-17
Publication date: 2004-03-17
Anticipated expiration: 2023-04-17
Also published as: EP1397483B1; AU2003222824A1; WO2003089565A1; ATE296347T1; DE50300574D1

Abstract

The invention relates to a micro fluid system comprising a substrate, at least two reservoirs and at least one channel which is integrated into the substrate connecting the two reservoirs, the outlet of at least one channel being arranged above the base surface of one reservoir.

Description

Microfluidic system

Description of the invention

The invention relates to a microfluidic system with a substrate, at least two reservoirs and at least one channel integrated into the substrate, which connects two reservoirs.

Many biological / chemical analyzes are carried out using (micro) -fluid components. These are characterized by simple or networked duct systems. Pumps are used to conduct the liquids through such channels. This is particularly necessary if a lot of liquid is to be passed through a channel system or if one and the same liquid is to be repeatedly passed through the channel system.

The pumps can be installed in or on the microfluidic system. However, pumps have many disadvantages. For example, the microfluid system can only be used in connection with an external energy source and must be permanently connected to it. (A power cord is required for an integrated pump, and tubing is required for an external pump).

If components of the pump come into contact with the sample solution, chemically undesirable reactions can occur. By using pumps there is a large dead volume, which leads to problems when using little sample material, especially when exchanging solutions. Many pumps pulsate, which leads to strong pressure fluctuations in the channel. This can lead to damage, especially with small ducts. Even small pumps lead to high flow rates in a relatively small duct system. This can detach or damage molecules or cells attached to the channel surface.

However, the continuous or cyclical change of solution in a channel system is of crucial importance for many applications of microfluidic systems. One example is the cultivation of cells in a microchannel. If these grow, they form toxic substances that cause the cells to die. In narrow channels, the geometric arrangement severely limits diffusion, so that the concentration of toxic substances in the area of the channel increases quickly. If the solution is not exchanged, cell growth can be observed to slow sharply towards the interior of the channel. A constant supply of nutrient medium and disposal of toxic substances can only be achieved through a time-controlled change of solution in the sewer. Pumps often have the disadvantages described above.

For the reasons mentioned above, it is also not possible to transport cells in a closed system or to store them for a longer period while maintaining their metabolism.

Another application of pumps is the purification of liquids. There are different methods for this. It is important for everyone that, firstly, a defined volume flow can be created and secondly, certain reaction times are made available to the reaction partners (the substances in the solution and the substances in the carrier).

Another application is the detection of certain substances in a channel system, in which it is necessary to let certain liquids run through the same channel in succession. Flow rates and response times must also be observed here. Pumps would have to work extremely precisely and be controlled by special electronics.

The object of the invention is to provide a microfluidic system with which liquids can be transported in a simple manner and without the microfluidic system requiring an external connection.

This object is achieved by the microfluid system according to claim 1. It is a microfluid system with a substrate, at least two reservoirs and at least one channel integrated into the substrate, which connects two reservoirs, the mouth of at least one channel above the base area of a reservoir is arranged. With such a microfluid system, liquids can be transported in a simple manner by tilting the microfluid system to one side, as a result of which a liquid flows from a reservoir (a higher reservoir) through a channel connected to it into a reservoir arranged at the other end of the channel. The mouth of the channel arranged above the base of a reservoir has various advantages. If a liquid flows through this mouth into a reservoir, this prevents the liquid from flowing back into the channel. If the liquid is in a reservoir into which a channel opens with a mouth arranged above the base area, the liquid can only flow out of the reservoir when the microfluidic system is sufficiently inclined so that the liquid can penetrate into the channel , In this way it can also be achieved that only a predetermined amount of liquid flows from a filled reservoir into a channel.

The microfluid system according to the invention can comprise a plurality of channels and / or reservoirs. Several channels can open into a reservoir. Advantageously, different channels open into a reservoir at different heights. Several reservoirs can be filled with different liquids. The channels can preferably cross and / or divide. In this way, different liquids can flow into the same channel from different reservoirs, possibly depending on the inclination. The reservoirs and channels can advantageously also be arranged in such a way that a closed liquid circuit is thereby formed. By tilting the microfluid system differently in different directions, for example, a liquid, starting from a first reservoir, can alternately flow through channels and reservoirs until it arrives again in the exit reservoir.

In a preferred development of all the microfluid systems described above, the base area of at least one reservoir can be inclined relative to a reservoir side wall. This means that the angle between the base and the side wall is not equal to π / 2. With a suitably chosen reservoir geometry, it is further possible to set at which angle of inclination of the microfluid system a liquid flows from a reservoir into a channel. According to an advantageous development, the microfluidic system can be closed off and comprise a device for pressure equalization, which connects each reservoir to at least one other reservoir. An airtight seal in particular can thus be made possible. Locking the microfluidic system prevents the liquid or liquids from coming into contact with the outside world. The device for pressure compensation can be designed in the form of channels integrated into the substrate, which open into the reservoirs in such a way that no liquid can penetrate and thus only serve for gas or pressure compensation. The device can preferably also be designed in the form of lines which connect the reservoirs but are not integrated into the substrate.

According to an advantageous alternative, the reservoirs can be closed with gas-permeable lids, so that the pressure is equalized with the surroundings.

According to an advantageous development of all the microfluid systems described above, at least one reservoir can be closed with a lid, the lid having a filter. Such a filter enables gas exchange with the environment or with another, connected reservoir without pollutants entering the reservoir or being able to escape from the reservoir.

In a preferred development of all the microfluid systems described above, at least one channel can have a charged surface, colloids, microfilters and / or a device for preventing backflow.

Is the surface z. B. positively charged, negatively charged organelles or molecules adhere to it. The electrostatic force increases with the charge of the organelle or the molecule. If, for example, the salt concentration in the sewer is increased, the length of the bye and the zeta potential decrease (Gouy-Chapmann theory). The less strongly charged organelles / molecules detach and can be removed by flow, for example. The highly charged organelles / molecules stick. In this way, organelles / molecules can be separated according to their charge. Thus, for example, DNA molecules can be purified if they form the most charged molecule and therefore remain the longest (with increasing ionic strength). The DNA can then be analyzed using known methods. Colloids of various sizes can be introduced into the channels. These can perform various tasks: purifying liquids; filtering out certain substances from liquids; the introduction of certain substances into liquids; the introduction of certain substances in the presence of specific substances in the liquid. It can be exploited that liquids can flow through the colloid barrier from different directions. In particular, colloids with a functionalized surface can be used.

Microfilters can be used to filter out certain substances. In a preferred development, the colloids can be used in conjunction with microfilters. The colloids can also be fixed by tapering a channel; the channel can be conical, for example. The colloids can e.g. can be fixed in the channel by membranes with a defined pore size or microfilter. It is also possible to arrange colloids of different sizes one after the other.

The device for preventing backflow enables corresponding channels to be used only for liquid transport in one channel direction. The device for preventing backflow can preferably be designed as a check valve.

According to an advantageous development of all of the microfluid systems described above, at least one channel and / or a reservoir can have at least one analysis substance. For example, an analysis substance can already be introduced into a channel during the manufacture of the microfluid system, so that later preparation of the microfluid system is simplified. The channel and / or the reservoir can preferably have an analysis substance in a shell, which dissolves only after a predetermined time or at predetermined environmental parameters.

In a preferred development of all the microfluid systems described above, at least one further channel can be provided in a microfluid system, which has a charged surface, solids, in particular colloids, microfilters and / or a device for preventing backflow. The different properties mean that different channels can perform different functions, for example when analyzing cells and / or cell compartments. According to an advantageous development of all the microfluid systems described above, these can have optically highly transparent regions and or optical components integrated into the substrate. This allows cells and / or cell compartments in the channels to be examined optically. In an advantageous development, the optically highly transparent areas or the integrated optical components can have a protective film. Such a film can prevent the areas or components from being scratched. The protective film can be removed before using the microfluid system.

In view of the above-discussed disadvantages a further object to provide a system for transporting fluids, in which liquids can be transported in a simple manner and without the microfluidic system requires an ex- ^'remote terminal.

This object is achieved by a system for transporting liquids in a microfluid system, comprising a microfluid system with a substrate, at least two reservoirs and at least one channel integrated in the substrate, which connects two reservoirs, and a device for pivoting the microfluid system about at least one axis in at least two directions. The microfluid system can advantageously be one of the microfluid systems described above.

This enables the targeted movement of liquids in a microfluid system without external connections or connections. After the substance to be examined has been introduced into the microfluidic system, it can be hermetically sealed. This represents a significant simplification and improvement in molecular and cell diagnoses, since in such a carrier analyzes, in particular long-term investigations, can be carried out without direct contact of the substances to be investigated with the outside world.

Swiveling is understood to mean rotating through an angle between 0 and 2π. The microfluidic system can be pivoted about an axis in two directions and / or about several axes in one direction. In an advantageous development, the microfluidic system can be periodically pivoted back and forth about an axis. The flow rate can be adjusted by the inclination of the substrate. In this way, liquids can be transported in the microfluid system due to gravitational force. The pivoting device is advantageously designed such that the microfluidic system can be pivoted about several axes. These pivots about different axes can in particular overlap; this can create a kind of tumbling movement of the microfluid system. In particular, a swiveling movement by an angle of less than 2π around a first axis can be superimposed with a complete circular rotation about a second axis. With a swiveling movement, in particular with a complete circular rotation, the centrifugal force with which the liquids are acted on can also be used for the liquid transport.

The swiveling movement allows liquids to flow through the same channel from different directions, depending on the inclination of the wearer. This is not possible when using a rotating base, in which the liquids are only moved by the centrifugal force. A chronological sequence of the flow direction and flow speed is also easy and precise to set. This is particularly advantageous when several liquids are brought together in a controlled manner.

The device for pivoting can, for example, comprise hydraulic devices with which the device is moved.

The device for pivoting can preferably have a device for fixing the microfluid system. This means that microfluidic systems are always arranged in the same place on the device. If the device thus performs certain pivoting movements automatically, examinations can be repeated with high accuracy.

According to the invention, a system for transporting liquids in a microfluid system is also provided, comprising a microfluid system with a substrate, at least two reservoirs and at least one channel integrated into the substrate, which connects two reservoirs, a first reservoir having a volume, and one at the first reservoir arranged device to pressurize the volume. The microfluid system is preferably one of the microfluid systems described above. Preferably, the device for pressurizing can be designed as an elastic membrane, which is arranged in the form of a cover on the reseπ / oir. The volume of the reservoir is pressurized by mechanical pressure on the membrane. The pressure can be applied, for example, by hand or using a microphone.

According to an advantageous development of the microfluid systems described above, the device for pressurizing can comprise a print cartridge or print cartridge.

According to an advantageous alternative, the device for pressurizing can have a device for heating and / or cooling the volume. By heating or cooling a gas in the volume, the gas expands or contracts, so that the volume has a positive or negative pressure is applied.

As described above, the microfluidic systems can have a plurality of channels and / or reservoirs. Several channels can open into a reservoir. The channels can also cross and / or split.

According to an advantageous development, the two previously described systems for transporting liquids can be combined with one another in a microfluid system.

The invention also provides a method for growing and / or analyzing cells, comprising the steps of: providing a microfluid system with a substrate, at least two reservoirs and at least one channel integrated into the substrate that connects two reservoirs, introducing at least one cell into at least one a channel, filling at least one reservoir with a liquid, and transporting the liquid through a channel connected to the filled reservoir by pivoting the microfluidic system about at least one axis in at least two directions. The microfluid system can be pivoted about an axis in two directions and / or about several axes in one direction. The microfluid system provided can preferably be one of the microfluid systems described above. In this way, cells can be grown in a microfluid system, for example, the nutrient medium supply and the product disposal being integrated into the microfluid system. By periodically swiveling the microfluid system back and forth, for example, liquid can flow through the channel from a respectively higher reservoir to a lower reservoir. The geometries of the channel and the reservoir can be coordinated so that there is significantly more liquid in the reservoirs than in the channel (typically a factor of 10-200). If toxic substances are formed in the channel or if the concentration of the gas dissolved in the liquid changes in the channel, this liquid can be directed into one of the reservoirs by tilting the carrier. Due to the large amount of liquid in the reservoir in relation to the channel, a corresponding dilution takes place there. Repeated seesaws guarantee the accumulation of toxic substances and the supply of nutrient media in the sewer. This allows cells to be kept alive in a channel system over a long period of time.

According to the invention, a method for growing and / or analyzing cells is provided, comprising the steps of: providing a microfluid system with a substrate, at least two reservoirs and at least one channel integrated in the substrate, which connects two reservoirs, introducing at least one cell into at least one channel Filling at least one reservoir with a liquid, and transporting the liquid through a channel connected to the filled reservoir by applying pressure to the liquid. The microfluid system provided can preferably be one of the microfluid systems described above.

According to an advantageous development, the previously described methods can be combined in one method.

According to an advantageous further development of the previously described methods, the filling of the reservoir also includes a filling with a gas. This ensures that a certain concentration of a certain gas or a gas mixture is present in solution in the liquid. The concentration of the gas in the liquid can be determined by the length of time the liquid remains in the reservoir become. A constant supply of dissolved gases in the liquid can be guaranteed by connecting the reservoirs with the corresponding gas.

By filling a reservoir, which is provided with a device for pressurizing, it can be achieved that there is a gas volume in particular above the liquid. Thus the device for pressurizing does not come into contact with the liquid and the pressurizing of the liquid takes place indirectly via the gas volume.

According to an advantageous development of the methods described above, a reservoir is filled with solids, in particular with colloids, and another reservoir with a liquid for functionalizing the solids. Through targeted filling. different reservoirs and subsequent swiveling of the chamber, the reservoirs filled in this way can be filled with liquid. The solids or parts of the solids located in the reservoirs can thus dissolve in the liquid. By swiveling again, the liquid with the dissolved substances can be led into a selected channel. This can e.g. serve to supply cells with nutrient medium in a chronologically defined sequence. The effect of various substances on cells can also be demonstrated in this way.

An advantageous method for the detection of molecules can comprise the steps: molecules in a first liquid can functionalize the surface of a colloid. Molecules of a second liquid bind to the functional groups on the colloid surface. A third liquid contains molecules which detach special components of the attached and / or functional group with attached molecule. In each of these sub-steps, certain reactions can be demonstrated.

The methods can preferably be used to separate and / or identify cell organelles (for example cell nucleus or mitochondria) or cell molecules (DNA, proteins). To do this, the cells in the substrate / channel are destroyed. This can be done, for example, by osmotic pressure, shear forces, compressed air, or special solvents. These methods are particularly easy to use in a microfluid system. The cell components released in this way can be filtered out, chemically bound, separated in the gel, electrophoretically moved and / or separated, immobilized or fixed on charged surfaces become. The components required for this can be introduced into the microfluid system or can be filled into the channel.

The methods can be used in particular for the following analysis methods: in-situ hybridization, FISH (fluorescence in situ hybridization) and CGH (comparative genomic hybridization).

In combination with temporally correlated pivoting movements, the above-described methods can be used to separate and analyze organelles and / or molecules in a microfluidic system with channels, reservoirs and the described components in a completely automated manner.

In a preferred development, all of the methods described above can be carried out in such a way that the microfluid system is a self-sufficient system due to the pivoting movements. This can be achieved by using the designs shown above. This ensures long-term supply to the cells. This enables an "instant test" on a cell basis. The carrier is preferably initially closed by a pull-off cover. This is opened in use so that the test can be carried out. This enables a storable test with living cells.

According to a preferred development of all the methods described above, these comprise the further step: fluorescence analysis of the cells and / or cell compartments. In this way, the cultivation and analysis up to the optical analysis can take place in a microfluid system.

Exemplary embodiments of the invention are explained in more detail below with reference to a drawing. It shows:

1 is a cross-sectional view of a microfluidic system with a channel and two reservoirs,

2 shows a cross-sectional view of a mirofluid system with a device for pressure equalization between two reservoirs,

3 shows a cross-sectional view of a channel with colloids arranged therein, and Fig. 4 is a cross-sectional view of a microfluidic system with two reservoirs with channel openings at different heights.

1 shows a microfluidic system with a substrate 1, two reservoirs 2 and 3 and a channel 4 integrated into the substrate 1. The orifices 5 and 6 of the channel 4 are arranged above base areas 7 and 8 of the reservoirs 2 and 3, respectively. Hollow cylinders 9 and 10 are formed on the base 7 and 8 within the reservoirs 2 and 3, through which the channel 4 is guided. The hollow cylinders 9 and 10 are arranged directly along the reservoir side walls 13 and 14 and merge into them. The orifices 5 and 6 are arranged at different heights, as seen from the base 7 and 8, so that, depending on the reservoir, a different angle of inclination of the microfluid system is required so that a liquid flows into the channel 4. The mouth-side surfaces 11 and 12 of the hollow cylinders 9 and 10 are chamfered so that the liquid does not splash upwards when flowing out of the mouth but flows to the side.

FIG. 2a shows a microfluid system, with cells 15 being arranged in the channel 4. The liquid reservoirs 2 and 3 each comprise liquid volumes 16 and 17 and - arranged above them - gas volumes 18 and 19. The liquid is a nutrient medium. Reservoirs 2 and 3 are hermetically sealed by covers 20 and 21. The reservoirs 2 and 3 are connected to each other by the cover via a gas channel 22, which enables gas exchange and thus pressure equalization. Toxic substances 23 arise during the cultivation of the cells 15.

The microfluidic system is swiveled to one side in order to dilute or remove the toxic substances from the cells and at the same time to supply nutrient medium. A microfluid system inclined in this way can be seen in FIG. 2b. Due to the inclination, liquid flows from the reservoir 2 into the lower reservoir 3. The toxic substances 23 are removed from the cells 15 and diluted in the reservoir 3; In addition, fresh nutrient medium is fed from the reservoir 2 to the cells. The gas from the reduced gas volume 19 flows through the gas channel 22 into the reservoir 2, whereby pressure equalization is created.

In an alternative embodiment, the channel system can be closed off with a movable membrane. This membrane can be placed in a cover, for example his. Pressing this membrane creates an overpressure on the corresponding side in the channel system and the liquid is moved in the channel. The pressure can be applied manually as well as transmitted via a microphone. By means of the transmission by means of a microphone, certain oscillation frequencies or superimpositions of oscillation frequencies of the pressure difference in the channel can be created. This can be used to determine the adhesive forces of particles, such as cells, on the inner walls of the channel system. The mixture of two substances can also be accelerated or the targeted transport of liquids can be carried out. This is particularly easy to implement in small channels. In a typical version, the microphone is attached directly above the carrier. In this way, a targeted movement of the liquid in the carrier can take place without a connection system.

In an advantageous alternative, the liquid can also be transported through the channel by applying an overpressure on at least one side of the channel. In one embodiment, the duct system is provided with individual plugs and / or a plug strip. These plugs can be connected to, for example, pressure cartridges via a hose system. A valve is advantageously attached between the print cartridge and the channel system. Opening the valve creates an overpressure in the duct system. The valve is advantageously an automatically controllable valve, for example a piezo valve. In a channel system with a plurality of channels connected to one another, a liquid can also be transported from a reservoir or channel simultaneously into several channels connected to it by pressurizing the reservoir or the channel. The most important advantages of such a design are, on the one hand, that there are no movable and therefore fault-prone components in the analyzer apart from the valve, and on the other hand that the sample substance only comes into contact with the carrier (reduction of the risk of contamination). Since only extremely low pressures (mm-cm water column) have to be used, the service life of a print cartridge used in this way is very long. In a further development, it is possible to use a pressure reducer between the print cartridge and the carrier system. This is advantageously also electrically controlled. The position of the sample substance can be determined by measuring the pressure applied. In a typical analysis protocol, the sample substance is first inserted into the carrier in this embodiment. filled and then the sample holder connected to the connections via which the overpressure can be applied.

Various colloid arrangements in a channel 4 are shown in FIG. 3. In FIG. 3a, the channel comprises colloids 24, which are fixed by a microfilter 25. 3b, the channel 4 tapers in the direction of flow. Large colloids 24 accumulate before the narrowing. Smaller colloids 26 are then arranged behind them, as a result of which a colloid barrier is formed from colloids of different sizes. The colloid barrier in FIG. 3 c only comprises large colloids 24, which are fixed before a narrowing.

Substances (analysis substances) are used in almost all analytical or diagnostic tests, with the help of which the ones to be analyzed. Substances are characterized, purified, duplicated, detected and / or neutralized / killed, among other things. Examples are buffers, passivation buffers, fluorescent molecules, radioactive molecules, marker molecules, proteins or whole cells. In a preferred further development, substances which are required for the analysis of the sample are already in the channel system (analysis substances). These can be solid, liquid or gaseous. In particular, the analysis substances can be dried in the sewer system. The analysis substances can be located directly in a channel or in a reservoir which is connected to the channel. A water-soluble barrier layer can be located between the reservoir and the channel. This has the advantage that certain substances can only act in the reaction and / or analysis area of the channel system after a certain time. The analysis substances can also be enclosed in shells which only dissolve after a certain time or with certain parameters (e.g. pH value, pks value) (as with certain medications). In this way, analysis processes can be carried out in a certain chronological order without external control, such as a solvent exchange. The carriers or microfluidic systems can be delivered with the already integrated substances.

4a shows a cross section of a microfluid system with a substrate 1, in which two reservoirs 27 and 28 are arranged one above the other. The reservoirs can be accessible from the side in that a further channel (not shown) opens into the side of each reservoir. The bases of the reservoirs are inclined relative to the side surface, the angle of inclination being different. If the microfluidic idsystem pivoted by a first, smaller angle (Fig. 4b), only liquid in the lower reservoir 27 reaches the channel mouth and flows into the channel. At a greater angle of inclination of the microfluid system (FIG. 4c), the liquid from the second reservoir 28 reaches the channel mouth and can flow through this channel.

Claims

claims

1. Microfluidic system with a substrate, at least two reservoirs and at least one channel integrated into the substrate, which connects two reservoirs, the mouth of at least one channel being arranged above the base area of a reservoir.

2. The microfluidic system of claim 1, wherein the microfluidic system is closed and comprises a pressure compensation device that connects each reservoir to at least one other reservoir.

3. Microfluid system according to claim 1 or 2, in which at least one channel has a charged surface, solids, in particular colloids, microfilters and / or a device for preventing backflow.

4. Microfluid system according to one of the preceding claims, in which at least one further channel is provided which has a charged surface, solids, in particular colloids, microfilters and / or a device for preventing backflow.

5. Microfluidic system according to one of the preceding claims, in which at least one channel and / or a reservoir has at least one analysis substance.

6. Microfluidic system according to one of the preceding claims, in which the substrate has at least one optically highly transparent region and / or at least one integrated optical component.

7. The microfluidic system according to claim 6, in which each optical highly transparent region and / or each integrated optical component has a protective film.

8. System for transporting liquids in a microfluidic system a microfluid system, in particular according to one of the preceding claims, with a substrate, at least two reservoirs and at least one channel integrated into the substrate, which connects two reservoirs, and

a device for pivoting the microfluidic system about at least one axis in at least two directions.

9. System for transporting liquids in a microfluidic system

a microfluid system, in particular according to one of claims 1-7, with a substrate, at least two reservoirs and at least one channel integrated into the substrate, which connects two reservoirs, a first reservoir having a volume, and

a device located on the first reservoir to pressurize the volume.

10. A method for growing and / or analyzing cells comprising the steps:

Providing a microfluidic system, in particular according to one of claims 1-7, with a substrate, at least two reservoirs and at least one channel integrated into the substrate, which connects two reservoirs,

Introducing at least one cell into at least one channel,

Filling at least one reservoir with a liquid, and

Transporting the liquid through a channel connected to the filled reservoir by pivoting the microfluidic system about at least one axis in at least two directions.

11. Method for growing and / or analyzing cells with the steps:

Introducing at least one cell into at least one channel, Filling at least one reservoir with a liquid, and

Transporting the liquid through a channel connected to the filled reservoir by pressurizing the liquid.

12. The method of claim 10 or 11, wherein filling the reservoir further comprises filling with a gas.

13. The method according to any one of claims 10-12, wherein a reservoir with a solvent for cell destruction and / or a reservoir with a liquid for cleaning Molecules is filled.

14. The method according to any one of claims 10-13, wherein a reservoir is filled with solids, in particular colloids, and a further reservoir with a liquid for functionalizing the solids.

15. The method according to any one of claims 10-14 with the further step:

Fluorescence analysis of the cells and / or cell compartments.