Patent Application
20130156643
The present invention relates to a device and a method for manipulating and/or analysing a liquid sample according to the pre-characterising clause of claim 1, 3 or 23.
The present invention is particularly concerned with diagnostics in microfluidic samples, most preferably in so-called point of care systems (POC Systems). In particular, the present invention relates to preferably minaturised immunoassays, i.e. the investigation of samples using antibodies. Particularly preferably, the present invention relates to so-called cartridge concepts, i.e. small, particularly card-like devices, for manipulating and/or analysing a liquid sample or for carrying out immunoassays.
In “Kontinuierlich arbeitende Mikrofluidik-Plattform zur Aufreinigung von Biomolekülen” by Karle, Marc; Miwa, Junichi; Roth, Günter; Haeberle, Stefan; Zengerle, Roland; von Stetten, Felix; which appeared in VDE Verlag GmbH, Paper 31, 12 Oct. 2009, pages 1 to 4, ISDN: 978-3-8007, 3183-1, a continuously operating microfluidic platform for purifying biomolecules is disclosed. The platform comprises channels and a rotatable permanent magnet acting on the platform. Magnetic particles moving along lines of magnetic force transport biomolecules that are to be purified through the channels. The magnetic field applied from outside causes the magnetic particles to be transferred by magnetophoresis over a phase boundary of a laminar flow from one buffer to the next. The transfer of the particles from a liquid phase into a gas space is not disclosed, only the transfer from one liquid or one liquid phase into another.
US 2009/0227044 A1 discloses a device with a microchannel in which magnetic luminescent nanoparticles serve as carriers for antibodies and act as an internal luminescence standard. The carriers are set in oscillation by an external magnetic field which is produced by electromagnets, so as to achieve better diffusion during an incubation step. Moreover, the electromagnets are used to keep the particles in the channel for washing steps and luminescence measurements.
WO 99/49319 discloses a microsystem for manipulating magnetic particles. The particles are coated with a reagent or antibody and moved by magnetic forces in the microfluidic system. Different particles can be provided with different reagents. Evaluation is carried out magnetically and/or by fluorescence measurement.
US 2002/0064866 A1 discloses fibrous spherical particles as carriers of substances for carrying out tests. For this purpose substances are bound to the jagged surface of the particles or incorporated in the surface thereof. The particles are applied as a suspension and if necessary held in place by magnetic forces.
U.S. Pat. No. 7,105,357 B1 discloses a method using a pipette to produce tiny droplets, while substances are bound to magnetic particles and these are held in the pipette by means of permanent magnets.
U.S. Pat. No. 5,135,720 A discloses a reaction vessel with a spherical substrate which comprises a magnetic core and a coating to which proteins or peptides can be attached. The substrate is held suspended by means of electromagnets and a regulating mechanism in order to improve the efficiency of an Edman reaction with substances located on the surface of the substrate.
U.S. Pat. No. 5,439,650 A discloses a similar device, in which part of the reaction vessel is movable with the suspended substrate and the associated electromagnets.
It has been found that the plurality of small magnetic, essentially spherical particles, or particles having some other undefined form, do not allow optimum manipulation or analysis of a liquid sample. In particular, optimum optical detection of the binding of analytes or complexes to the substrate particles is not possible.
The aim of the present invention is to provide a device and a method for manipulating and/or analysing a liquid sample, particularly in a microfluidic system or channel, in which optimised manipulation or analysis and/or an improved, preferably optical detection—particularly for detecting a reaction or ascertaining an analyte—is made possible and/or wherein a plurality of analyses can be carried out in parallel on a sample under defined conditions and/or at separate locations.
The above problem is solved by a device according to claim 1 or claim 3 or by a method according to claim 23. Advantageous further features are the subject of the sub-claims.
The present invention is concerned with the manipulation and/or analysis of a liquid sample, particularly in a microfluidic channel or system. By “microfluidic” is meant, according to the invention, volumes of preferably less than 10 ml, most preferably less than 1 ml, and/or channel or liquid cross-sections (maximum diameter) of preferably less than 2 mm, particularly preferably less than 500 μm.
In the present invention a substrate that is movable in a preferably microfluidic channel or system is used, comprising at least one detection or binding region for a substance. This substance may be an analyte of the sample or a complex formed thereby or reaction product dependent thereon and/or a reagent that interacts or binds with the sample, an analyte of the sample, a complex thereof or the like. For example, the reagent may interact or bind with a complex of the analyte or with a reaction production dependent on the analyte. Preferably the reagent itself is fixed or immobilised in or on the detection or binding region. In particular the detection or binding region may contain or comprise an immobilised antibody which interacts, most preferably binds, with an analyte of the sample or a complex containing the analyte. In this case the reagent is formed by the antibody that interacts with the analyte indirectly, namely with a complex or the like that contains the analyte. However, other interactions or reactions can be achieved, for example modification of the reagent or detection or binding region or dissolving of the reagent. The term “interaction” is accordingly preferably to be interpreted broadly in the present invention.
The movement of the substrate over the liquid/gaseous phase boundary, i.e. by movement into or through the gas space, ensures that as little liquid as possible is transported with the substrate. Rather, the liquid is at least largely held back in the respective liquid chamber. The liquid is, so to speak, wiped away as the substrate moves through the phase boundary into the gas space.
According to one aspect of the present invention the substrate is movable, particularly by means of a manipulating device, out of the sample, particularly in a gas space, or over or through a liquid/gaseous phase boundary or vice versa. This in turn contributes to optical detection, in particular.
By the term “detection” is preferably meant, according to the present invention, the detection of an interaction, modification or reaction in the detection or binding region and/or the binding of a substance such as an analyte, complex or the like, in the detection or binding region, to allow preferably qualitative and/or quantitative analysis of the sample, more particularly a qualitative and/or quantitative detection of at least one analyte of the sample. The detection may be carried out, in particular, optically, particularly preferably by luminescence or fluorescence measurement.
According to another aspect of the present invention the substrate is moved out of the sample into a gas space of the system for detecting a substance bound in the detection or binding region. This allows optimised, especially optical detection, most preferably by luminescence or fluorescence measurement.
Alternatively or additionally, the substrate may be moved through the gas space into a liquid that is different from the sample, or vice versa. Thus, different steps such as incubation steps, washing steps or the like may be carried out, particularly with minimal volumes of liquid.
In particular, the movement of the substrate across the phase boundary from liquid to gaseous has the effect that very little or no liquid is carried or transported by the substrate. Therefore the movement into the gas space or through it allows or causes, in particular, at least substantial separation of the substrate from the liquid that forms the liquid phase.
Particularly preferably, in the present invention, the term phase boundary denotes a transition from liquid to gaseous or vice versa.
According to one aspect of the present invention the substrate is at least substantially planar or plate-shaped and/or provided with an at least substantially flat or planar top with a detection or binding region. This contributes to a good or defined detection or measurement of an analyte.
According to another aspect of the present invention the substrate is guided in or by the channel in a defined alignment. In particular, the substrate is not rotatable in the channel but preferably faces with a flat side and/or its side containing the detection or binding region towards one side (particularly the flat side) of the device or of the channel. This allows particularly good, especially optical detection from the latter side and thus simplifies the construction of the device or the detection generally.
According to another aspect of the present invention the substrate has a length which is greater than the maximum cross-section of the channel. This allows a substantially more defined movement and/or alignment of the substrate in the channel or microfluidic system than in the prior art. Thus a substantially more defined progress and/or a better or more defined, particularly optical detection can be obtained.
According to another aspect of the present invention the substrate fills the cross-section of the channel by more than 30%, preferably more than 40%, more particularly more than 50% by area. In this way the volumes required can be minimised and/or the diffusion lengths or reaction times that occur can be reduced.
According to another aspect of the present invention the substrate is of dimensionally stable configuration with a defined shape. This again contributes to a defined detection.
According to another aspect of the present invention only a single substrate is provided for the detection and/or in the channel. This contributes to a defined detection.
According to another aspect of the present invention the substrate is configured as a plastics component and/or injection moulding. This allows simple, inexpensive manufacture and/or makes it possible to achieve defined properties and/or to facilitate mass production.
According to another aspect of the present invention the detection or binding region is at least partly microstructured. By “microstructuring” is preferably meant an, in particular, repeating structure and/or a structure with an average structural width of 10 nm to 500 μm, preferably less than 10 μm, and/or with elevations or depressions ranging from 10 nm to 500 μm, preferably less than 10 μm. The microstructuring serves to enlarge the surface area and/or assist with the binding of a substance such as an analyte or reagent, particularly an antibody or the like, in the detection or binding region.
According to a further aspect of the present invention the substrate may be movable into different liquids, for example from the liquid sample into a different liquid or vice versa, and/or into different reaction chambers, as selected. This in turn contributes to an optimised reaction process, the optimised performance of various steps and/or optimised detection.
The above-mentioned aspects of the present invention and the other aspects and features of the present invention that arise from the following description and claims may be implemented independently of one another but also in any desired combination.
Further aspects, features, properties and advantages of the present invention will become apparent from the claims and the following description of preferred embodiments by reference to the drawings, wherein:
In the Figures, the same reference numerals have been used for identical or similar components and parts, where similar or corresponding advantages and properties are achieved, even if the related description has not been repeated.
The device 1 preferably has (at least) one in particular microfluidic channel 3 for receiving the sample 2. The channel 3 or the device 1 forms, in particular, a microfluidic system or a part thereof.
As indicated in a schematic longitudinal section of the device 1 according to
The base part 1A is preferably embodied as a plastics part and/or injection moulding.
The base part 1A is preferably at least substantially flat, planar, plate-shaped and/or rigid in its configuration. The channel 3 or the microfluidic system is preferably formed in or along a flat side of the base part 1A and/or open towards a flat side. The channel 3 or the depression or flat side is preferably at least substantially completely covered by the cover 1B. However, other design solutions are also possible.
The cover 1B is preferably embodied as a film. The cover 1B is preferably stuck on, laminated on and/or welded on. Preferably, the cover 1B is formed by an adhesive film or heat-sealable film or the like.
The cover 1B is preferably transparent, at least in parts, in particular in order to permit optical detection, as will be described in more detail hereinafter.
The device 1 comprises at least one substrate 4 that is movable in the channel 3 or system and/or in the sample 2.
The substrate 4 is preferably movable into the sample 2 and/or movable within the sample 2, as indicated by the position A shown by broken lines in
The substrate 4 preferably comprises at least one detection or binding region 5, in this embodiment two or more detection or binding regions 5. The function of the detection or binding region or regions 5 is explained in more detail hereinafter, while reference is made in particular to
Each detection or binding region 5 preferably interacts with only one substance. The substance is preferably an analyte of the sample 2 or a product formed therefrom or dependent thereon, such as a complex or a complex or a reagent that interacts or binds with the analyte or a product dependent thereon. In particular, a detection or binding region 5 serves to bind a specific substance such as a constituent or analyte of the sample 2 and/or a complex 6 formed therefrom and/or to bind a reagent which interacts with the sample 2 or a constituent or analyte of the sample 2 or a complex 6 formed therefrom. Particularly preferably, the reagent or substance in the embodiment shown consists of one or more possibly different antibodies 7, which interact or bind with an analyte or a complex 6 or some other product of the analyte.
Preferably the substance that is arranged in the detection or binding region 5 or that binds the latter or the reagent is an antibody 7 or contains antibodies 7. Most preferably, the detection or binding region 5 is formed or covered by immobilised antibodies 7.
The expression “detection or binding region” in the present invention is preferably to be interpreted very generally as meaning that it interacts directly or indirectly (e.g. after pre-treatment or application of the antibodies 7) with the substance, particularly an analyte. The interaction may consist in particular in a reaction, a binding, a complex formation, an adhesion, an accumulation, a catalytic influence or any desired combination thereof.
Preferably, the substrate 4 comprises several detection or binding regions 5, which are in particular spatially separated from one another, which interact particularly with different substances or serve to detect different analytes of the sample 2, as shown in the embodiment by way of example.
The proposed device 1 and the proposed method thus preferably serve for the investigation or diagnosis of the sample 2, particularly the qualitative and/or quantitative determination of at least one analyte of the sample 2. In particular, an immunoassay is carried out, most preferably as a sandwich immunoassay. However, other reactions may also take place or other substances, reaction products, properties or the like may be determined or measured or detected.
Alternatively or additionally, the proposed device 1 and the proposed method may also be used to manipulate the sample 2, particularly by moving the substrate 4 within the sample 2 and/or by moving the substrate 4 out of the sample 2 and/or by moving the substrate 4 into the sample 2. Thus for example it is possible to mix the sample 2 or transfer a certain amount of the sample 2 or a constituent of the sample 2. It is also possible to transfer into the sample by moving the substrate 4.
For moving the substrate 4 the device 1 preferably has a manipulating device 8. The manipulating device 8 may however also be associated with the device 1, in particular may form part of a test device, as will be explained in more detail hereinafter.
The movement of the substrate 4 preferably takes place magnetically, in the embodiment shown, particularly by the variation of a magnetic field M acting on the substrate 4. In particular, the magnetic field M is generated externally. In the embodiment shown, the manipulating device 8 comprises for this purpose at least one external magnet and/or electromagnet or a plurality of electromagnets, as shown in
Moreover, the substrate 4 is at least partly made of magnetic or magnetisable material, or provided therewith, which forms in particular a magnet 9 as shown in
The magnetic material or the magnet 9 may for example be accommodated in the substrate 4, as shown in
The use of the magnetic material or the magnet 9 may ensure, for example, that the substrate 4 is movable along a gradient of the magnetic field M acting on the substrate 4, i.e. in particular the magnetic field M generated by the manipulating device 8, also in particular into an area of high magnetic field strength.
The substrate 4 can also be moved in a specified direction, into a specified region and/or in opposite directions, by suitably changing the magnetic field M or the gradient of the magnetic field M. By correspondingly rapid changes, particularly a change in the direction of the gradient of the magnetic field M, it is also possible to implement a possibly very rapid back and forth motion or reciprocating movement of the substrate 4 or oscillation of the substrate 4.
Alternatively or additionally, the substrate 4 may also be moved by any other method, for example by the action of gravity, for example during pivoting or rotation, or by the action of some other force or acceleration, for example during rotation of the device 1, by electrical attraction or repulsion, by frictional or interlocking engagement on the substrate 4, for example by means of a filament or other tensioning or compression means, by ultrasound or the like.
The substrate 4 is preferably movable in the longitudinal direction or along the channel 3 at least in one direction or in both directions.
In order to achieve or assist an easy and/or defined movement of the substrate -particularly in the channel 3 or along the channel 3 or on the bottom of the channel 3 or on the base part 1A—the substrate 4 optionally comprises at least one guide means 10 which, in the embodiment shown, is formed by, in particular, a plurality of projections or runners. The guide means 10 or the projections or runners may for example assist with the sliding of the substrate 4 on the base part 1A or on the bottom of the channel 3, i.e. may reduce the sliding resistance, in particular, and/or if necessary may engage in a channel or longitudinal groove or the like (not shown), in particular in order to guide the substrate 4 as it moves for example in the longitudinal direction of the channel 3. The guide means 10 may alternatively or additionally guide or hold the substrate 4 or the region or regions 5 at a spacing from the bottom of the channel 3, from the side walls of the channel 3 and/or from the cover 1B, and/or may hold it at a spacing from the bottom of the channel 3, from the side walls of the channel 3 and/or from the cover 1B.
The substrate 4 is preferably guided in a defined alignment in the channel 3 or by means of the channel 3. This makes detection easier.
The substrate 4 is preferably at least substantially planar or plate-shaped in configuration and/or preferably comprises an at least substantially planar top with the detection or binding region 5 or the detection or binding regions 5. In particular, this makes detection easier.
Preferably, the substrate 4 has a length which is greater than the maximum cross-section of the channel 3. Accordingly, the substrate 4 is guided through the channel 3 at least in the longitudinal direction.
Preferably the channel 3 is at least substantially flat or flattened in cross-section, in particular so that the substrate 4 is not rotatable in the channel 3, in particular because of the greater width compared with the relatively small internal height of the channel 3.
Particularly preferably, the substrate 4 fills the cross-section of the channel 3 by more than 30% by area, more particularly by more than 40% or 50%, as schematically shown in the cross-section according to
The substrate 4 is preferably dimensionally stable with a defined shape. This in turn contributes to a defined detection.
Particularly preferably, the substrate 4 or at least its surface with the region and/or regions 5 is made from, or coated with, a non-luminescent or non-fluorescent material. This assists detection of substances that are bound in the region 5.
Preferably, the substrate 4 or its surface with the at least one region 5 is at least substantially dark or, most preferably, black. This contributes to optical detection.
The substrate 4 is preferably very thin in its configuration. In particular, its thickness (without guide means 10) is substantially 10 μm and 500 μm. The length and/or extent in the direction of movement V of the substrate 4 is preferably between 1 mm and 10 mm. The width of the substrate 4 is preferably between 0.5 mm and 5 mm. Preferably the length of the substrate 4 corresponds to its main extent.
Preferably the width of the substrate 4 is greater than its thickness by at least a factor 2, particularly by a factor 3 or more. Moreover, the length of the substrate 4 preferably corresponds at least to 1.5 times, more particularly at least two times or more, the width of the substrate 4.
The longitudinal extent of the channel 3 is preferably at least twice the length of the substrate 4, particularly between 10 and 100 mm. The channel 3 preferably at least substantially has a rectangular cross-section, or forms such a section. The internal height of the channel preferably corresponds at least to the thickness of the substrate 4 or, more particularly, at least 10% more than this thickness, particularly between 10 μm and 1 mm. The internal width of the channel 3 corresponds at least to the width of the substrate 4, particularly at least 10% more. In particular the internal width of the channel may be between 0.5 and 10 mm.
The substrate 4 is preferably at least substantially movable in its main plane of extent, particularly in the channel 3 or in the system. The relatively thin configuration of the substrate 3 helps to ensure a low flow resistance.
The substrate 4 is preferably at least substantially movable in a straight line in the channel 3 and/or is movable back and forth in the channel 3. However, it is also possible for the channel 3 not to run in a straight line but to coil or meander, for example, or to bend or extend in some other way. The substrate 4 can then preferably be moved along the channel 3 or in a section or region of the channel 3.
Preferably, only a single movable substrate 4 is provided in the device 1 or channel 3 or system. This contributes to a defined movement and/or detection.
The substrate 3 is preferably made of plastics or is a plastics component. This favours the manufacturing process or allows the use of material with desired properties. For example, the substrate 4 is at least substantially made from PMA or PE.
The substrate 3 is preferably an injection moulded part or is produced by injection moulding; This allows cheap and easy manufacture and/or shaping.
Preferably, at least the top or flat side of the substrate 4 with the at least one detection or binding region 5 is of at least substantially flat or smooth configuration. The top or the detection or binding region 5 may, however, also be microstructured or structured in some other way, as required. The structuring is shown for example in the regions 5 in the schematic section in
With regard to the binding of the antibodies 7 or other reagents it should be noted that, particularly preferably, plasma treatment of the substrate 4 or its top or flat side and/or of the regions 5 and most preferably immobilisation of the antibodies 7 takes place on the substrate 4 or in the regions 5.
The substrate 4 is preferably movable into different parts of the channel 3 or microfluidic system and/or into different liquids. This will be explained in more detail hereinafter.
In the embodiment shown the substrate 4 is preferably initially movable within the sample 2, for example it can be moved back and forth or reciprocated and/or set oscillating. The region a of the channel 3 or of the system in which the sample 2 is located, preferably forms a reaction chamber. Preferably the sample 2 is located only in this channel region a, i.e. is restricted to part of the channel 3.
The channel 3 or the system preferably has a region b which forms a gas space, i.e. is not filled with the sample 2 or any other liquid. The substrate 4 is preferably movable from the region a into the region b, i.e. from position A to position B, and/or vice versa, preferably by means of the manipulating device 8. In particular, the substrate 4 can be moved out of the liquid sample 2 or into it or across a phase boundary from liquid to gas or gas to liquid 11, as shown in
In order to keep the sample 2 or liquid in the region a, means 12 are preferably provided for holding back liquid. The means 12 comprise in particular a capillary stop or an abrupt increase in cross-section, for example provided by a trench 13 which is formed, in particular, both in the bottom and in side walls of the channel 3. The means 12 may, however, additionally or alternatively comprise a surface region with different surface properties which may for example be of a hydrophobic nature, if the sample 2 or other liquid that is to be held back is hydrophilic, or vice versa.
Alternatively or additionally the means 12 serve to “wipe off” the liquid or sample 2 from the substrate 4 when the latter is moved out of the liquid or sample 2, more particularly directly into a gas space and/or over a phase boundary 11. The means 12 may in particular comprise, for this wiping off, a succession of several regions of reduced capillarity or different surface properties, for example a plurality of channels, grooves 14 or the like, which are preferably smaller than the trench 13 in the embodiment shown. However, here again, different design solutions are possible.
The means 12 are preferably disposed between the regions a and b or in the region of the transition from the region a or sample 2 to the region b or the gas space. Preferably, the trench 13 with the significantly reduced capillarity or the capillary stop is arranged directly and immediately adjacent to the reaction chamber or the region b or the sample 2, while the further means for wiping off liquid or grooves 14 or the like are then preferably adjacent to the trench 13 or the like on the side opposite the liquid or sample 2, i.e. the side nearest the gas space.
As already mentioned, the substrate 4 is preferably movable into different positions. Particularly preferably, the substrate 4 can be brought into contact with different liquids. Instead of changing the liquid, it is proposed that preferably the substrate 4 is moved accordingly or that the position of the substrate 4 is changed. In particular, the substrate 4 is movable from the sample 2 into a different liquid 15, for example a washing liquid, or vice versa, as shown in
In the embodiment shown the liquid 15 is preferably contained or held in a region c of the channel 3 or microfluidic system or device 1. The region c preferably forms another reaction chamber.
The substrate 4 is preferably movable, starting from position a or from the sample 2, particularly through the region b or the gas space, into the additional fluid 15 or reaction chamber and/or into the region c, as indicated by position C in
Between the gas space and the region c, means 12 for holding back the liquid 15 are preferably provided, as shown in
In the embodiment shown the region b or the gas space is arranged between the region a or the reaction chamber formed thereby and/or the sample 2, on the one hand, and the region c or the reaction chamber formed thereby and/or the liquid 15, on the other hand. However, the gas space or region b arranged in between may also be omitted or greatly shortened, for example formed only by means 12 or only by a larger trench 13 or the like. In particular, the gas space or the means 12 between the regions a and c may be shortened to such an extent that the region b virtually disappears and/or is shorter than the length of the substrate 4.
Alternatively, it is also possible for the means 12 or the grooves 14 or the like to extend over a distance in the direction of movement of the substrate 4 or the longitudinal extent of the channel 3 which substantially corresponds to the length of the substrate 4 or, more particularly, is significantly longer.
If required, the means 12 between the sample 2 and liquid 15 or between two other liquids may be omitted entirely, particularly if the liquids are not miscible and form a phase boundary, for example by corresponding coating, through which the substrate 4 is movable.
In the first embodiment the device 1 or the channel 3 or the system preferably or optionally comprises a further liquid 16, in addition to the liquid 15 that differs from the sample, into which the substrate 4 can be moved (if required). For this purpose the device 1 or the channel 3 or the system preferably comprises another region e for receiving or holding the liquid 16.
In the embodiment shown the substrate 4 is movable, starting from position C or out of the region c through a region d, which forms an optional additional gas space, or through position D into the region e or position E, namely into the additional liquid 16.
Between the regions c and d on the one hand and the regions d and e on the other hand, means 12 or trenches 13 and/or grooves 14 or the like are preferably also arranged.
It should be noted that the means 12 of the device 1 or of the channel 3 may theoretically be at least substantially identical or may differ as necessary, for example because of the use of different liquids or the like.
In the embodiment shown the substrate 4 is preferably movable starting from position A via positions B, C and D into position E. Depending on the reaction taking place or for other purposes the retention time of the substrate 4 in the individual positions or regions and/or in the individual liquids or in the sample 2 can be varied at will. It is also possible for the substrate 4 to be additionally moved as desired within the respective region or around the respective position, i.e. for example in the sample 2 and/or in the liquids 15 or 16.
As already pointed out, the detection particularly preferably takes place in a gas space. In the embodiment shown, detection may take place in the region b or d or in position B or D. If desired, detection may also take place in both regions or positions, i.e. detection may be carried out in several different regions or positions. If required, different detections may also be carried out in the different regions or positions. Thus, for example, multi-step or different reactions may be carried out, detected or monitored one after another.
As already mentioned, in the embodiment shown, the substrate 4 is preferably moved into different reaction chambers. If necessary, the respective liquid in a reaction chamber or in a region may be changed. For example, instead of the sample 2 that forms a first liquid, a different liquid may be introduced into the region a beforehand or afterwards. In order to change the liquid, or during the changing of the liquid, the substrate 4 may then either remain in region a, as desired, or be moved into another region, particularly into the gas space or adjacent region b.
The individual regions may be of different lengths. Preferably, individual regions or all of the regions filled with liquid, in this case regions a, c and e, may each be matched to the size or length of the substrate 4, so that, if desired, the substrate 4 is sufficiently movable within the respective region or within the respective liquid but on the other hand the volume of liquid required is low or even minimised. This can be achieved by correspondingly adapting the length of the respective region. Particularly preferably, the length of the respective region and/or the longitudinal extent of the means 12 is preferably at least substantially between 1.2 times and 10 times, most preferably between 1.5 times and 3 times the length of the substrate 4 or the extent of the substrate 4 in the direction of movement.
As a result of the displacement of the volume by the substrate 4 it may be possible or useful to make the dimensions of the required volume of liquid such that, in particular, the preferred longitudinal dimensions or at least substantially total filling the respective regions are reached (particularly first) in the state where the substrate 4 is located in the liquid. This can also lead to a minimising of the volume of liquid required in each case.
Alternatively or additionally the required volumes of liquid can be minimised if the substrate 4 fills the channel 3 over at least 30% of its cross-sectional area, preferably more than 40%, especially more than 50%. In other words, if the width and/or height of the channel 3 is or are matched to the corresponding dimensions of the substrate 4, a reduction in the necessary volumes of liquid and/or the guiding of the substrate 4 in the channel 3 as described previously can also be obtained. Particularly preferably, the substrate is configured to at least substantially match the cross-section of the channel 3 or vice versa.
The substrate 4 is preferably movable in the longitudinal direction of the channel 3 or along the channel 3, as schematically indicated by case V in
In order to enable the desired mobility of the substrate 4, the manipulating device 8 preferably comprises a plurality of correspondingly positioned electromagnets, the electromagnets being, in particular, controllable in the manner required to achieve the desired magnetic field M or the desired gradient.
Preferably the position of the substrate 4 in the device 1 or in the channel 3 or microfluidic system can be detected. This is done inductively, in particular. For example, this can be done inductively by means of a corresponding coil or a plurality of coils and/or by means of the electromagnets or the coils of the electromagnets of the manipulating device 8. However, the detection of the position of the substrate 4 may additionally or alternatively be achieved by some other method, for example optically.
The channel 3 preferably has an at least substantially constant cross-section and/or an at least substantially smooth or flat bottom or other guide surface for the substrate 4. The means 12, trenches 13 and/or grooves 14 or the like preferably constitute only relatively short interruptions and/or cross-sectional enlargements in the direction of movement V of the substrate 4, i.e. they have a particularly short extent in this direction, in particular, in order to enable or ensure good guiding of the substrate 4 and/or ease of movement or sliding of the substrate 4.
However, other design solutions are also possible. For example, guide means may also be formed by the base part 1A or in the channel 3, which are embodied, for example, in the manner of strips, rails or runners and/or which project in order to reduce the sliding resistance of the substrate 4 and/or to guide the substrate 4 to be movable for example in the longitudinal direction.
The device 1 preferably comprises at least one detection device 17 which comprises, in particular, one or more measuring or sensor devices 18, as shown in
The detection device 17 is configured in particular for detecting an analyte or substance, such as a complex 6 of the analyte, optionally also of various substances or complexes 6 of different analytes, which are present on, particularly bound to, a region 5 or in various substances or regions 5 of the substrate 4.
The detection device 17 preferably operates optically, particularly by luminescence or fluorescence measurement. This is possible, for example, by virtue of the fact that the substances or complexes 6 or the analytes that are to be detected contain corresponding optical features or markers. For example, using the detection device 17 or corresponding measuring or sensor devices 18, it is also possible to determine a plurality of different or varying substances or analytes, particularly in or on different areas 5, as shown purely schematically in
The detection is thus preferably carried out optically, particularly by fluorescence or luminescence measurement. However, any other type of detection may be carried out additionally or alternatively.
The measurements of the detection device 17 can be evaluated and/or processed and particularly also displayed or issued in some other way in the detection device 17 and/or separately from it, in a separate evaluating unit (not shown). For example, the content of a particular analyte or the contents of different analytes in the sample 2 can be stored, displayed and/or provided as an output.
As already mentioned, qualitative and/or quantitative detection or measurement may be carried out. The proposed device 1 is particularly suited to or intended for the adaptation and carrying out of miniaturised immunoassays.
The proposed device 1 is, more particularly, a cartridge design.
The proposed device 1 is particularly intended for or suited to measuring parameters or analytes in blood plasma, blood serum or the like as a sample 2.
The device 1 preferably comprises a receptacle 19a for the sample 2 or blood or other body fluid or the like. The receptacle 19a is formed for example as a receiving opening in the cover 1b, but may also take any other form. The receptacle 19a is for example connected to the channel 3 or its region a or the reaction chamber thus formed via a connecting channel 2, in order to convey the sample 2 or the like, more particularly automatically by means of capillary forces, into the reaction chamber or into the region a and fill the latter with the sample 2. The receptacle 19a or device 1 may additionally contain a device for blood separation such as a filter, a membrane or the like, particularly in order to separate off any blood placed therein or for example blood placed in the receptacle 19a or blood introduced by some other method, for example the blood plasma or blood serum, and to convey it as sample 2 into the region a or the reaction chamber formed thereby.
The device 1 makes it possible to minimise the amount of sample and reagent required.
The proposed device 1 allows a simple construction, a simple design, a simple assembly and/or simple handling.
The device 1 provides the possibility of miniaturised detection, particularly by fluorimetry.
The proposed device 1 enables a number of parameters or analytes to be analysed or determined simultaneously.
The proposed device 1 forms an inherently self-contained system, in particular. Preferably, apart from the sample 2 or the blood or other fluids, there should not be any need to add any other reagents, substances or the like.
Most preferably, the device 1 makes it possible to carry out conventional, modified and/or miniaturised immunoassays, most preferably in the form of sandwich assays or competitive immunoassays.
A fundamental idea of the proposed device 1 or of the proposed method is to move a solid body, namely the substrate 4, through a cavity, particularly in the form of the channel 3 or other chamber or in some other, particularly microfluidic system. The movement is carried out in particular by means of a magnetic field gradient, as explained previously.
A particularly preferred process sequence will be described in more detail hereinafter:
A defined volume of the sample 2, preferably blood plasma, is added to the device 1 through the receptacle 19a. The sample 2 is transported or conveyed through the connecting channel 20a into the reaction chamber or into the channel 3 or region a, e.g. driven by pressure and/or by capillary forces.
The reaction chamber or the region a is preferably filled with a defined volume of the sample 2.
The reaction chamber or the region a may if necessary simultaneously serve as a chamber for the reaction and detection.
In the channel 3 or region a or in the reaction chamber the sample 2 is preferably brought into contact with a reagent 21 which is most preferably kept in dry form or is already located in the channel 3 or region a or in the reaction chamber. For example, the reagent is arranged in the cover 1B or applied thereto and dried on.
The reagent 21 is dissolved by the sample 2 and is able to react with the sample 2 or analyte of the sample 2. The dissolving of the reagent 21 may if necessary be assisted by corresponding movement of the substrate 4 in the region a or in the reaction chamber.
If necessary, the substrate 4 may already be in the reaction chamber or in the region a when the sample 2 is introduced, or may only be moved into it subsequently after the sample 2 has been placed therein.
Preferably, the reagent 21 contains a conjugate for an analyte of the sample 2 that is to be analysed. The reagent 21 may if necessary consist at least substantially of only the conjugate and/or may contain other substances and materials. Preferably the reagent 21 or the conjugate has, at least to some extent, a particularly fluorescent and/or luminescent and/or other marker quality.
The reagent 21 or conjugate which is initially stored in dry form is reconstituted with the sample 2. It can then react with the analyte in the sample 2 and, more particularly, form an analyte/conjugate complex 6.
In the embodiment shown, most preferably, catching antibodies 7 or other substances that react with the complex 6 are preferably immobilised on the surface 4A which is preferably visible and/or in particular capable of being analysed by the detection device, or in the preferably visible detection or binding region or regions 5. A number of handling and functional possibilities arise as a result of the mobility of the substrate 4, the geometric freedom in the configuration and arrangement of the regions 5 and/or the controlled movement, particularly by means of a magnetic field M or magnetic field gradients.
Thanks to the uniform, defined and/or reproducible movement possibilities of the substrate 4, the sample 2 and the conjugate may for example ideally be homogenised and reacted. Furthermore, an optimum complex formation or other reaction may be carried out. At the same time the substrate 4 may take on the function of a mixer.
In a second reaction step a sandwich complex may also be formed. The analyte/conjugate complex 6 reacts with the immobilised antibodies 7 on the substrate 4 and forms a sandwich complex (catching antibody-analyte, detector-antibody). This state is schematically shown in
The conjugates may also be referred to as detector antibodies or may contain these or be formed from them, in particular as they are supposed to react or bind specifically with the analyte of the sample 2 that is to be determined.
The reagent 21 or conjugates preferably contain an optical marker or an optically active substance which may optionally also be formed by the detector antibody itself.
After the sandwich reaction or binding of the complexes 6 to the substrate 4 or some other detection reaction, there is usually or always an excess of reagent 21 or of conjugates, markers, optically active substances or the like in the sample 2 and/or on the substrate 4. This excess which is not (correctly) bound should be removed for later detection, particularly for precise quantitative measurement of the analyte. This can preferably be done by washing or by means of a washing liquid, particularly a washing buffer.
According to an alternative embodiment, the washing liquid is introduced into the reaction chamber or the region a and in this way the sample 2 is displaced or removed, for example through an overflow channel (not shown) into an overflow chamber or the like (not shown). In this process the substrate or its surface 4A or the region 5 is washed at the same time. Moving the substrate 4 in the washing liquid, particularly moving it back and forth, can achieve particularly effective washing.
The washing liquid may be introduced, for example, by means of compressed air and/or capillary force and/or by some other method.
The sample 2 may if necessary also be removed or discharged beforehand, i.e. before the introduction of the washing liquid.
According to another alternative embodiment the washing liquid is stored or contained in a separate reaction chamber or in a separate region c of the device 1. In this case the liquid 15 and/or 16, for example, forms a washing liquid of this kind or some other liquid that is different or distinct from the sample 2. The substrate 4 is then washed accordingly, or subjected to other reactions or treatments, by moving into the liquid 15 and/or 16 or into the corresponding regions c and/or e.
The introduction of the liquids 15 and 16, respectively, is preferably carried out through corresponding receptacles 19c and 19e and connecting channels 20c and 20e. The filling may in particular be carried out in a similar manner to the filling of the device 1 with the sample 2, so that the remarks on this subject apply here. The receptacles 20c and 20e are preferably in turn formed by fill openings in the cover 1b. The transfer of the liquids 15 and 16, respectively, into the corresponding regions c and e or reaction chambers formed thereby is in turn preferably carried out by capillary forces, possibly by compressed air and/or by some other suitable method.
The liquids 15 and 16 may theoretically be different liquids. However, they may also be the same liquid, for example a washing liquid. In this case the two regions c and e or the reaction chambers formed by them may if necessary also be capable of being filled with the liquid through a common receptacle.
After the corresponding reactions and the washing and/or other treatments that are preferably provided, detection takes place. The detection is carried out particularly optically, most preferably from the side which is faced by the surface 4a and the region 5 on which the detection is being carried out. It should be noted in this context that, with regard to the plurality of detection or binding regions 5 which are preferably provided, these are most preferably arranged on a common flat side, surface 4a or top of the substrate 4. However, a fundamentally different arrangement is also possible. For example, separate or different regions 5 may also be formed on opposite sides of the substrate 4. If desired, detection may also be carried out from different sides and/or at different points. Moreover, the different regions 5 may be used simultaneously or consecutively for reactions or for binding analytes or the like and/or for the detection.
In the proposed device 1, detection is theoretically possible, as desired, through the base part 1A or through the cover 1B. In the embodiment shown the detection preferably takes place through the cover 1B. The cover 1B is accordingly of sufficiently transparent configuration at least in the detection region required. The substrate 4 preferably faces with its region 5 or regions 5 towards the cover 1B in corresponding manner.
In order to carry out as few phase transitions as possible (gaseous-solid-liquid-solid) during the detection, the substrate 4 or its detection or binding region 5, or several or all of the detection or binding regions 5, may be pressed against the preferably transparent wall. In particular, they may be pressed against the cover 1A or vice versa. The cover 1A may, for example, be elastically deformable for this purpose, and in particular may be in the form of a film. This makes it possible, in particular, to reduce or minimise the effect of the liquid on the optical detection when the substrate 4 for the detection is still in the liquid or is still covered with (traces of) liquid. Disruption caused by transitions from and/or into the gaseous phase can also be avoided. At the same time this results in a particularly precise positioning and/or focusing of the surface for the optical measurement, particularly the preferred measurement of luminescence or fluorescence.
According to a particularly preferred variant the detection or the optical measurement take place in a gas space, i.e. when the substrate 4 has previously been moved out of the respective liquid, such as the sample 2, or liquid 14 or 16. This also helps to minimise unwanted effects of the liquid. Moreover, once again, it is possible for the substrate 4 with its region or regions 5 to be pressed against the, more particularly, transparent wall or cover 1A provided for the measurement.
The detection is preferably carried out optically or by measuring or determining the luminescence, preferably the photoluminescence, particularly the fluorescence. The detection device 17 or its measuring or sensor device 18 is of suitable construction and comprises, for example, a CCD sensor or other photosensor or the like. Moreover, the detection device 11 preferably also comprises a corresponding light source or the like.
In the embodiment shown the detection that preferably takes place in a gas space may occur, for example, in the region b and/or d. Alternatively or additionally, detection may also take place in liquid, i.e. particularly in region a, c and/or e. For calibration purposes and/or to determine the position of the substrate 4, the substrate 4 may be provided with corresponding markings, optically detectable regions, particularly fluorescent regions or the like.
The detection or measurement values may be evaluated in the detection device 11 itself and/or in a subsequent evaluation and may be given out in particular in the form of the content of the analyte in the sample 2 that is to be detected. Where different analytes are to be detected, correspondingly different contents or other different parameters are issued.
The pressing of the substrate 4 against the transparent wall or the cover 1B may also be carried out by means of the manipulating device 8 or magnetically.
The proposed device 1 and the proposed method result in different advantages:
The substrate 4 is preferably movable in contactless manner, particularly magnetically.
The substrate 4 is movable in all directions in space and/or in rotation.
The substrate 4 is movable in a defined manner.
The substrate 4 makes it possible to have very large detection or binding regions 5.
The proposed substrate 4 allows particular sensitivity during detection.
Optimum reaction and process conditions are made possible, particularly for an immunoassay.
There is no need for precise handling of liquid reagents.
A plurality of parameters or analytes can be examined, particularly measured or detected, simultaneously, particularly by partially different immobilisations or antibodies 7, region 5 and/or using different substrates 4.
A simple and/or inexpensive set up is made possible.
A simple assembly and simple introduction of reagents, liquids or the like is made possible.
The substrate 4 can very easily be moved or transferred into different liquids and/or reaction chambers.
Different liquids can be separated from one another by gas phases or gas spaces.
The substrate 4 can be used for different functions, particularly as a transporter and/or substrate for molecules and other substances, particularly biofunctional and/or specific molecules.
Different binding reactions may take place on the substrate 4, particularly for removing interfering substances, for binding molecules, analytes or the like that are to be measured. In particular, there may be any desired interactions between, for example, a coating of the substrate 4 and constituents of the sample 2 that is to be analysed, which are detectable.
The substrate 4 may be used in particular by means of a back and forth or reciprocating movement and/or a rotary movement as a mixer or mixing element for liquids, for phase homogenisation or the like.
The substrate 4 forms a movable surface for carrying out, in particular, multi-step reactions and/or for detection.
The substrate 4 may be used as a valve element with a barrier function for controlled opening and closing of sections and regions, particularly to release liquid in a controlled manner and/or regulate its flow.
The proposed method and the proposed device 1 are universally usable and allow, in particular, the controlled progress of multi-step reactions and/or the detection of all kinds of substances or analytes and/or the determination of all kinds of parameters.
The device 1 or the proposed method may also form part of a larger microfluidic system or process.
For example, it is also possible for the device 1, the channel 3 or the, in particular, microfluidic system to comprise several, particularly two or more substrates 4, which are selectively movable for example at the same time or successively into the sample 2 or reaction chamber or the region 2 and/or into a gas space or other reaction chamber or liquid 15 or 16. The substrates 4 then serve in particular for the detection or determination of different analytes and/or parameters or the like.
The device 1 or the channel 3 is preferably provided with suitable ventilation. In particular, at least one ventilation channel 1C is provided, which is connected in particular to a gas space. In the embodiment shown a separate ventilation channel 1C is connected to a gas space, in this case to the regions b and d. Depending on the sequence of filling of the device 1 or channel 3 with the sample and/or the liquids 15 and 16, a single ventilation channel 1C may also be sufficient.
The ventilation channel 1C may be formed, or terminate, in the base part 1A and/or cover 1B, as selected.
In order to improve the detection and/or determination of the position of the substrate 4, the base part 1 A or the bottom of the channel 3, in particular, may have favourable properties for this purpose such as a specific colour or other surface properties.
According to an alternative embodiment not shown here, the substrate 4 may also have depressions or other means for transporting and/or accommodating the sample 2, for example, especially a defined volume of the sample 2.
Moreover, according to an alternative embodiment (not shown) the substrate 4 may itself also be configured as a microfluidic system with at least one channel, one chamber and/or one reservoir for a reagent or the like.
According to another alternative embodiment the substrate 4 may also serve to transport at least one reagent, for example the reagent 21. For example, the substrate 4 may be provided with the dried-in reagent 21 which is dissolved in desired manner when the substrate 4 is placed in the sample 2 or other liquid, and can then react, for example.
Particularly preferably, the substrate 4 is provided or equipped with all the reagents 21, antibodies 7 and/or other materials or substances which are needed for determining or detecting a particular analyte or several particular analytes or parameters or the like. Thus, depending on the requirements of the desired detection, a suitable substrate 4 may be inserted in the device 1 or channel 3. In this way, different substrates 4 may also be used in a channel 3 or a microfluidic system for determining different analytes, parameters or the like. In particular, the different substrates 4 can then be moved successively into the sample 2 or brought into contact with it.
As required, the detection or binding regions 5 on the substrate 4 may also be omitted, particularly if the substrate 4 is, for example, used only or essentially only for mixing or transporting purposes and/or forms another reaction system or microfluidic system or the like.
It should also be noted that the detection or binding regions 5 may particularly preferably be microstructured, as mentioned previously. However, such microstructuring is purely optional.
Particularly preferably, the immobilising of the antibodies 7 on the substrate 4 or on its surface 4 a or in the respective detection or binding region 5 or in different detection or binding regions 5 may be carried out by prior plasma coating and/or silanisation of the substrate 4, at least in the desired regions. Alternatively or additionally, so-called photolinking of the antibodies 7 may also take place, thus substantially increasing the density of the bound antibodies 7. However, any desired or suitable treatment is theoretically possible. This is a further advantage of the proposed device 1, the proposed substrate 4 and the proposed method.
Further embodiments will be explained in detail hereinafter with reference to
The movement of the substrate 4 is preferably controlled by the manipulating device 8, not shown here.
The device 1 or the channel 3 or the preferably microfluidic system may also comprise a number of branches or one branch with different channel regions or channel branches or the like.
If necessary, a plurality of substrates 4 may also be used in the device 1 or the channel 3 or the system, which may be arranged in desired manner depending on the number of branches, reaction chambers or the like, particularly in order to carry out desired processes, manipulations and/or analyses.
The test device 22 preferably comprises the detection device 17 with the measuring or sensor devices 18 for the detection explained previously, particularly optical detection, most preferably by luminescence and/or fluorescence measurement.
The test device 22 preferably comprises the manipulating device 8 or the electromagnets preferably provided for this purpose and most preferably an associated control or the like (not shown). By means of the manipulating device 8 the substrate 4 is moved in the apparatus or in the channel 3 or microfluidic system in the desired manner as explained previously.
The measured values from the detection device 17 are preferably captured by an evaluating device 24, picked up, further evaluated, stored and/or further processed and given out, for example, through an output device 25 of the test device 22. The output device 25 may be, in particular, a display but could also be an interface or the like.
The test device 22 constitutes a preferably mobile device which can be used together with the device 1 as a POC system. The proposed POC system consisting of test device 22 and device 1 is accordingly suitable for universal use.
The device 1 may for example initially be filled with the sample 2 and then introduced into the receiving region 23 of the test device 22 in order to carry out the desired reaction, detection and evaluation.
It should be noted that the substrate 4 in the device 1 is preferably securely held in a predetermined region, for example the region a or the region b, until the device 1 is actually used, in particular until the sample 2 has been added. For this purpose the device 1 and/or the substrate 4 may be provided with corresponding retaining means. The retaining means may be, for example, attachment means, adhesion means or the like. The retaining means may also be formed by a mechanical or chemical bond or the like. The retaining means may also be formed by a frictional or interlocking connection, for example a clamp or the like. The substrate 4 may also be secured magnetically in a desired position, for example by the installation of a permanent magnet in the device 1 or the base part 1A at a desired location.
Individual features of the various embodiments and the embodiments themselves may be combined as desired.
| Number | Date | Country | Kind |
|---|---|---|---|
| 10002685.5 | Mar 2010 | EP | regional |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/EP11/01254 | 3/14/2011 | WO | 00 | 11/7/2012 |
