WO2013072110A1

WO2013072110A1 - Microfluidic filter element for separating sample components from a biological sample fluid

Info

Publication number: WO2013072110A1
Application number: PCT/EP2012/068901
Authority: WO
Inventors: Martina Daub; Thomas BRETTSCHNEIDER; Christian Dorrer
Original assignee: Robert Bosch Gmbh
Priority date: 2011-11-14
Filing date: 2012-09-26
Publication date: 2013-05-23
Also published as: DE102011086235A1

Abstract

The present invention relates to a microfluidic filter element for separating sample components from a biologic sample fluid, said filter element having at least one substrate, various channels, at least one filter chamber arranged in the substrate and containing a filter material, and, immobilised on the filter material, capture molecules which the filter material is interspersed with.

Description

Description title

Microfluidic filter element for separating sample components from a biological sample fluid

State of the art

The present invention relates to a microfluidic filter element for

Separating sample components from a biological sample fluid.

For the diagnosis and choice of the correct therapy of many diseases, the detection of certain sample components, e.g. Pathogenic cells, bacteria, viruses, endotoxins or other, the detection methods accessible molecules, from different sample fluids such as blood, urine or sputum essential. In the past, for the detection of cells, e.g. used by bacteria, mostly microbiological processes. However, these cell-culture-based determinations are very time-consuming procedures, since the cultivation of the cultures and their evaluation require two to three days. In order to obtain reliable results, it is also a necessary condition that the to be detected

Components of the sample fluid can be separated from this. However, the often low concentration of these ingredients is one

Challenge in isolation.

In order to separate the constituents to be detected, the prior art uses a multiplicity of different filters which, on the one hand, are selected depending on the size of the constituents to be separated on the one hand and the remaining constituents contained in the sample fluid on the other hand. The size of the components of the sample to be detected must therefore be sufficiently different from the remaining components of the sample liquid. In the case of bacteria in whole blood, this requirement is not met because the size of the bacteria is comparable to the size of the cellular components of the blood. One way to circumvent this problem is to use microchannel-based separation techniques, which are the different hydrodynamic ones

Exploiting properties of, for example, erythrocytes and bacteria. Another known possibility of filtration is provided by analyte-specific binding molecules which are immobilized on various filter materials. GB-2401942 A discloses a filter element for use in a

Detection assay in which specific binding molecules are immobilized on the surface of a porous membrane. The desired target components are trapped using the binding molecules and thus fixed on the underside of the membrane.

Another possibility is the separation of specific sample components from a sample fluid with the help of functionalized beads of a few

Micrometer diameter. For this purpose, for example, antibodies are immobilized on the bead surface. After addition of the beads to the sample fluid, they bind to the associated antigens and in this way collect the desired sample constituents, such as, for example, pathogenic cells. The separation of the beads including the pathogenic cells from the sample fluid is then carried out by filtration or, in the case of magnetic beads, by external magnetic fields.

Disclosure of the invention

The subject of the present invention is thus a microfluidic

Filter element for separating certain sample components from a biological sample fluid, comprising

at least one substrate,

at least one filter chamber arranged in the substrate,

at least one channel disposed in the substrate for supplying the biological sample fluid and at least one in the substrate

arranged channel for deriving the remaining after the filtering process components of the biological sample fluid,

arranged in the filter chamber filter material and

filter molecules immobilized on the filter material for the particular sample components, wherein the filter material is interspersed with the capture molecules. The specific capture molecules such as antibodies or enzymes, but also polynucleic acids, polymers, etc. are on the surface of the

Filter material immobilized so that the desired sample components are bound to the capture molecules when flowing through the sample liquid. In this case, the deposition of the sought sample components is distributed over the entire filter material, since the filter material is interspersed with the capture molecules and thus faces the sought sample components an extremely high number of possible binding sites. Because of this property, the filter element of the invention operates extremely efficient and highly specific. The pores of the filter material are dimensioned so that components of the sample liquid, which do not interact with the catcher molecules, can pass the filter substantially. A mechanical filtration through the pure filter material therefore plays a minor role.

The term "certain sample constituents" is understood to mean all desired specific constituents of the biological sample fluid which can be purposefully separated with the aid of the microfluidic filter element. These are in particular both pathogenic and non-pathogenic cells or viruses and their constituents, such as, for example, endotoxins, nucleic acids, proteins or other cellular constituents.

With the microfluidic filter element according to the invention, the sample components can be separated very efficiently and highly specifically from a biological sample fluid. The size of the to be filtered

Sample components and the remaining components of the sample be comparable. This is partly due to the fact that a mechanical filtration is completely dispensed with and thus the pores of the filter can be chosen to be relatively large, so that a much higher throughput is possible. In addition, due to the large pores, filtration of large quantities of sample fluid is possible without the filter tending to clog. The

Microfluidic filter element according to the invention thus represents a clear further development of known microfluidic filter systems, since they were hitherto only suitable for low throughput quantities. Likewise, the use of beads can be dispensed with. The use of immobilized capture molecules that permeates the entire filter material enables highly specific filtration compared to conventional filters or methods based on hydrodynamic effects. On a dilution of the sample fluid in advance of the filtration can be dispensed with.

The absence of beads also saves further process steps for mixing or separating the beads with or from the sample fluid and the associated additional use of filters or external magnetic fields. This results in enormous cost savings compared to

conventional bead handling systems.

The binding of the particular sample components to the capture molecules may be accomplished by suitable solutions, e.g. appropriate buffer solutions are repealed. In this case, the filter element can be rinsed both with the original filter flow and in the opposite direction. In contrast, other buffer solutions that allow the specific binding of the desired

Do not affect sample components on the capture molecules, flush over the filter without rinsing out the desired sample components.

This allows a further purification of the sample components can be achieved.

The term substrate is understood in the following to mean a carrier material in which the individual constituents of the microfluidic filter element are arranged. The microfluidic filter element has at least one filter chamber arranged in the substrate, which forms a cavity in which the actual filter material is arranged. This filter material in turn is with the immobilized capture molecules to bind the desired

Interspersed with sample components. To the filter chamber at least one channel disposed within the substrate for supplying the biological sample fluid and at least one channel for discharging after the filtering process

remaining components of the biological sample fluid. In one embodiment of the microfluidic filter element, this is composed of a plurality of superimposed substrates in which at least one filter chamber and the channels for supplying the biological sample fluid and for deriving the remaining after the filtering process

Components of the biological sample fluid at least partially in

different substrate layers are arranged and the filter material is flowed transversely.

By the construction of several substrate layers, a transverse onflow is only possible because the channels for supplying the biological

Sample fluids and for diverting the remaining components of the sample fluid at different levels, so that the intervening

Filter material in the filter chamber can be flowed transversely over a large area. The multi-layer substrate structure is also advantageous in the production, since the individual chambers and channel components can be better matched by their arrangement in different layers.

In a further embodiment, the microfluidic filter element is constructed from a substrate having at least one filter chamber and channels for supplying the biological sample fluid and for deriving the remaining components of the biological sample fluid after the filtering process, and the filter material is flowed laterally. This simplified embodiment allows working in a plane, which may be advantageous for various applications in the microfluidic area.

In a further embodiment, the microfluidic filter element has at least one further channel, at least one valve and / or at least one collecting chamber for the remaining components of the biological sample fluid.

As already mentioned above, the further channels are used, for example, for rinsing with buffer solutions or for eluting the bound sample components. The valves regulate the flow rates within the channels. For this purpose, individual or all channels can be equipped separately with valves. Since the filter effect does not depend crucially on the flow rate, it is possible for generating the flow use cheap, integrated pumps, such as peristaltic pumps that produce, for example, no continuous flow. Alternatively, however, are also external, ie outside the

Microfluidic system mounted, pumps for controlling the flow rate possible. Furthermore, within the substrate one or more collection chambers for the remaining components of the biological

Be arranged sample fluid. In addition, collection chambers for the buffer solutions or the already eluted specific sample components are conceivable.

In one of the embodiments, the microfluidic filter element has channels with a height and a width of in each case 5 μm to 5 mm, preferably 50 μm to 2 mm, and the filter element is altogether for receiving a biological sample fluid having a volume of 1 μm to 20 ml , preferably 10 μΙ to 10 ml.

Generally, however, the dimensions are easy to examine

Sample fluid and the components contained therein.

In a further embodiment of the microfluidic filter element, the capture molecules are selected from the group of proteins, preferably antibodies or enzymes, polynucleic acids, preferably DNA or RNA, and polymers. However, other catcher molecules are possible. The catcher molecules are, for example, by silanization,

Aminopropyltriethoxysilane (APTES), covalent bonds by means of a

EDC / NHS chemistry or protein A immobilized.

The catcher molecules are in each case specific to the sought

Sample components are matched and enter into a reversible or irreversible binding with these. In addition, a direct implementation of the sought sample components by the capture molecules is possible. This is the case, for example, when the capture molecule is an enzyme which filters a particular enzyme substrate from the sample fluid. The

Immobilization of the catcher molecules is carried out according to common methods such as Example of silanization. The filter material is complete with the

Catcher molecules is interspersed.

In one embodiment of the microfluidic filter element is the

Filter material selected from the group of fibers, preferably silica fibers; Membranes, preferably capillary membranes of a polymer material or cellulose; Beds of particles, preferably of colloids, more preferably of silica, polymethyl methacrylate or melamine; and metal meshes, preferably transmission electron microscopy grids.

As already shown, the separation of the desired

Sample components from the sample fluid specific catcher molecules in the

Filter material to be immobilized. For this purpose, depending on the filter material, different methods are used. In the interests of high efficiency of the filter, the surface available for the interaction must be as large as possible. As a filter, every material comes with controllable

Pore sizes in question. For this purpose, in particular fiber filters,

preferably based on silica, or membrane filters, both as

Pore membrane filter or can be made as a capillary membrane filter of polymer material or cellulose. In addition to the classic filter materials, however, also beds of particles such as colloids for use in the filter element according to the invention are suitable. The function of the colloids is to produce a filter with adjustable pore size. However, the actual separation of the desired sample components continues to take place via the catcher molecules. As preferred materials here silica, polymethyl methacrylate or melamine are used.

In a preferred embodiment, the filter material consists of metal nets or metal grids in the manner as they are for example for

Transmission electron microscopy can be used. The so-called TEM grids are commercially available and have defined hole sizes of approx.

several 100 μηη to about 10 μηη on. The superimposition of several networks or grids is provided in a further embodiment of the filter element. In a further embodiment of the microfluidic filter element, the particles and metal mesh are coated with gold or platinum. As a result, the functionality of the filter material can be increased and a coupling of catcher molecules can be simplified.

In a further embodiment of the microfluidic filter element, the filter material has a thickness of 100 μηη to 10 mm, preferably 30 μηη to 7 mm, more preferably 50 μηη to 5 mm and a pore size of 0.5 to 1000 μηη, preferably 1 to 500 μηι, particularly preferably 5 to 300 μηι, on.

In the case of the mentioned TEM grids, the minimum thickness of the filter material is dictated by the thickness of a single TEM grid.

The pores of the filter material are dimensioned so that components of the sample liquid that do not contain any of the sample components sought can pass the filter substantially. In the case of bacteria in whole blood, this results in e.g. a minimum pore size of several μηη. Larger pores reduce the fluid resistance. At the same time, however, the efficiency of the filter material is lowered. This results depending on the application

Pore sizes up to several 100 μηη. The size of the filter chamber is adjusted in advance to the desired filter material and its extent. However, the diameter of the filter chamber is generally between 1 and 30 mm.

In a further embodiment of the microfluidic filter element, the substrate consists of a polymer, preferably a polycarbonate,

Polypropylene, cycloolefin copolymer, copolymer or polymethylmethacrylate; a glass or silicon containing material.

In principle, however, all materials which can be formed by extrusion, deep drawing or embossing on a microscale are suitable.

In a preferred embodiment of the microfluidic filter element, the substrate has a thickness of 0.2 to 8 mm, preferably 0.5 to 3 mm and an outer dimension of 10 x 10 to 100 x 100 mm ² . Again, the dimensions of the substrate must be adapted to use in a microfluidic system.

In a further aspect of the invention, the use of the above-described microfluidic filter element takes place in a lab-on-chip system or in a purification tube.

With the use of the filter element according to the invention thus the transition from the range of large sample volumes (milliliters) in the area becomes smaller

Sample volumes (microliters) facilitated. The application not only relates to the field of classical microfluidics, but also to other applications in which such a purification is to be achieved, e.g. by means of purification tubes. Such art-known kit systems based on purification tubes are often used in in vitro diagnostics.

drawings

Further advantages and advantageous embodiments of the microfluidic filter element according to the invention are illustrated by the figures and explained in the following description. It should be noted that the pictures have only descriptive character and are not intended to limit the invention in any way. The following symbols are used in the figures:

Substrate 1

Feeding channel 2

of the biological sample fluid

Discharge channel after filtering 3

remaining components of the biological sample fluid

other channels 4,5

Valves 6, 7, 8, 9

Collecting chamber 10

Filter chamber 1 1

Filter material 12

Catcher molecules 13 specific sample components 14 other sample components 15

Fig. 1 shows schematically the filter principle of the filter element according to the invention

Fig. 2 shows the filter element according to the invention in plan view

FIG. 3 shows the microfluidic filter element as a layer system comprising a plurality of substrates 1

Fig. 1 shows schematically the filter principle of the filter element according to the invention using the example of a fiber filter. At the filter material 12 catcher molecules 13 are immobilized. At these immobilized capture molecules 13, the particular sample components 14 are separated by binding from the sample fluid. Other sample components 15 pass through the filter material 12 due to the large pores in the filter material 12 substantially unhindered. The figure is based on the example of a 13-functionalized fiber filter. In doing so, a biological sample fluid, e.g. Blood, bacteria 14 filtered. The other sample components 15, e.g.

Erythrocytes, the filter material 12 can pass largely unhindered. However, the principle can be applied to a variety of applications.

Fig. 2 shows a simple realization of the filter element according to the invention. Starting from a polymer substrate 1 in this substrate 1, the channels for supplying the biological sample fluid 2 and for deriving the remaining after the filtering process components of the biological sample fluid 3, more channels 4/5 for the supply and removal of the buffer solution, as well as in the channels 21 3, 4/5 placed valves 6 / II 8/9 arranged. In addition, the substrate 1 has a filter chamber 1 1 with therein befindlichem filter material 12 and a collecting chamber 10. With closed valves 8/9 sample fluid is introduced via channel 2 into the filter chamber 1 1 and flows through the

Filter material 12 laterally. While the specific sample components 14 bind to the respective capture molecules 13, the remaining

Sample components 15 of the sample liquid via the channel 3 in the

Passage chamber 10. To all remaining components 15 of the

Sample liquid from the filter material 12 to remove, can with a appropriate buffer solution rinsed. For this purpose, the valves 8/9 are opened and the valves 6/7 are closed so that the channels 4/5 are opened. By flushing with the buffer solution through the channels 4/5, the other sample components 15 can now be removed. In the same way, the sought-after specific sample components 14 can be further

Elute processing from the filter material 12. Alternatively, the concentrated sample components 14 may be placed directly on the filter material 12 for analysis and diagnostics, e.g. for a PCR analysis, continue to be used. 3 shows the microfluidic filter element as a layer system comprising a plurality of substrates 1. With this construction, a transverse flow through the filter material 12 becomes possible.

Claims

claims

1 . Microfluidic filter element for separating certain

Sample components (14) of a biological sample fluid, comprising at least one substrate (1),

at least one in the substrate (1) arranged filter chamber (1 1), at least one arranged in the substrate (1) channel (2) for supplying the biological sample fluid and at least one in the substrate (1) arranged channel (3) for deriving the after the filtering process remaining components of the biological sample fluid,

in the filter chamber (1 1) arranged filter material (12) and

on the filter material (12) immobilized catcher molecules (13) for certain sample components (14), wherein the filter material (12) with the

Catcher molecules (13) is interspersed.

2. Microfluidic filter element according to claim 1, characterized in that the filter element of a plurality of layered layers

Substrates (1) is constructed, the at least one filter chamber (1 1) and the channels for supplying the biological sample fluid (2) and for deriving the remaining after the filtering process components of the biological sample fluid (3) are at least partially disposed in different substrate layers and the Filter material (12) is flowed transversely.

3. Microfluidic filter element according to claim 1 or 2, characterized

in that the filter element is constructed from the substrate (1) with the at least one filter chamber (1 1) and the channels for supplying the biological sample fluid (2) and for deriving the components of the biological sample fluid (3) remaining after the filtering process, and Filter material (12) is flowed laterally.

4. Microfluidic filter element according to one of the preceding claims, characterized in that the filter element at least one further channel (4, 5), at least one valve (6, 7, 8, 9) and / or at least one collecting chamber (10) for the remaining Components of the biological sample fluid has.

5. Microfluidic filter element according to one of the preceding claims, characterized in that the channels (2,3,4,5) have a height and a width of 5 μηη each to 5 mm, preferably 50 μηη to 2 mm, and the filter element as a whole for receiving a biological

Sample fluid having a volume of 1 μΙ to 20 ml, preferably 10 μΙ to

10 ml, is designed.

6. Microfluidic filter element according to one of the preceding claims, characterized in that the capture molecules are selected from the group of antibodies, enzymes, polynucleic acids and polymers.

7. Microfluidic filter element according to one of the preceding claims, characterized in that the filter material (12) is selected from the group of fibers, preferably silica fibers; Membranes, preferably capillary membranes of a polymer material or cellulose; Beds of particles, preferably of colloids, more preferably of silica, polymethyl methacrylate or melamine; and metal meshes, preferably transmission electron microscopy grids.

8. Microfluidic filter element according to claim 7, characterized in that the particles and metal mesh are coated with gold or platinum.

9. Microfluidic filter element according to one of the preceding claims, characterized in that the filter material (12) has a thickness of 100 μηη to 10 mm, preferably 30 μηη to 7 mm, more preferably 50 μηη to 5 mm, and a pore size of 0.5 to 1000 μηη, preferably 1 to 500 μηη, particularly preferably 5 to 300 μηη having.

10. Microfluidic filter element according to one of the preceding claims, characterized in that the substrate (1) consists of a

Plastic polymer, preferably a polycarbonate, polypropylene, cycloolefin copolymer, copolymer or polymethyl methacrylate; a glass or silicon containing material.

1 1. Microfluidic filter element according to one of the preceding claims, characterized in that the substrate (1) has a thickness of 0.2 to 8 mm, preferably 0.5 to 3 mm and an outer dimension of 10 x 10 to 100 x 100 mm ² has.

12. Use of a microfluidic filter element according to one of

Claims 1 to 1 1 in a microfluidic lab-on-chip system or a Aufreinigungsröhrchen.