EP2624954B1 - Method for washing a microfluid cavity - Google Patents

Description

The invention relates to a microfluidic component: according to claim 1 for washing a cavity in the microfluidic component.

In recent years, biotechnology and genetic engineering have grown in importance. A basic task of this technology is the detection of biological molecules such as DNA (deoxyribonucleic acid) or RNA (ribonucleic acid), proteins, polypeptides, etc. For many medical applications, molecules in which genetic information is encoded are of particular interest. Their detection, for example in a blood sample from a patient, enables pathogens to be detected, which makes it easier for the doctor to make a diagnosis.

In biotechnology and genetic engineering, microfluidic components or microfluidic cartridges are increasingly used.

Microfluidic cartridges are used in a variety of ways in the form of single-use tests, with so-called lateral flow cartridges being used as a rule, the components of which have length and width measurements that are a few millimeters to centimeters.

To carry out tests, an analysis fluid to be tested (e.g. blood, urine or saliva) is fed to a cartridge equipped with a biosensor. The sample is added to the cartridge before or after the cartridge is inserted into an analysis device. The analyte is added in an opening in the cartridge, with the liquid being fed through microchannels to corresponding sample preparation chambers and sample examination chambers.

The term "micro" is intended to imply that the channels and / or cavities (chambers) have a dimension on the micrometer scale at least in one geometrical direction of extent, ie. H. the dimensions in at least one dimension are less than one millimeter.

The term “microfluidic” is understood to mean that a pressure-induced and / or capillary liquid flow occurs through and in the microchannels and / or microcavities.

The term “microfluidic component” is understood to mean a component which has at least such microchannels or microcavities for the storage and transport of liquids or fluids and gases.

The term “microfluidic cartridge” is understood to mean a device (possibly consisting of several microfluidic components) for the analysis of liquids.

The detection of low concentrations of biological and inorganic substances in biological samples is often difficult. The tests (assays) for this type of detection in microfluidic cartridges are usually associated with several process steps, which include the binding of a primary antibody, multiple washing steps, the binding of a second antibody, further washing steps, and (depending on the type of detection system ) If necessary, also include enzymatic and washing measures.

The number of steps that are usually required when using such microfluidic cartridges to obtain a desired, specific signal are time-consuming and labor-intensive. The need for modern microfluidic cartridges, however, aims to shorten the measurement time between the application of the sample liquid and ultimately the appearance of the measured value. This time is lengthened by frequent washing steps, which, however, are mostly desirable and necessary in order to increase the sensitivity and reduce background values.

In a washing step of a chamber, a liquid previously introduced into the chamber (for example reaction liquid) is usually washed out by a washing liquid introduced into the chamber immediately thereafter. Specifically, a quantity of washing liquid is passed through the chamber, the liquid to be washed from the chamber mixing with the washing liquid (diffusion) and being removed from the chamber with the washing liquid.

Since the washing process in a microfluidic system usually takes place in the form of a laminar flow with no significant turbulent component, the liquid to be washed away is not sufficiently captured by the washing liquid, especially in the corner areas of chambers. This leaves residues in the chamber. This usually requires a multiple repetition of washing steps, which, however, is counterproductive in terms of achieving the shortest possible measurement time. This also drives demand Washing liquid and thus also the space required for reservoir and waste increase, which is undesirable in a volume-minimized microfluidic system.

From the DE 697 37 857 T2 for example, it can be seen that the need for multiple washing steps is known in the art and is considered to be time consuming and labor intensive.

Also the DE 601 31 662 T2 it can be seen that washing steps are often necessary, but extend the measurement time with microfluidic cartridges.

The US 2008/0069739 A1 describes a software control for chemical process systems in which internal elements are automatically cleaned with solvents or drying gases, which means that devices can be reused. One focus here is on flexible, re-configurable, reusable multi-purpose "lab-on-a-chip" systems that can be reconfigured using software. Schematically controllable valve subsystems are discussed, by means of which reactants can be selected and directed to controlled reaction chambers, whereby the reaction chambers and transport lines can be cleaned of solvents and gases by pumping through them. For example, valve complexes with several inlets (e.g. for two sample liquids / reactants, liquid solvent and gas) and one outlet are presented. The valves in such a complex are, for example, two-way valves and are formed by microvalve structures. By energetic control of the valves, selected reactants, cleaning solvents and gas for drying are released from the outlet of the valve complex. Here, gas can be used both for the final drying and for a "clearing" between the passage of a reactant and a flushing solvent.

The US 2007/0207063 A1 shows a device for controlling a fluid sequence running into (and out of) an integrated work area. The device comprises a base plate on which a first fluid channel with an inlet and an outlet is arranged. A plurality of valve elements are arranged in the channel in order to divide it into several segments. The segments for their part are connected to injection tanks and the valve elements to venting tanks, these tanks being "open-type" in contrast to the duct and valves, ie having a side that is open at the top. The individual working fluids are each injected into an inlet tank connected to the inlet and the injection tanks and flow by capillary force to the valve elements, which form a pressure barrier. The injection tanks and the ventilation tanks are closed and the pressure barriers of the valves are overcome by pressure at the inlet tank or by negative pressure at the outlet, excess air trapped in the duct is pressed into the ventilation tanks and the flow of the fluid sequence in the duct into or through the work area is started.

The US 2004/0063217 A1 shows a fluidic miniature cassette made of plastic, which contains a reaction chamber with a multiplicity of immobilized species, a capillary channel and a pump structure with an external actuator. Various reservoirs (one for washing liquid, one for rinsing air, one for solution and one for an antibody conjugate) are connected to the pump structure, so that the contents can be pumped from the connected reservoir into the reaction chamber.

The US 2008/038839 A1 shows a method of sequentially feeding liquid or gaseous fluids (reagents, solvents, reactants) to a chemical, biological or biochemical process or a reaction site (specific example: immunoassay method). Rinsing liquids can be used intermittently to remove unwanted reactant residues or to prepare the reaction site. Different fluids are stored in the same vessel, a third fluid separating a first from a second fluid. As an example of such a vessel, a tube (or tube) is given, which sequentially contains a reagent, an air cushion, a rinsing liquid, a further air cushion and a second rinsing liquid, the respective fluids regionally depending on the size and material of the tube or are arranged one behind the other like a plug and the air cushions prevent the liquids from contacting one another. Such a vessel with plug-like fluid sections is fluidically connected to a reaction site (for example a microfluidic immunoassay) and the fluids are pressed to the reaction site by means of a pump, syringe or other pressure source or drawn to the reaction site by means of negative pressure.

The invention is based on the object of providing a microfluidic component for washing a cavity in the microfluidic component.

According to the invention it is now provided that a gas is supplied to the cavity before the washing liquid is supplied. This “prewashing” makes it possible to significantly reduce the need for washing liquid to be subsequently supplied, which is necessary in order to bring about a desired reduction in the residual concentration of the liquid to be washed out in the cavity. The need for washing liquid can therefore be reduced and, under certain circumstances, a reduction in the washing time or washing steps is also possible.

It is very useful here if the gas is passed through the cavity in the form of a bubble, that is to say with a defined volume. This enables the method to be implemented in a microfluidic component or a microfluidic cartridge even without an external gas connection, so that, for example, the gas bubble with a defined volume can be provided in a cavity of the microfluidic component itself.

With regard to the necessary reduction of space and material requirements, it is very useful if the gas bubble has a volume which is smaller than the volume of the cavity. Of course, the volume should still be large enough for efficient washing.

It is therefore expedient to select approximately 40% to 60%, preferably approximately 50% of the volume of the cavity to be washed out as the volume of the gas bubble. This significantly reduces the need for gas to be stored, but is still completely sufficient to achieve the desired functionality or effect.

The gas bubble spreads continuously when it is introduced into the cavity to be washed by means of excess pressure and immediately becomes so wide that it touches the side walls of the cavity. It can thus displace a large part of the liquid to be washed out, located in the cavity, through an outlet opening to be provided in the cavity. Subsequent washing liquid in turn displaces the gas bubble in the direction of the outlet opening. The gas bubble works like a barrier layer between the first liquid to be washed out and the subsequent washing liquid. Finally, the gas bubble is pressed completely out of the cavity by the washing liquid.

Because a very high percentage of the liquid to be washed out has already been displaced from the cavity by the gas bubble, the washing liquid can easily absorb any remaining, small residual portion of liquid to be washed out by diffusion and carry it out of the cavity as it is transported further. Under certain circumstances, a single washing step is sufficient to achieve a desired residual concentration.

Although, of course, many gases (such as nitrogen or noble gases) are possible, air is very useful as a gas to be used because it is inexpensive and technically simple to provide.

Under certain circumstances it can be useful if the supply of gas or air and subsequent liquid for washing is repeated several times.

As already mentioned, the invention would also like to provide a microfluidic component for carrying out the washing process.

The invention is based on a microfluidic component containing at least one first cavity which is filled with a liquid for washing at least one second cavity and means for establishing a fluidic connection between the at least one first and the at least one second cavity.

According to the invention, at least one further cavity, which is filled with a gas, is now arranged between the first and the second cavity, viewed in the direction of flow of the liquid.

If the cavity containing the washing liquid is now pressurized, the washing liquid flows in the direction of the cavity containing the gas and, if necessary, only pushes the gas bubble into the cavity to be washed after releasing a corresponding fluidic connection (for example by means of corresponding valves) .

To reduce the space requirement for the cavity to be kept or to reduce the gas requirement, it is very useful if the at least one further cavity filled with gas has a volume which is smaller than the volume of the at least one second cavity to be washed. This is because it has been shown that a significantly smaller gas volume than the volume of the cavity to be washed is already sufficient to achieve the desired effect.

It has proven to be very advantageous if, viewed in the direction of flow of the liquid, at least one valve is connected upstream and at least one valve is connected downstream of the gas-filled cavity. In this way, undesired gas or liquid flows can be excluded. It is very advantageous if the valves can be controlled. As a result, the flow of the liquid or gas can be controlled even better, which among other things can also reduce the risk of undesired bubble or foam formation. Control can preferably be carried out by means of electrical signals or pulses.

To increase the washing efficiency, it is also conceivable to design the cavity to be washed in such a way that it has a first section in the direction of flow in which its cross section continuously expands and a second section in which the cross section of the cavity tapers again continuously. A section with a constant cross section is then expediently arranged between these sections with a changing cross section. The first section should expediently be arranged in the area of the inlet opening and the second section in the area of the outlet opening, viewed in the direction of flow.

There can be applications in which it is advantageous if the cavity filled with gas can be fluidically connected to at least one further gas reservoir. Here too, a controllable valve can expediently be provided for releasing or interrupting a fluidic connection.

In this way it is possible to repeat the steps described (introduction of a gas bubble into the cavity to be washed - pressing out the gas bubble by subsequent washing liquid) several times with the microfluidic component or the microfluidic cartridge, if necessary.

Air is expediently used as the gas here, too, and the ambient air can be used as a further gas reservoir.

Fig. 1

eine prinziphafte Draufsicht auf einen Teil eines mikrofluidischen Bauteils gemäß einer ersten Ausführungsform,

Fig. 2

eine prinziphafte Draufsicht auf einen Teil eines mikrofluidischen Bauteils gemäß einer zweiten Ausführungsform,

Fig. 3a

eine prinziphafte Einzeldarstellung einer Kavität, welche gewaschen wird, in einer ersten Ausführungsform,

Fig.3b

eine prinziphafte Einzeldarstellung einer Kavität, welche mit einer Waschflüssigkeit gewaschen wird, in einer zweiten Ausführungsform,

Fig. 4

eine prinziphafte Darstellung des Verfahrens am Beispiel einer Kavität gemäß Fig. 3b.

Further advantages and refinements of the invention become clear on the basis of exemplary embodiments, which will be explained in more detail with the aid of the accompanying figures. Mean

Fig. 1

a basic plan view of part of a microfluidic component according to a first embodiment,

Fig. 2

a basic plan view of part of a microfluidic component according to a second embodiment,

Fig. 3a

a basic individual illustration of a cavity which is washed, in a first embodiment,

Fig.3b

a basic individual representation of a cavity which is washed with a washing liquid, in a second embodiment,

Fig. 4

a basic illustration of the method using the example of a cavity according to Figure 3b .

In Fig. 1 a section of a microfluidic component 1 can be seen. In concrete terms, several microfluidic functional elements can be seen which, for the sake of illustration, have one microfluidic function group 90 (outlined by dashed lines) are to be assigned. The microfluidic functional group 90 comprises a first, preferably circular, chamber 10 filled with washing liquid F2. Furthermore, a second, approximately rectangular chamber 20 can be seen, which is filled with a liquid F1.

The liquid F1 has triggered a specific detection reaction in the chamber 20. Some of the biomolecules contained in F1 are bound in the chamber 20. The remainder of F1 is now to be washed out of the chamber 20 with the washing liquid F2. The chamber 20 can be, for example, a PCR chamber (PCR = polymerase chain reaction). The type of detection reaction caused in the chamber 20 by the liquid F1 is, however, of no further importance for understanding the invention and therefore does not need to be explained further.

A further chamber 30, which is filled with air L in the exemplary embodiment, is arranged between the chamber 10 and the chamber 20. Instead of air, other gases, for example nitrogen or the like, can of course also be used. The chambers 10, 20 and 30 are fluidly connected to one another by means of microchannels 40, with a preferably electrically controllable valve 50a or 50b being provided between the chambers 10 and 30 or 30 and 20, with which the fluidic connection can be released or interrupted .

Furthermore, a further microchannel 80 is provided with which the fluidic connection from the chamber 20 to other, not shown microfluidic functional elements, e.g. a waste area can be produced.

From the Fig. 1 it can also be seen that the air-filled chamber 30 is connected to a microchannel 60. The microchannel 60 establishes a fluid connection between the chamber 30 and a further gas reservoir. Here, too, the fluidic connection can be interrupted or released by means of a preferably electrically controllable valve 70. The mentioned gas reservoir itself can be realized by one or more further cavities or chambers (not shown).

When using air in the chamber 30, it is advisable to also fill the gas reservoir accessible via the microchannel 60 with air or merely to provide access to the ambient air or to an air pump (not shown) via the microchannel 60.

A film, preferably bonded to the component 1, for covering or sealing the aforementioned chambers and channels is not shown or numbered. The component 1 itself is a plastic plate, which is preferably manufactured by injection molding.

To initiate a washing process, the chamber 10 in the application example is now subjected to a pressure of approximately 0.4 bar to 0.8 bar. This is preferably done by means of suitable actuators of a microfluidic cartridge into which the component 1 is installed (not shown).

At the same time as the application of pressure, the valves 50a and 50b are activated, which thus release the fluidic connection between the chambers 10, 20 and 30. As a result of the pressure build-up, the washing liquid F2 is now pressed in the flow direction S into the chamber 30 and also pushes the air L located in the chamber 30 in front of it in the flow direction S, in the direction of the chamber 20 first the air L is pressed in in the form of a defined air bubble. This leads to a very efficient "prewashing" of the chamber 20. Specifically, a large part of the liquid F1 located in the chamber 20 is already displaced by the air L, so that the washing liquid F2 following the air bubble L only contains the remaining liquid F1 must remove from the chamber 20.

In this way, at least the requirement for washing liquid F2 to be kept available, which is necessary for generating a required residual amount of liquid F1 that is to remain in the chamber 20 as a maximum, can be significantly reduced.

If a single washing process is not sufficient, it is conceivable to repeat the described washing process in the desired number. The valve 50a is closed again for this purpose. The valve 70 is then opened and a fluidic connection between the chamber 30 and the air reservoir mentioned is released.

In this way, the chamber 30 can be filled with air L again, for example by a pump. After valve 70 is closed, valve 50a is opened again and pressure is built up on chamber 10, as already described. If necessary, the size and shape of the chamber 10 can be varied as required. Several chambers 10 are also conceivable, each of which is assigned to a washing step.

The washing process in chamber 20 is described below in connection with FIG Fig. 4 explained in more detail.

In Fig. 2 Another embodiment 1 'of a microfluidic component according to the invention is now shown in principle. In contrast to the execution according to Fig. 1 If the microfluidic component 1 ′ has a plurality of microfluidic functional groups 90 (as in FIG Fig. 1 described). Correspondingly, several further microchannels 80 are also provided. For example, they can be connected to a shared waste area.

This embodiment 1 'can serve, for example, to combine the reaction and washing steps to be carried out in the functional groups 90, to cascade them or to run several assays at the same time.

Now in Fig. 3 two possible geometries of the chamber 20 to be washed are shown, although other geometries are of course also conceivable. The chamber geometry according to Figure 3b represents according to the geometry Fig. 3a represents an improvement in terms of washing efficiency and can expediently be combined with the method according to the invention.

In Fig. 3a it can be seen that the chamber 20 as in FIG Fig. 1 shown, is formed. It thus has an approximately rectangular plan in plan view, the inlet (microchannel 40) and the outlet (microchannel 80) also being recognizable. The chamber 20 is already penetrated by washing liquid F2 in the flow direction S here.

The diagonal arrangement of the inlet and outlet (40 and 80) in the direction of flow S can improve the washing efficiency somewhat, but significant residues of liquid F1 are unavoidable in the corner areas not assigned to the inlet or outlet, since this method of washing diagonally leaves out the opposite corners.

An improvement in washing efficiency simply by redesigning the chamber geometry is in Figure 3b shown.

A chamber 20 ′ can be seen therein, which has an inlet opening 21 and an outlet opening 22 in the direction of flow S. A first section 23 with a continuously widening cross section of the chamber 20 'adjoins the inlet opening 21. Concrete In this section 23, in plan view, the opposite walls of the chamber 20 ′ diverge in a V-shape. The section 23 is followed by a section 24 with a constant cross section of the chamber 20 '. The opposing walls of the chamber 20 ′ here run approximately parallel. The section 24 is in turn adjoined by a section 25 in which the cross section of the chamber 20 'is continuously reduced. The opposite walls of the chamber 20 'run towards one another in the direction of the outlet opening 22 in a V-shape.

The chamber geometry is thereby optimized with regard to the flow course of the washing liquid F2. Nevertheless, certain residues of liquid F1 to be washed away in the corner areas are unavoidable here as well.

Fig. 4 now shows in detail how the process leads to a significant improvement in washing efficiency:
The chamber 20 'is initially filled with the liquid F1 to be washed away ( Figure 4a ). After the washing process has been initiated (as described above), the air bubble L previously driven by the washing liquid F2 is first pressed into the chamber 20 ', specifically in the area of the inlet opening 21 ( Figure 4b ), until the entire air bubble L has been pushed into the chamber 20 '( Figure 4c ). It can be seen that the air bubble L spreads outward very quickly in the direction of the side walls of the chamber 20 ′ and forms contact areas 26 with them.

As the washing process continues, the washing liquid F2 following the air bubble L penetrates into the chamber 20 '( Figure 4d ). Due to the air bubble L or the contact areas 26, there is on the one hand a very good displacement of the liquid F1 in the direction of the outlet opening 22 and, on the other hand, a very good separation between the liquid F1 and the subsequent liquid F2.

There is therefore essentially no diffusion between the liquid F1 and the subsequent liquid F2, with the exception, if appropriate, of residual amounts of liquid F1 still remaining (extremely small) behind the contact areas 26, viewed in the direction of flow S.

In particular to the Figures 4d and 4e it can be seen that the size of the air bubble L in no way has to correspond to the volume of the chamber 20 '. It should only be ensured that the defined amount of air L in the chamber 30 is so large that an air bubble L can be generated which is so large that they form the mentioned contact areas 26 with the chamber 20 'and thus quasi as a barrier layer between the liquid F1 and the subsequent liquid F2 can serve.

In Figure 4e it can be seen that the air bubble L, which in turn has been displaced by the subsequent liquid F2 in the direction of the outlet opening 22, has displaced a very high percentage of the liquid F1 from the chamber 20 '.

The Figures 4f and 4g it can be seen that the air bubble L is pressed into the outlet opening 22 by the subsequent liquid F2 and finally only the liquid F2 is located in the chamber 20 '. With the washing liquid F2, only an extremely small residue of the liquid F1 remaining in the chamber 20 'to be washed away then has to diffuse with the washing liquid F2.
As a result, the requirement for washing liquid F2, which is necessary in order to achieve the required residual concentration of liquid F1 in the chamber 20 ', can be significantly reduced. The low residual concentrations of liquid F1 that can be generated can therefore be flushed out within a short time by the washing liquid F2 flowing in.

In the present exemplary embodiment, good results could be achieved with a chamber geometry of the chamber 20 'of approximately 32 mm ² in plan, combined with a height of a few hundred μm at volume flows of approximately 4 μl / sec. Volume flows from 2 µl / sec to around 10 µl / sec could be achieved. A pressure of approximately 0.4 bar has proven to be extremely useful as the initial pressure for triggering the washing process, although significantly higher pressures of up to approximately 0.8 bar have also been used.

List of reference symbols

1, 1 '

Microfluidic component

10

first chamber for receiving washing liquid

20, 20 '

second chamber with liquid to be washed away

21st

Entrance opening

22nd

Exit opening

23

first section with widening cross-section of the chamber

24

Section with constant cross section of the chamber

25th

second section with a decreasing cross section of the chamber

26th

lateral contact areas of the air bubble with the wall of the second chamber

30th

another chamber for the intake of air

40

Microchannels

50a, b

controllable valves

60

Microchannel

70

controllable valve

80

Microchannel

90

microfluidic functional group

F1

liquid to be washed away

F2

Washing liquid

L.

Air or air bubble

S.

Direction of flow

Claims (6)

1. Microfluidic component (1, 1'), containing at least one first cavity (10), which is filled with a liquid (F2) for washing at least one second cavity (20, 20'), and means (40, 50a, 50b) for providing a fluidic connection between the at least one first (10) and the at least one second cavity (20, 20'), wherein, viewed in the direction of flow (S) of the liquid (F2), at least one further cavity (30) is arranged between the first (10) and the second cavity (20, 20'), cavity (30) being filled with a gas (L),
   wherein the cavity (20') to be washed comprises, in the direction of flow (S), a first section (23) in which the cross-section of the cavity (20') widens out continuously and a second section (25) in which the cross-section of the cavity (20') tapers continuously and wherein a section (24) of constant cross-section is arranged between the sections (23 and 25) of varying cross-section and
   wherein the at least one further cavity (30) filled with gas (L) has a volume that is smaller than the volume of the at least one second cavity (20, 20') to be washed,
   characterised
   in that a pressure can be applied to the first cavity (10) so that the liquid (F2) for washing the second cavity (20,20') flows in the direction of the cavity (30) containing the gas and pushes the gas as a gas bubble along in front of it, into the cavity (20') that is to be washed, wherein the gas bubble is so large that, when pushed into the cavity (20') that is to be washed, it can form, in the section (24) of constant cross-section, contact regions (26) with the side walls of the cavity (20') that is to be washed, and
   in that, viewed in the direction of flow (S) of the liquid (F2), at least one valve (50a) is connected upstream of the cavity (30) filled with gas (L) and at least one valve (50b) is connected downstream of the cavity (30) filled with gas (L), wherein the valves (50a, 50b) are actuatable.
2. Microfluidic component (1, 1') according to claim 1, characterised in that the cavity (30) filled with gas (L) is fluidically connectable to at least one further gas reservoir.
3. Microfluidic component (1, 1') according to claim 2, characterised in that the fluidic connection can be provided by an actuatable valve (70).
4. Microfluidic component (1, 1') according to one of claims 1 to 3, characterised in that the gas (L) is air.
5. Microfluidic component (1, 1') according to one of claims 1 to 4, characterised in that the volume of the gas bubble is about 40 % to 60 % of the volume of the cavity (20, 20') that is to be washed.
6. Microfluidic component (1, 1') according to one of claims 1 to 5, characterised in that a liquid which is to be washed out is contained in the second cavity (20, 20').

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