VAN LIESHOUT RON M L (NL)
WITKAMP JAN F (NL)
PUNYADEERA GARDIYE H P K C (AU)
VAN LIESHOUT RON M L (NL)
WITKAMP JAN F (NL)
| CLAIMS:
1. A device comprising a porous substrate, wherein on one surface of the porous substrate one or more capture probes are immobilized a flexible membrane and - a holder which holds the porous substrate and flexible membrane together, wherein the holder exhibits at least two openings for applying a sample and for letting the sample pass through said porous substrate, wherein the porous substrate and the flexible membrane are arranged such that the flexible membrane is placed on the side of the porous substrate which is opposite of the surface on which the capture probes are immobilized.
2. A device according to claim 1, wherein the capture probes are independently selected from the group comprising an antibody, a nucleic acid molecule, a carbohydrate, a protein, such as an enzyme, a structural protein, a transmembrane protein, a receptor protein, a surface receptor protein, an immunoglobulin, a signaling protein, a peptide, a cryptand, a crown ether, small molecules, metabolites, cells.
3. A system comprising one or more devices according to any of claims 1 to 3, wherein the system further comprises a means for applying a force to the flexible membrane, so that the flexible membrane is deformed such that a compartment is formed between the porous substrate and the flexible membrane, and wherein the system comprises an opening through which a sample may be applied to the device, and whereby said opening may furthermore allow the detection of binding of a target to the capture probe.
4. A system according to claim 3, whereby the system furthermore comprises a means for qualitatively and/or quantitatively detecting the presence of one or more targets bound to the capture probes and/or recording the signals.
5. A system according to claim 4, whereby the system furthermore comprises a means for analyzing the recorded signals.
6. A method of detecting an analyte in a sample comprising the steps of: - providing a device according to any of claims 1 to 2 -or a system according to any of claims 3 to 5, applying a sample to porous substrate, applying a force to the flexible membrane which is thereby deformed such that a compartment is formed between the porous substrate and the flexible membrane, and whereby the sample is drawn through the porous substrate into said compartment by the vacuum force generated upon the formation of the compartment, detecting target molecules bound to the capture probes.
7. A method according to claim 6., wherein upon removal of the force the sample is pushed back through the porous substrate by the force generated by the relaxation of the flexible membrane.
8. A method according to claims 6 or 7, wherein the sample is moved back and forth through the porous substrate several times.
9. A device according to any of claims 1 to 2, a system according to any of claims 3 to 5 or a method according to any of claims 6 to 8 for analyzing a sample for the presence of an analyte.
10. A device according to any of claims 1 to 2, a system according to any of claims 3 to 5 or a method according to any of claims 6 to 8. for the detection and/or diagnosis of a disease or condition by analyzing a sample obtained from a patient. |
Flow-through biosensor
FIELD OF THE INVENTION
The invention is in the field of detection of bio molecular and/or chemical targets with immobilized capture probes.
BACKGROUND OF THE INVENTION
The quantity of available sample material for many tests and in particular tests with samples that have to be taken from living creatures (human or animal) is very small, meaning that the sample volume is 100 μl or less. Thus, the device to carry out the tests has to be relatively small, reliable and easy to use and it should be possible to integrate a detection system in the same housing. Miniaturized assays have been developed, in which detection of target molecules is achieved by hybridization with molecules which are immobilized on the surface of a medium, such as the so called DNA chips.
SUMMARY OF THE INVENTION This invention relates to a device, a system, and a methods for detecting one or more analytes in a fluid. It can for example be used in agricultural, food industry, forensic, chemical, biochemical and medical applications.
The objective of the present invention is to provide a novel device, which allows for miniature scale analysis of liquid samples. An embodiment of the present invention is a device comprising a porous substrate, wherein on one surface of the porous substrate one or more capture probes are immobilized a flexible membrane and a holder which holds the porous substrate and flexible membrane together, wherein the holder exhibits at least two openings for applying a sample and for letting the sample pass through said porous substrate, wherein the porous substrate and the flexible membrane are arranged such that the flexible membrane is placed on the side of the porous substrate which is opposite of the surface on which the capture probes are immobilized.
A further embodiment of the present invention is a system comprising one or more membrane assays according to the present invention, wherein the system further comprises a means for applying a force to the flexible membrane, so that the flexible membrane is may be deformed such that a compartment is formed between the porous substrate and the flexible membrane, and wherein the system comprises an opening through which a sample may be applied to the membrane assay, and whereby said opening may furthermore allow the detection of binding of a target to the capture probe. A further embodiment of the present invention is a method of detecting an analyte in a sample comprising the steps of: providing a device according to the present invention or a system according to the present invention, applying a sample to porous substrate, - applying a force to the flexible membrane which is thereby deformed such that a compartment is formed between the porous substrate and the flexible membrane, and whereby the sample is drawn through the porous substrate into said compartment by the vacuum force generated upon the formation of the compartment, detecting target molecules bound to the capture probes A further embodiment of the present invention is the use of a device according to the present invention, a system according to the present invention or a method according to the present invention for analyzing a sample for the presence of an analyte.
A further embodiment of the present invention is a device according to the present invention, a system according to the present invention or a method according to the present invention for the detection and/or diagnosis of a disease or condition by analyzing a sample obtained from a patient.
BRIEF DESCRIPTION OF THE DRAWINGS
Fig. 1 is a schematic representation of the combined substrate-membrane assembly of the device according to the present invention. a) Dedicated markers are applied onto one side of a porous substrate, e.g. a nitrocellulose membrane (2). A very thin flexible membrane (3) is positioned against the opposite side of this porous substrate. These two elements are clamped together by a holder,
which in this specific case consists of an upper part (1) called the "membrane cap" and a lower part (4) called the "membrane ring". b) shows the assembled state.
Fig. 2A is a schematic representation of the part of the system according to the present invention by which the membrane assay is operated. This representation shows the setup of a device wherein the membrane is operated by vacuum. The device (5) is placed into a small chamber in the body of the system (11). On both sides there is a sealing (10) mounted to prevent any leakage between lid and body of the system. Lid (9) is placed on top of the device (5 + 10) to clamp it into the body (11). In the lid there is an opening (6) into which one can apply the sample material. This opening also makes continuous camera observation of the process possible allowing real time detection of the analyte capture probe binding. A vacuum can be applied via vacuum connection (8) in the body, by which the flexible membrane will be deformed into cavity (7).
Fig. 2B is a conceptual schematic drawing of a particular embodiment of the device according to the invention. The device is composed of an antibody printed nitrocellulose membrane and a flexible foil to pump solutions through the nitrocellulose membrane. The plastic rings containing both the flexible foil (yellow in colour) and the nitrocellulose membranes are clamped together between rings which have been placed beforehand. When applying vacuum on the system the flexible foil will deform drawing the samples through the nitrocellulose membrane. To increase reaction kinetics the samples will be pumped a number of times through the nitrocellulose membrane enhancing replenishment of the solution.
Fig. 3 is a schematic representation of the working modes of the system as represented by Fig. 2. In the initial situation (A), the vacuum connection (compare Fig. 2) is under atmospheric pressure or slightly above atmospheric pressure. A sample is applied into the compartment above the porous substrate. If a vacuum is applied through the connection, the flexible membrane is deformed into the cavity below (B) and the sample is drawn into this cavity.
Fig. 4 shows the CRP Dose response curve performed on the optical integrated device (sample volume 70μl, flow-through 7 min assay - no wash)
Fig. 5 shows the flow-through (left column) versus no-flow (right column). Detection of 50 pM of CRP in 70 μl of sample within 7 minutes.
Fig. 6 shows the real-time Kinetics performed using an assay where all the reagents were added at the same time (all-in-one). In (all-in-two) CRP and detection
antibodies and the CRP analytes were added at the same time, followed by an incubation with Streptavidin
Fig. 7 shows the variation of the CRP sandwich assay (100 pM).
Fig. 8 shows the detection of 100 pM of CRP in PBS buffer. A sandwich assay was performed using (A) sample volume 70 μl and (B) sample volume 300 μl. CRP was detected using a secondary biotinylated antibody with streptavidin Alexa ® 633. The signal intensity for 300 μl of sample volume is higher than that for the 70 μl of sample
Fig. 9 shows the effect of using different pump cycle programmers on the signal intensity. Assay was performed using CRP 100 pM. (3X3 = 3 seconds vacuum and 3 seconds no vacuum; 1X2 = 1 sec vacuum and 2 seconds no vacuum; etc)
Fig. 10 shows the CRP dose response curve in serum. Detection of 1 pM of CRP in 70 μl of sample volume in 11 minutes
Fig. 11 shows the CRP analyte incubation kinetics (analyte concentrations are I nM, lOO pM and lO pM)
DETAILED DESCRIPTION OF EMBODIMENTS
This invention relates to devices, systems and methods for analyte detection..
These can be used to determine the concentration of a target by using target sensor modules
(capture probes) immobilized on a porous medium. In addition, the devices and methods of the present invention allow for very rapid analysis of samples, leading to results in minutes.
The invention is based on a container (cell) with a porous medium and a flexible membrane and a system that may cause a flow of the sample through the porous medium in two opposite directions. The flow can be actuated by a vacuum force caused by the deforming and relaxation of a flexible membrane which is positioned against one side of the porous medium. Furthermore the porous medium can easily be placed into and taken out of this cell by the person operating the device. Depending on the desired tests these cells can be placed next to each other forming a multiple linear or circular array in which tests can be carried out simultaneously. A detection unit which may be integrated into the system makes immediate results (diagnosis) possible. Due to the novel and straightforward design of the assay of the present invention, miniature scale applications become accessible. However, the device and or system according to the present invention can be scaled up, therefore larger volumes are not meant to be excluded by this description.
An embodiment of the present invention is a device comprising
a porous substrate, wherein on one surface of the porous substrate one or more capture probes are immobilized a flexible membrane and a holder which holds the porous substrate and flexible membrane together, wherein the holder exhibits at least two openings for applying a sample and for letting the sample pass through said porous substrate, wherein the porous substrate and the flexible membrane are arranged such that the flexible membrane is placed on the side of the porous substrate which is opposite of the surface on which the capture probes are immobilized. Preferably, the flexible membrane and the porous substrate are in direct contact to one another. Furthermore preferably, the porous substrate should be less flexible than the flexible membrane. This means that the force needed to deform the porous substrate is significantly higher than the force needed to deform the flexible membrane. In a preferred embodiment, the flexible membrane is a rubber membrane. Preferably, the flexible membrane is composed of an elastic material, which enables the membrane to essentially regain its original form, once the deforming force is withdrawn.
The flexible membrane used in the present invention may be composed of any material which ensures sufficient flexibility and which is impermeable and inert for the sample to be used in the assay. Depending on the specific conditions of the assay (e.g. reactive substances, organic solvents), a skilled person will be aware from which materials the material of the flexible membrane may be selected. Non- limiting examples of such materials may be latex, PE, nitrite, PU, in particular such which are suitable for the preferred uses of the assay. For instance, the flexible membrane should be compatible with the conditions of biochemical assays which are conducted in aqueous media. Preferably the thickness of the flexible membrane is 0.025 to 0.5 mm, preferably, 0.05 to 0.25 mm, more preferably 0.075 to 0.15 mm, most preferably about 0.1 mm. The diameter of the flexible membrane is preferably 0,5 mm to 10 mm, more preferably 1 mm to 7,5 mm, most preferably about 2 mm or about 5 mm.
The porous substrate may for example be selected from the group comprising a non- woven fabric, a substrate based on polished and etched hollow fibres of glass or other materials, an electroformed substrate, nitrocellulose, a hydro gel, nylon, PVDF, a polyacrylamide gel, or aluminum oxide. Preferably, the substrate is at least partly transparent for radiation, preferably optical radiation, such as ultraviolet, visible light or infrared. This improves the detection possibilities for the substrate. It is obvious that several of the above-
mentioned materials for the porous substrate may be combined in one substrate. In certain cases, this will be mandatory, such as for instance for hydro gels and polyacrylamide gels, which are too fragile themselves and will have to be supported on a more solid porous substrate, such as nitrocellulose, nylons, PVDF, aluminum oxide or glass. The thickness of the porous substrate is preferably 25 μm to 1000 μm, more preferably 50 μm to 500 μm, most preferably about 150μm. The diameter of the porous substrate is preferably 0,5 mm to 10 mm, more preferably 1 mm to 7,5 mm, most preferably about 2 mm or about 5 mm.
In another preferred embodiment, the one or more capture probes are independently selected from the group comprising an antibody, a nucleic acid molecule, a carbohydrate, a protein, such as an enzyme, a structural protein, a transmembrane protein, a receptor protein, a surface receptor protein, an immunoglobulin, a signaling protein, a peptide, a cryptand, a crown ether, small molecules, metabolites, cells.
According to the invention, only one type of capture probe or multiple types of capture probes may be immobilized on one surface of the porous substrate. Capture probe according to the invention indicates one or more molecules with specificity for one target. Hereby, different capture probes may be selected from each of the above-mentioned entities. For instance, two or more different antibodies, or any of the other above-mentioned entities, may be immobilized on the same porous substrate in order to simultaneously capture and detect two targets. However, the detection of only one analyte with two different capture probes is also envisioned. In embodiments where multiple different capture probes are used, it is preferred to print multiple, more preferably up to 100 distinct spots onto the porous substrate, whereby each spot comprises only one specific type of capture probe.
In another embodiment, the one or more types of capture probes comprise a spacer and a reactive group which possesses a specific reactivity towards a target in a sample.
In another preferred embodiment, the concentration of capture probes immobilized at least on one surface of the porous substrate is 80-150 μg/cm 3 . In this context, "μg/cm 3 is to be understood as being directed to μg/cm 3 for substrates where the capture probes may also be bound inside the porous substrate, whereas for porous substrates where the binding rather only takes place on the surface the expression is directed to the more appropriate term μg/cm 2 . Thus, "μg/cm 3 " and μg/cm 2 " may be used interchangeably in this context.
Another embodiment of the present invention is a system comprising one or more devices according to the present invention,
wherein the device further comprises a means for applying a force to the flexible membrane, so that the flexible membrane is may be deformed such that a compartment is formed between the porous substrate and the flexible membrane, and wherein the device comprises an opening through which a sample may be applied to the device, and whereby said opening may furthermore allow the detection of binding of a target to the capture probe.
In a preferred embodiment, the force applied to the flexible membrane is gas pressure and the means for applying a force to the flexible membrane comprised in the device is a vacuum pump or a line to which a vacuum pump or other means to create a vacuum may be attached, or alternatively, a line through which a pressurized gas can be applied to the device. The vacuum or gas pressure force applied is 200 to 900 mbar abs., preferably 400 to 600 mbar abs., most preferably about 425 mbar abs or about 475 mbar abs. The same ranges may be applied for the hydraulic force.
Preferably, the system according to the present invention furthermore comprises a vacuum and/or a pressure gauge. Said gauge(s) may be used in order to control the pressure ranges at which the device is operated. The gauges may produce an electronic readout which serves to keep the minimum and maximum pressure during operation of the device in a predetermined range. In this way, it can be ensured that the parts of the device (e.g. the porous substrate or the flexible membrane) are not stressed beyond their respective capacities and are thus protected from rupture during operation.
The system according to the present invention may comprise more then one membrane assays according to the present invention so that multiple samples may be analyzed in parallel.
In a preferred embodiment of the system according to the present invention, the system furthermore comprises a means for qualitatively and/or quantitatively detecting the presence of one or more targets bound to the capture probes and/or recording the signals. In the context of the present invention, the detection may afford to add further molecules (e.g. antibodies) or chemicals (buffers, dyes, etc) onto the porous substrate in order to detect binding of the target to the porous substrate. For instance, the detection may be performed using a labeled antibody. Said label may for instance be a fluorescent, radioactive, biotin or other suitable label commonly used in biochemical assays. The signal that can be generated by/with this label (e.g. by irradiation, by radioactive decay, or by other means which are usual in commonly used biochemical assays, which may also comprise the addition of other compounds necessary for detection) can be measured on top of the membrane.
The detection method may be based on optical phenomena selected from the group comprising, UV/vis absorption, IR absorption, fluorescence absorption or emission, FRET, phosphorescence, chemiluminescence, electroluminescence, chemo-luminescent, magnetic detection. The detection may also take place in a time-resolved manner. In another preferred embodiment of the system according to the present invention, the system furthermore comprises a means for analyzing the recorded signals by means of an analysis software.
The system may be connected to a computer system or may itself comprise a computer system, whereby the computer system comprises a software which analyzes the recorded signals. This analysis may be used by a skilled person to base a diagnosis or other evaluation of the results thereon.
Another embodiment of the present invention is a method of detecting an analyte in a sample comprising the steps of: providing a device according to the present invention or a system according to the present invention, applying a sample to porous substrate, applying a force to the flexible membrane which is thereby deformed such that a compartment is formed between the porous substrate and the flexible membrane, and whereby the sample is drawn through the porous substrate into said compartment by the vacuum force generated upon the formation of the compartment, detecting the amount of target molecule which is bound to the capture probes. According to the present invention, said force may be applied to the flexible membrane by various means. Non- limiting examples of this force are a vacuum or gas pressure force, an electrostatic force, a magnetic force, a hydraulic force. The flexible membrane must be selected such, that it is suitable for use with the various forces described above. For instance, magnetic particles might be contained in the flexible membrane or a metal membrane may be attached to the flexible membrane in order to make it susceptible to a magnetic force. Preferably however, a hydraulic or gas pressure or vacuum force is used. Gas pressure hereby means that, instead of a vacuum which is applied to the flexible membrane on the side which is opposite of the porous substrate, a pressurized gas may be applied from the side of the porous substrate on the side which is opposite of the flexible membrane in order to push the sample through the porous substrate and thereby deform the flexible membrane.
The device and/or the system according to the present invention are conceived such that upon the application of a force, the flexible membrane may be deformed such that a compartment is formed between the porous substrate and the flexible membrane, and whereby the sample is drawn through the porous substrate into said compartment by the vacuum force generated upon the formation of the compartment. The device and/or the system according to the present invention are furthermore conceived such that upon removal of the force the sample is pushed back through the porous substrate by the force generated by the relaxation of the flexible membrane. Thus, by repeated application and removal of the force, the sample may be moved back and forth through the porous substrate several times. According to the present invention, unbound molecules are pulled into the compartment below the membrane, and thus removed from the surface where detection takes place. In this way, an enhanced signal-to-background/noise ratio is obtained.
In a preferred embodiment of the method according to the present invention, upon removal of the force the sample is pushed back through the porous substrate by the force generated by the relaxation of the flexible membrane. If necessary, an additional force (for instance by gas pressure) may be applied in order to ensure and/or speed up the transfer of the sample back through the porous substrate.
In a preferred embodiment of the method according to the present invention the sample is moved back and forth through the porous substrate several times. By sequentially repeating the abovementioned processes of drawing and pushing the sample through the porous substrate, the sample may be moved back and forth through the porous substrate twice, three, four, five, or even more times. In this way, a longer contact between the sample and the capture probes immobilized on the porous substrate may be ensured.
A further embodiment of the present invention is the use of a device according to the present invention, a system according to the present invention or a method according to the present invention for analyzing a sample for the presence of an analyte.
A further embodiment of the present invention is a device according to the present invention, a system according to the present invention or a method according to the present invention for the detection and/or diagnosis of a disease or condition by analyzing a sample obtained from a patient.
Diseases or conditions which may be detected and/or diagnosed according to the present invention are in principle only limited by the availability of an appropriate disease marker for the disease or condition. Mainly, these are diseases or conditions where the disease markers are proteins, peptides, nucleic acids or metabolites. For instance, such
diseases may be selected from cancer, conditions or diseases caused by inflammatory processes, hereditary diseases or conditions, conditions or diseases caused by bacterial, viral or protozoal infections. In general, the embodiments of the present invention may be used in applications where rapid diagnosis is needed, or where the early diagnosis of a disease is necessary (e.g. an infectious disease), as well as in the determination of a prognosis and/or staging of sepsis.
The material of which the mechanical parts of the device (such as for instance the membrane cap and the membrane ring) are composed may be any material or combination of materials which a skilled person may recognize to be appropriate to yield the desired mechanical and functional properties and which are inert under the conditions of the assay. Non- limiting examples of such materials are metals, such as steel or aluminum, ceramic materials, glass or plastics.
In the context of the present invention, the target may be a bio molecule, such as a protein, a nucleic acid molecule, a carbohydrate, a peptide, an antibody, a glycoprotein, a receptor molecule or a ligand of a receptor molecule, or fragments of the above-mentioned targets/analytes. The Target/ Analyte may also be a human, animal or plant cell, a bacterial cell, a virus, a virus capsid, or fragments of the above-mentioned targets. The Target may also be a chemical or biochemical substance contained in a sample. Non-limiting examples of such substances are metal ions, organic chemicals, in particular toxic or environmentally harmful chemicals, metabolites, drugs and drugs of abuse, cells.
A "sample" in the context of the present invention may originate from a biological source. Encompassed are biological fluids such as lymph, urine, cerebral fluid, bronco leverage fluid (BAL), blood, saliva, serum, feces or semen. Also encompassed are tissues, such as epithelium tissue, connective tissue, bones, muscle tissue such as visceral or smooth muscle and skeletal muscle, nervous tissue, bone marrow, cartilage, skin, mucosa or hair.
A "sample" in the context of the present invention may also be a sample originating from an environmental source, such as a plant sample, a water sample, a soil sample, or may be originating from a household or industrial source or may also be a food or beverage sample.
A "sample" in the context of the present invention may also be a sample originating from a biochemical or chemical reaction or a sample originating from a pharmaceutical, chemical, or biochemical composition.
The amount of sample is preferably 1000 μl or less, more preferably 500 μl or less, even more preferably 100 μl or less, most preferably 50 μl or less.
Where appropriate, as for instance in the case of solid samples or viscous suspensions , the sample may need to be solubilized, homogenized, or extracted with a solvent prior to use in the present invention in order to obtain a liquid sample. A liquid sample hereby may be a solution or suspension.
Liquid samples may be subjected to one or more pre-treatments prior to use in the present invention. Such pre-treatments include, but are not limited to dilution, filtration, centrifugation, pre-concentration, sedimentation, dialysis, lysis, eluation, extraction. Pre-treatments may also include the addition of chemical or biochemical substances to the solution, such as acids, bases, buffers, salts, solvents, reactive dyes, detergents, emulsifϊers, chelators, enzymes, chaotropic agents.
A gas-pressure operated system according to the present invention may comprise a means of altering the pressure (such as a vacuum pump) in order to actuate the deforming and relaxation of the flexible membrane. In an other embodiment, said gas- pressure operated system may not comprise a means of altering the pressure, whereby in this embodiment the system is used with vacuum and compressed gas which is supplied by the operator, e.g. in the form of vacuum lines or pumps and/or gas bottles or lines which are present in the operator's facility. In the case that only compressed gas is available to the operator, the device will have to comprise or to be fitted with a vacuum ejector to create a vacuum.
In the context of capture probes, targets and molecules described herein, expressions like "the" "a" or "one" and equivalent expressions are not to be understood as referring to a single entity, but to a plurality of identical entities, unless specified otherwise. The singular is used herein for the convenience of the reader.
It is to be noted that the holder, device and assay according to the present invention can have a large variety of parts and shapes, and that the assemblies shown herein by means of figures represent only one possibility and are not meant to be limiting the assay or devices according to the present invention to a specific shape.
EXAMPLES
A particular embodiment of the device according to the present invention is shown in Fig. 1. The combined substrate-membrane assembly is clamped together by an upper part (1) called the membrane cap and lower (4) part called the membrane ring. Specific
capture probes are applied onto one side of a porous medium (2), which may for instance be a nitrocellulose membrane. A thin flexible membrane (3) (approx is positioned against the opposite side of the porous medium.
Fig. 2 shows a particular setup of the system according to the present invention, wherein the device (5) is placed into a small chamber in the body (11). On both sides there is a sealing (10) mounted to prevent any leakage between lid and body. Lid (9) is placed on top of the membrane assay (5 + 10) to clamp it into the body (11). In the lid there is an opening (6) into which the operator can apply the sample material. This opening also makes continuous detection of the binding process possible. The size of this opening (diameter of the hole and thickness of the lid) depends of the desired quantity of sample material.
When vacuum is applied to connection (8) in the body of the system, the flexible membrane will be deformed into cavity (7). This creates a volume for the sample material underneath the device as well as a small vacuum force that causes the sample material to flow from the upper side of the porous medium to the lower side of the porous medium. When the vacuum is taken away the flexible force in the flexible membrane causes it to contract, whereby the sample material will be pressed upwards through the porous medium back into opening (6). Depending of the material of which the flexible membrane is composed, its quality and the thickness of the flexible membrane it might be necessary to apply a compressed gas (e.g. compressed air) onto connection (8) to ensure the flexible membrane will be pressed back against the porous medium. Alternating switching between vacuum and compressed air will then be needed. When vacuum is applied onto connection 8, all sample material will be below the porous medium. This makes it possible to detect the markers on the porous medium with a detection unit which is mounted above the opening (6) in the lid (9). This detection can be carried out continuously during the pumping cycles, making a time-resolved analysis possible.