Receiving Unit for Receiving a Fluid, Method and Apparatus for Producing a Receiving Unit, Method and Apparatus for Operating a Receiving Unit, and Receiving Device

Abstract

A receiving unit for receiving a fluid has a receiving element with a receiving face and at least one micro-cavity that is arranged and formed in the receiving element on the receiving face in order to receive the fluid. The receiving face further has a hydrophilic surface characteristic in at least one subregion adjoining the at least one micro-cavity.

Claims

1. A receiving unit for receiving a fluid, the receiving unit comprising: a receiving element having a receiving face and at least one micro-cavity defined in the receiving element on the receiving face, the at least one micro-cavity being shaped to receive the fluid, wherein the receiving face has a hydrophilic surface character in at least one subregion bordering the at least one micro-cavity.

2. The receiving unit as claimed in claim 1, wherein the at least one micro-cavity has a side wall aligned substantially perpendicular to the receiving face.

3. The receiving unit as claimed in claim 1, wherein the receiving face is configured at least partly as a silicon nitride layer or silicon oxide layer or a silane layer.

4. The receiving unit as claimed in claim 1, wherein the receiving element is formed of a silicon substrate.

5. The receiving unit as claimed in claim 1, wherein: a plurality of further micro-cavities, are defined in the receiving element on the receiving face and are shaped to receive the fluid, and the micro-cavity and the plurality of further micro-cavities are aligned in an arrangement region in a square, rectangular, round, oval, circular or hexagonal form.

6. The receiving unit as claimed in claim 1, wherein the micro-cavity contains at least one stored reagent and/or additive.

7. The receiving unit as claimed in claim 1, wherein the receiving face has an optically detectable feature which has a predefined position relative to an arrangement of the at least one micro-cavity.

8. A receiving device comprising: the receiving unit as claimed in claim 1; a housing in which the receiving unit is accommodated; a chamber configured to introduce a fluid into the receiving unit; and at least one channel configured to carry the fluid to the receiving unit and/or from the receiving unit.

9. A method for producing a receiving unit, the method comprising: introducing a micro-cavity into a receiving face of a receiving element of the receiving unit, the micro-cavity configured to receive a fluid, so as to produce the receiving unit, wherein the receiving face has a hydrophilic surface character in at least one subregion bordering the micro-cavity.

10. The method for producing the receiving unit as claimed in claim 9, the introducing of the micro-cavity comprising using a deep reactive ion etching method.

11. A method for operating a receiving unit having a receiving element with a receiving face and at least one micro-cavity defined in the receiving element on the receiving face, the at least one micro-cavity being shaped to receive a fluid, the receiving face having a hydrophilic surface character in at least one subregion bordering the at least one micro-cavity, the method comprising: filling the at least one micro-cavity with the fluid; sealing the filled micro-cavity with a second fluid; implementing to enable at least one reaction in the micro-cavity and to obtain a reaction result; and evaluating the reaction result.

12. An apparatus configured to perform and/or activate the steps of the method as claimed in claim 11 in corresponding units.

13. The method as claimed in claim 11, wherein the method is implemented in a computer program configured to perform and/or activate the filling of the at least one micro-cavity, the implementing of the at least one reaction, and the evaluating of the reaction result.

14. The method as claimed in claim 11, wherein the computer program is stored on a machine-readable storage medium.

15. The receiving unit as claimed in claim 5, wherein the receiving face has the hydrophilic surface character between the micro-cavity and the plurality of further micro-cavities.

16. The receiving unit as claimed in claim 7, wherein the optically detectable feature has a predetermined character in relation to its size, shape and/or optical properties.

Description

[0035] Exemplary embodiments of the approach presented here are represented in the drawings and explained in more detail in the description hereinafter. In the drawings:

[0036] FIG. 1 shows a schematic side representation of a receiving device according to one exemplary embodiment;

[0037] FIG. 2A shows a schematic side representation of a receiving unit according to one exemplary embodiment;

[0038] FIG. 2B shows a schematic representation in plan view of a receiving unit according to one exemplary embodiment;

[0039] FIG. 3 shows a schematic side representation of a receiving unit according to one exemplary embodiment;

[0040] FIG. 4 shows a schematic representation of different stages of intermediates in a possible production process for a receiving unit according to one exemplary embodiment;

[0041] FIG. 5 shows a flow diagram of a method for producing a receiving unit according to one exemplary embodiment;

[0042] FIG. 6A shows a perspective representation of a receiving unit according to one exemplary embodiment;

[0043] FIG. 6B shows a perspective representation of a receiving unit according to a further exemplary embodiment;

[0044] FIG. 7 shows a representation for explaining the procedure for ascertaining a reaction result of a polymerase chain reaction obtained in a receiving unit according to one exemplary embodiment;

[0045] FIG. 8 shows a representation of a reaction result obtained in a receiving unit according to one exemplary embodiment, after an entrainment test;

[0046] FIG. 9 shows a representation for explaining the procedure for ascertaining a reaction result of a multiplex test obtained in a receiving unit according to one exemplary embodiment;

[0047] FIG. 10 shows a flow diagram of a method for operating a receiving unit according to one exemplary embodiment; and

[0048] FIG. 11 shows a block diagram of an apparatus according to one exemplary embodiment.

[0049] In the description hereinafter of useful exemplary embodiments of the present invention, the same or similar reference symbols are used for the elements represented in the various figures and acting in a similar way and the description of these elements is not repeated.

[0050] FIG. 1 shows a schematic side representation of a receiving device 100 according to one exemplary embodiment. The receiving device 100 is designed to introduce a fluid into a receiving unit 105 and/or to overlayer the receiving unit 105 with a further fluid, the so-called sealing liquid, on the receiving face 130 at least in subregions thereof and more particularly in the arrangement region of the cavities and more particularly after introduction of the fluid into the receiving unit 105. For this purpose the receiving device 100 has the receiving unit 105 for receiving the fluid, a housing 110 for accommodating the receiving unit 105, a chamber 115 designed to introduce the fluid into the receiving unit 105, and at least one channel 120, designed to carry the fluid to the receiving unit 105 and/or to carry it from the receiving unit 105 and/or to enable venting of the chamber 115 and of the micro-cavities 135, 150. The receiving device 100 optionally has a pump device which is designed to pump the fluid and optionally the sealing liquid through the at least one channel 120. The receiving unit 105 has a receiving element 125 having a receiving face 130 with a hydrophilic surface nature, and at least one micro-cavity 135, which in the receiving element 125 is arranged on the receiving face 130 and is shaped to receive the fluid.

[0051] According to this exemplary embodiment, the receiving element 125 is formed, for example, of a silicon substrate. The receiving face 130 is designed for example at least partly as a silicon nitride layer, silicon oxide layer and/or as a silane layer—for example as a polyethylene glycol-silane layer—and this facilitates, for example, penetration of the fluid into the micro-cavity 135. According to this exemplary embodiment the micro-cavity 135 has side walls 140, which are aligned substantially perpendicular to the receiving face 130, being aligned for example at an angle 145 of between 80° and 100° with respect to the receiving face 130. According to an alternative exemplary embodiment, the micro-cavity 135 has a virtually cylindrical shaping. Moreover, optionally, according to this exemplary embodiment the receiving unit 105 has a plurality of further micro-cavities 150, which in the receiving element 125 are arranged on the receiving face 130 and are shaped to receive the fluid. In this case the micro-cavity 135 and the plurality of further micro-cavities 150 are arranged in an arrangement region not represented here, more particularly in a square, circular, rectangular or oval region of the receiving face, more particularly with a mandated distance from the edge of the receiving face, more particularly according to a hexagonal, square or rectangular scheme; more particularly, between the micro-cavity 135 and the plurality of further micro-cavities 150, the receiving face 130 has a hydrophilic surface character. Additionally, optionally, the receiving unit 105 according to this exemplary embodiment has an optically detectable feature 155, which has a defined position relative to the arrangement of the at least one micro-cavity 130. This means that the optically detectable feature 155 according to this exemplary embodiment has a predetermined character in relation to size and optical properties.

[0052] In other words a micro-cavity array chip, meaning the receiving unit 105, for aliquoting of a fluid, which is also referred to as sample liquid, i.e., for the filling of the micro-cavities 135, 150 in the receiving unit 105 with the sample liquid, by wetting of the receiving face 130 with the sample liquid and successive wetting of the receiving face 130 with a sealing liquid, where the sample liquid remains at least partly in the micro-cavities 135, 150 of the receiving unit, the implementation of mutually independent reactions in the aliquots, i.e., in the sample liquid partitions present in the micro-cavities 135, 150 after aliquoting of the sample liquid, where these partitions may each contain specific reagents stored in the micro-cavities 135, 150, and also a method for producing the receiving unit 105, are presented. The approach presented here relates, accordingly, to an apparatus for distributing a sample liquid over a multiplicity of compartments, which are also referred to as micro-cavities 135, 150, and implementation of a multiplicity of mutually independent reactions in the micro-cavities 135, 150. More particularly there is distribution of the fluid and implementation of the reactions for example in an automated procedure in a microfluidic system, which according to this exemplary embodiment is referred to as receiving device 100.

[0053] As a result of the approach described here, accordingly, a solution is likewise provided which according to this exemplary embodiment, by means of the receiving unit 105, permits simple introduction and storage of, for example, dried reagents in the micro-cavities 135, 150, sufficiently reduces entrainment of the stored reagents during the controlled distribution of the fluid over the micro-cavities 135, 150, exhibits only very low crosstalk of reactions between the various micro-cavities 135, 150, enables (automatable) microfluidic aliquoting of the fluid in the micro-cavities 135, 150, can be produced cost-effectively and/or can be integrated into a receiving device 100, so that fully automated microfluidic processing is achieved.

[0054] The receiving device 100 according to this exemplary embodiment possesses in particular the chamber 115 with advantageously predetermined dimensions 160, which is envisaged for the introduction of the fluid into the micro-cavities 135, 150 and/or for the sealing of the micro-cavities 135, 150 with a second fluid which is not miscible with the fluid. The microfluidic chamber 115 according to this exemplary embodiment possesses at least one channel 120, which is also referred to as a supply and/or removal channel and which is intended for controlled carrying of the fluid or fluids to and from the receiving unit 105. In one advantageous configuration of the receiving device 100, it further comprises a channel system, not represented here, and/or a pump apparatus, not represented, to enable fully automated microfluidic processing of the receiving unit 105.

[0055] This receiving unit 105 according to this exemplary embodiment has the receiving face 130, which is also referred to as a planar surface and which possesses an arrangement of micro-cavities 135, 150 introduced on the receiving element 125, which is formed from a substrate material. According to this exemplary embodiment this receiving face 130, in particular in an immediate environment around the micro-cavities 135, 150, possesses hydrophilic wetting properties. The micro-cavities 135, 150 according to this exemplary embodiment are notable in particular for virtually perpendicular side walls 140, with the receiving face 130 at the micro-cavities 135, 150, or at openings thereof, enclosing in particular an angle 145 of virtually 90° with respect to the side walls 140 of the micro-cavities 135, 150. Located optionally in the micro-cavities 135, 150 is in particular at least one stored substance, also referred to as reagent or additive. The micro-cavities 135, 150 optionally have a virtually cylindrical shape, which permits particularly simple fabrication of the receiving unit 105. The arrangement of the micro-cavities 135, 150 follows, in particular, a square, hexagonal or rectangular scheme, to enable, optionally, a standardized introduction of reagents into the micro-cavities, more particularly with use of an array spotting system, more particularly a piezo-based precision dispensing system. Merely optionally the receiving face 130 according to this exemplary embodiment additionally possesses the optically detectable feature 155, which, for example, has a defined position relative to the arrangement of micro-cavities (20) and has a suitable character in relation to size and optical properties. As a result the feature 155 is detectable in particular by an optically sensitive apparatus such as a camera of an array spotting system and can be used for defined, fully automated introduction of reagents into the arrangement of micro-cavities 135, 150. Alternatively or additionally to this, the feature 155 can be used for a relative positional determination of the micro-cavities 135, 150, especially in the case where reactions implemented in the micro-cavities 135, 150 are evaluated optically.

[0056] The approach presented here describes in summary a receiving unit 105 having a combination of a hydrophilic receiving face 130, with which the fluid comes into contact at least in subregions for the filling of the micro-cavities 135, 150; at least partly perpendicular side walls 140 of the micro-cavities 135, 150, which in particular counteract entrainment of reagents stored in the micro-cavities 135, 150; stored reagents which permit implementation of different, specific detection reactions in the micro-cavities 135, 150; and/or at least one stored additive, such as, for example, a substance which ensures wetting and complete filling of the micro-cavities 135, 150, so that no air is included in the micro-cavities 135, 150, and/or leads to a reduction in the entrainment of the aforementioned reagents stored in the micro-cavities 135, 150; and a use of cavities 135, 150 with a solid bottom. This means no through-holes, thereby facilitating storage of reagents and/or at least one additive in the cavities 135, 150.

[0057] According to this exemplary embodiment, the approach presented here, in addition to a microfluidic functionality in relation to the filling and/or the sealing of the reaction compartments, ensures low crosstalk between reactions implemented in the compartments, meaning micro-cavities 135, 150. The approach presented here further describes wetting properties of the receiving face 130, composed for example of silicon nitride, silicon oxide or a hydrophilic silane layer, more particularly a polyethylene glycol-silane layer, the micro-cavities 135 (for example, with polyethylene glycol as dried additive and primers and probes for a molecular DNA detection reaction as dried reagent and/or a silicon oxide layer, silicon nitride layer or a silane layer as hydrophilic surface), and, for example, a flow cell made of polymer, such as of polycarbonate, for example.

[0058] Alternatively, for example, a component produced in an alternative method not described here may likewise be used for providing the functionalities stated here, but in that case the receiving unit 105 is somewhat more costly and inconvenient to produce, with, for example, two lithography steps, than the receiving unit 105 produced according to the approach described here.

[0059] For preventing or reducing the fluidic crosstalk between adjacent compartments, the prior art makes use in particular of apparatuses which have a hydrophobic surface character between the compartments. This brings with it, however, the disadvantage that the hydrophobic top side makes it more difficult to fill the compartments in the substrate. In particular, according to the prior art—with a hydrophobic top side and a small size of the compartments, in the case, for example, of a lateral dimension/diameter of the compartments in the sub-millimeter range—cavities having sloping side walls, or through-holes, or recesses with a low aspect ratio are used in order to enable simple filling of the compartments with aqueous phases. Measured on the basis of their area, however, cavities having sloping side walls possess a comparatively low volume. This is a disadvantage for implementation of high-multiplex amplification reactions—especially with optical evaluation of the reactions—since on the one hand the desire is for a maximum surface density of reactions proceeding in parallel and on the other hand—because of the small volume of the compartments—only a comparatively weak fluorescence signal is generated, which in the case of optical evaluation leads to a reduced signal-to-noise ratio. Cavities having sloping side walls also make it more difficult to store reagents in them, since the flow profile that develops therein when the compartments are filled with a sample liquid leads preferentially to unwanted entrainment of the stored reagents. Through-holes in turn bring with them the disadvantage that introduction and storage of reagents in the individual reaction compartments is more difficult, as the reagents can be deposited only on the side walls of the through-holes.

[0060] FIG. 2A shows a schematic side representation of a receiving unit 105 according to one exemplary embodiment. The receiving unit 105 represented here may correspond to or be similar to the receiving unit 105 described in FIG. 1. According to this exemplary embodiment, the receiving unit 105 is represented merely in an enlarged form, so that at least one stored reagent 200 in accordance with this exemplary embodiment can be perceived in the micro-cavity 135. This means that according to this exemplary embodiment the receiving unit 105 has at least one stored reagent.

[0061] Moreover, in accordance with this exemplary embodiment, there is emphasized representation of the fact that a center point of the micro-cavities 135, 150 has the same distance 205 from an adjacent micro-cavity 135, 150 as a center point of the optically detectable element 155 has to the center point of the respectively adjacent micro-cavity 135, 150.

[0062] Described in other words is the receiving unit 105 which enables the distribution of the fluid over the micro-cavities 135, 150 and the implementation of a multiplicity of mutually independent reactions in the micro-cavities 135, 150, there being dried reagents 200 stored in the micro-cavities 135, 150. Additionally presented is a method, described in one of the subsequent figures, for producing the receiving unit 105. The receiving unit 105 permits, in particular, reliable introduction of reagents 200 into the micro-cavities 135, 150; reduces entrainment of the reagents 200 stored in the micro-cavities 135, 150 during distribution of the fluid over the micro-cavities 135, 150 sufficiently, to <10%, for example; the receiving device 105 exhibits only very little (<1%) crosstalk of reactions between the various micro-cavities 135, 150 after sealing of the micro-cavities with a suitable sealing liquid; it allows automatable microfluidic aliquoting of the fluid in the micro-cavities 135, 150; and it can be integrated into a microfluidic system, such as the receiving device 100.

[0063] According to this exemplary embodiment, therefore, the receiving unit 105 has the micro-cavities 135, 150 which serve to form microfluidic compartments. These micro-cavities 135, 150 have virtually perpendicular side walls, especially at an interface to a side of the receiving unit 105 that comes into contact with the fluid, and possess, in particular, stored reagents 200 and also a restricted aspect ratio, in order, for example, to prevent unwanted inclusion of air in the micro-cavities 135, 150 during the filling of the micro-cavities 135, 150 with the fluid and to enable complete filling of the micro-cavities 135, 150 with the fluid. The receiving face of the receiving unit 105, which comes into contact with the fluid and via which the micro-cavities 135, 150 are filled, has, according to this exemplary embodiment, a hydrophilic surface character, especially in the immediate environment around the micro-cavities 135, 150, to enable the fluid to penetrate the micro-cavities 135, 150. In a particularly advantageous way, the receiving unit 105 may be part of a receiving device as described in FIG. 1, in order, for example, to enable fully automated microfluidic processing and optionally implementation of reactions in the micro-cavities 135, 150. As a result, this exemplary embodiment enables reliable filling as a result of the hydrophilic surface character of the receiving face, bordering the micro-cavities 135, 150, of the receiving unit 105, storage of reagents 200 and/or of an additive in the micro-cavities 135, 150, and also a suitable aspect ratio of the micro-cavities 135, 150 with the suitably charactered fluid.

[0064] Furthermore, by virtue of the (virtually) perpendicular side walls of the micro-cavities 135, 150, in combination, for example, with a suitable additive, entrainment of the stored reagents 200 during the filling with the fluid can be minimized to <10%, for example. This is the case in particular in comparison to micro-cavities 135, 150 having sloping side walls, with a geometry which, in conjunction with the flow profile that develops, leads in principle to greater entrainment of reagents 200 stored in the micro-cavities 135, 150. Furthermore, with the approach presented here, by means, for example, of sealing of the micro-cavities 135, 150, after the micro-cavities 135, 150 have been filled, for example, with a suitable second fluid, which is not miscible with the fluid, it is possible to achieve only a low level of crosstalk, <1% for example, between adjacent reaction compartments during the implementation of chemical reactions in the micro-cavities 135, 150. Accordingly it is possible to implement mutually independent, spatially separate reactions in the micro-cavities 135, 150. Because of the resultant geometric multiplexing, for example, when suitable target-specific detection reagents are stored in the micro-cavities 135, 150, a fluid can be studied for a multiplicity of different targets. Furthermore, according to this exemplary embodiment, by virtue of the receiving device, a fully automated microfluidic processing is enabled in a particularly advantageous way. The receiving device used for processing the receiving unit 105 may in particular be produced cost-effectively from a polymer or from a combination of polymer materials. In this way the functionality provided by the receiving unit 105 is realized in compact lab-on-chip systems which can be used in molecular laboratory diagnostics.

[0065] FIG. 2B shows a schematic representation of the plan view of a receiving unit 105 according to one exemplary embodiment. The receiving unit 105 possesses micro-cavities 135, 150 which are arranged in a circular arrangement region 600 according to a hexagonal scheme. The outer border (marked by the dot-dashed line) of the arrangement region 600 of the micro-cavities 135, 150 also has a mandated minimum distance from the edge of the receiving face of the receiving unit 105. This edge region can be utilized in particular to enable easy handling of the receiving unit 105 with an automatic charger (pick-and-place robot) and so, for example, to enable easy fabricatability of an above-described receiving device 100, for example. In this exemplary embodiment the receiving unit 105 additionally possesses optically detectable features 155, or, otherwise designated, reference markings, which can be used, for example, for unambiguous assignment and/or symbolic identification of the micro-cavities 135, 150 and/or can be employed, for example, for positional determination of the receiving unit 105 in processing apparatuses having optical detection systems, such as for example for positional determination in a precision dispensing system for the automated introduction of reagents into the micro-cavities 135, 150 and/or, for example, for positional determination in a processing apparatus which by means of an optical system can be used in particular for the detection of fluorescence signals and which is able for example to detect the fluorescence signal profile of, for example, biochemical reactions in the micro-cavities 135, 150.

[0066] FIG. 3 shows a schematic side representation of a receiving unit 105 according to one exemplary embodiment. The receiving unit 105 represented here may correspond to or be similar to the receiving unit 105 described in FIG. 1 or 2. The only difference is the enlarged representation according to this exemplary embodiment, such that the optically detectable element is not depicted.

[0067] FIG. 4 shows a schematic representation of different stages of intermediates in a possible production process 400 for a receiving unit 105 according to one exemplary embodiment. The receiving unit 105 in this case may correspond to or be similar to the receiving unit 105 described in one of FIGS. 1 to 3 and can therefore also be used in a receiving device as described in FIG. 1.

[0068] Serving here as substrate material according to this exemplary embodiment is a receiving element 125 of silicon, which is also referred to as a silicon wafer. First of all the hydrophilic surface character on the substrate material is produced on the receiving face 130. This surface according to this exemplary embodiment is more particularly a silicon nitride surface, which can be generated on the silicon substrate by means, for example, of a method for the deposition of silicon oxide, silicon nitride and polysilicon, and also of metals, said method also being referred to as low-pressure chemical vapor deposition (LPCVD). Especially suitable here in accordance with this exemplary embodiment is a layer system composed for example of 50 nm SiO.sub.2 and 140 nm Si.sub.3N.sub.4 to produce a low-strain Si.sub.3N.sub.4 layer with effective attachment to the silicon substrate. Silicon nitride is suitable as a surface coating according to this exemplary embodiment because it has, in particular, hydrophilic wetting properties. After pretreatment with hexamethyldisilazane (HMDS), for example, a photoresist 405 is applied, which serves as a mask for etching of the micro-cavities into the silicon substrate. According to this exemplary embodiment, after exposure 410 of the photoresist 405 for definition of the structures to be etched, the photoresist is developed. Subsequently, according to this exemplary embodiment, by means of dry CF.sub.4 etching 415, for example, the Si.sub.3N.sub.4 and SiO.sub.2 on the exposed regions 420 are removed. By means of deep reactive ion etching 425, for example, the micro-cavities 135, 150 are introduced into the silicon substrate. Deep reactive ion etching 425 is optimized advantageously in process terms for production of microstructures having virtually perpendicular side walls. By treatment in, for example, an oxygen plasma 430, the remaining photoresist 405 is removed. According to this exemplary embodiment, one or more reagents 200 are introduced into the micro-cavities 135 by means, for example, of a piezo-based precision dispensing system or an array spotting system. With particular advantage a production process 400 of this kind can take place at wafer level, thereby enabling particularly cost-effective and parallelized production of the receiving unit 105. Singularization of the receiving units 105 produced in a parallelized process may be accomplished, for example, by means of sawing, breaking, or another singularization method, for example a laser-based method, such as, for example, the method referred to as Mahoh dicing, in particular after the introduction of the reagents 200 into the micro-cavities 135.

[0069] FIG. 5 shows a flow diagram of a method 500 for producing a receiving unit according to one exemplary embodiment. The method 500 represented here may comprise eight substeps 502, in accordance with the production process 400 described in FIG. 4, and is able to produce a receiving unit as was described in one of FIGS. 1 to 3. According to this exemplary embodiment, the method 500 comprises a step 505 of providing the receiving face and a step 510 of introducing the at least one micro-cavity into the receiving face for receiving the fluid, to produce the receiving unit.

[0070] According to one exemplary embodiment the steps 505, 510 and/or substeps 502 of the method 500 may, in one advantageous version, be omitted and/or implemented in a changed order.

[0071] FIG. 6A shows a perspective representation of a semifinished product during the production of receiving units 105 according to one exemplary embodiment. The receiving unit 105 represented here may correspond to or be similar to the receiving unit 105 described in one of FIGS. 1 to 3. According to this exemplary embodiment, the plurality of further micro-cavities 150 is shaped to receive the fluid. According to this exemplary embodiment, the micro-cavity 135 and the plurality of further micro-cavities 150 are arranged in a virtually square arrangement region 600, in a way such that they follow a square scheme. The receiving face 130 here, in particular between the micro-cavity 135 and the plurality of further micro-cavities 150, has a hydrophilic surface character.

[0072] According to this exemplary embodiment it becomes clear, moreover, that the receiving unit 105, as well as the micro-cavity 135 and the plurality of further micro-cavities 150, has a further plurality 605 of micro-cavities shaped to receive the fluid. According to this exemplary embodiment, the further plurality 605 of micro-cavities is arranged in a further arrangement region 610 such that they form a square, rectangular or hexagonal form, more particularly wherein, between the arrangement region 600 and the further arrangement region 610, a spacing region 615 is arranged in which there are no micro-cavities 135, 150, 605. According to this exemplary embodiment, the spacing region 615 has a width which corresponds, for example, at least to twice the minimum distance of adjacent micro-cavities of the arrangement region 600 or to the further arrangement region 610.

[0073] In order words, according to this exemplary embodiment, a representation of a processed silicon wafer having micro-cavities 135, 150, 605 is reproduced, after, for example, implementation of the method described in FIG. 5 for producing a receiving unit 105.

[0074] FIG. 6B shows a perspective representation of a silicon substrate having predetermined breakage points introduced, for forming a plurality of receiving units before singulation of the substrate. The receiving units each have a micro-cavity and a plurality of further micro-cavities, which are arranged in a (virtually) circular arrangement region according to a hexagonal scheme. Additionally the receiving units each possess optically detectable features for the purpose, for example, of introducing reagents into the micro-cavities by means of a precision dispensing system and/or for determining position of the receiving unit in a detector and/or for unambiguously identifying the micro-cavities.

[0075] FIG. 7 shows a representation for explaining the procedure for ascertaining a reaction result 700 of a polymerase chain reaction, obtained in a receiving unit 105 according to one exemplary embodiment. A reaction result 700 of this kind may be obtained in a receiving unit 105 as has been described in one of the above-presented FIGS. 1 to 3.

[0076] In other words, for example, the sample liquid used, also referred to as fluid, was a so-called PCR master mix, which contained a target gene with a concentration of 10 initial copies per micro-cavity (25 nl). The PCR master mix additionally contained a target-specific TaqMan fluorescent probe (Cy3) which indicates amplification of the target gene.

[0077] Illustrated schematically in FIG. 7a is a fluorescence micrograph of the fluid distributed over the micro-cavities of the receiving unit 105, which may also be referred to as apparatus, the micrograph having been recorded during temperature cycling for the implementation of the polymerase chain reactions. The micro-cavities containing fluid in which a significant quantity of the PCR product has already been generated appear pale, owing for example to the cleaving of the fluorescence probe. According to this exemplary embodiment, the micro-cavities without a significant quantity of the PCR product appear dark.

[0078] FIG. 7b shows a signal profile belonging to the micro-cavity “F3”, which exhibits a sigmoidal rise, which is attributable to the process of a polymerase chain reaction in this micro-cavity.

[0079] FIG. 7c shows normalized, sigmoidally fitted amplification curves for the individual micro-cavities, collected together in a graph. According to this exemplary embodiment, 89 of the 96 micro-cavities show a rise in the fluorescence signal at a mean ci value—meaning PCR cycle at the inflection point of the sigmoidal signal rise—of 31.53 with a standard deviation of 0.81 temperature cycles. According to this exemplary embodiment, 4 micro-cavities show no significant rise in the fluorescence signal during temperature cycles. The remaining 3 micro-cavities exhibit a rise in the fluorescence signal at ci values>45 temperature cycles. FIG. 7d illustrates this using a histogram of the ci values.

[0080] FIG. 7e illustrates this using a map with a spatial distribution of the ci values in a suitable false-color representation. On the basis of FIGS. 7c, d, e it is clear that in a large proportion of the micro-cavities (92.71%), amplification takes place in a ci value range between 30 and 34 temperature cycles. The fluctuation in the ci values measured may be attributed partly to the statistical fluctuation of the number of copies initially present in the micro-cavities. On the basis of a binomial distribution, a fluctuation of between about 2 and 16 initial copies per micro-cavity, corresponding roughly to the 4 PCR cycles stated above, can be assumed for a mean initial copy number of 10 copies per micro-cavity. The number of negative cavities, on the other hand, cannot be attributed solely to the statistical fluctuation of the number of copies in the cavities on the basis of the binomial distribution. A decisive part is played here by the amplification characteristics of the detection reaction, especially the sensitivity, the limit of detection. The micro-cavities with a negative signal profile are due to the fact that there is not always amplification in the case of a small number of copies initially present in a micro-cavity. The sensitivity of the detection reaction selected is too low for this purpose. In additional measurements, a statistical detection limit for the reaction used here was ascertained, at what is called a limit of detection of about 2.5 initial copies per micro-cavity. According to this exemplary embodiment, moreover, the micro-cavities with a negative signal profile show that in these cavities, even after progression of the amplification reaction in the adjacent micro-cavities, there is no significant copy number generated by means of a PCR. Accordingly, these micro-cavities may be employed as an indicator of crosstalk between adjacent reaction compartments. In particular the 3 micro-cavities in which a delayed PCR takes place are potentially relevant for this purpose. The delaying of the sigmoidal rise by more than 10 PCR cycles, indeed, according to this exemplary embodiment, cannot be attributed to the initial statistical fluctuation of the copy numbers.

[0081] Instead, these are possibly delay-positive or false-positive amplification reactions, possibly initiated as a result of the crosstalk between amplification reactions in adjacent micro-cavities. On the basis of the fact that the array exhibits micro-cavities with a negative signal profile, and also of the fact that the rise of the false-positive reactions occurs only with a delay of 10 PCR cycles, it may be concluded, according to this exemplary embodiment, that the crosstalk between reactions implemented in adjacent micro-cavities is

[00001]$<\frac{1}{2^{10}}\approx 0.1\%$

per amplification cycle. In agreement with further, comparable tests, the experiment therefore shows illustratively that the receiving unit 105 is suitable for implementation of (geometrically) multiplex amplification reactions without significant crosstalk between adjacent reaction compartments, also referred to as micro-cavities.

[0082] FIG. 8 shows a schematic representation of a reaction result 800 obtained in a receiving unit 105 according to one exemplary embodiment, after an entrainment test. A reaction result 800 of this kind can be obtained in a receiving unit 105 as described in one of the above-presented FIGS. 1 to 3.

[0083] According to this exemplary embodiment, entrainment of reagents stored in the micro-cavities is studied, this entrainment possibly occurring in the course of so-called microfluidic processing of the receiving unit 105, meaning in the course of controlled filling of the micro-cavities with a fluid and subsequent controlled sealing of the micro-cavities with a second fluid. For this purpose, according to this exemplary embodiment, copies of a target gene, an ABL gene for example, were introduced into (almost) every second micro-cavity, in the form of a chessboard-like pattern, by means of a precision dispensing system/array spotting system, and were stored for example in dried form together with polyethylene glycol (PEG) as additive (see FIG. 8a).

[0084] FIG. 8b shows a schematic illustration of a fluorescence micrograph made during temperature cycling. The micrograph was made after a significant rise in the fluorescence signal was already apparent in a number of micro-cavities, indicating the generation of a PCR product. In the micro-cavities in which about 100 copies of template DNA of the target gene were stored (patterned filling in FIG. 8a), the fluorescence signal observable is greater than in the micro-cavities without stored template DNA (no filling in FIG. 8a). This points accordingly to selective amplification and hence to only a low level of entrainment of stored reagents during the microfluidic processing.

[0085] Shown additionally in FIG. 8c, according to this exemplary embodiment, is a spatial distribution of the ci values. In the micro-cavities in which in each case 100 copies of template DNA were stored, reliable amplification is observable at ci values between 26.8 and 28.8 temperature cycles. In the remaining micro-cavities, conversely, mostly no amplification can be observed within 50 temperature cycles. Only in 8 micro-cavities is there a delayed amplification, with a delay of more than 4 temperature cycles. Accordingly the entrainment of reagents stored in the micro-cavities of the receiving unit 105, occurring during the microfluidic processing of the receiving unit 105, can be quantified at a maximum of about ½.sup.4= 1/16=6.25%. The receiving unit 105 is therefore likewise suitable for the implementation of multiplex amplification reactions wherein target-specific reagents, such as primers and probes, for example, are stored in the micro-cavities.

[0086] FIG. 9 shows a representation for explaining the procedure for ascertaining a reaction result 900, obtained in a receiving unit 105 according to an exemplary embodiment, for a multiplex test. A reaction result 900 of this kind may be obtained in a receiving unit 105 as described in one of the above-presented FIGS. 1 to 3. According to this exemplary embodiment, the reaction result 900 represented is the result from a multiplex test with stored primers and probes. For this purpose, according to this exemplary embodiment, target-specific primers and probes were stored in respectively 12 micro-cavities of the receiving unit 105, in dried form, for example, together with polyethylene glycol as additive, these primers and probes addressing the two target genes “ABL” and “e13a2”. The probes possessed fluorophores corresponding to “Cy3” and “Cy5”, as outlined in FIG. 9a. The sample liquid introduced was a PCR master mix having a concentration of 100 copies of ABL template DNA per micro-cavity, 25 nl for example, and processing took place.

[0087] FIGS. 9b, c illustrate schematically two fluorescence pictures made before and after thermocycling. The representations shown are each made up of two individual fluorescence micrographs, made with the filter sets represented in the horizontal patterning corresponding to the fluorophore Cy3 and with the filter sets represented in the vertical patterning corresponding to the fluorophore Cy5. The pictures do not indicate any significant entrainment of reagents stored in the micro-cavities or crosstalk between reactions which occur in adjacent micro-cavities. Only the micro-cavities with stored primers and probes display a significant fluorescence signal. According to this exemplary embodiment, therefore, the pictures confirm the previous experiments described in FIG. 8 in terms of the low entrainment during the microfluidic processing and the negligible crosstalk between adjacent micro-cavities of the apparatus presented here.

[0088] Shown in FIG. 9d is the sigmoidal signal profile of the micro-cavity “G4”, which indicates a positive detection of the ABL template DNA in the sample liquid by means of polymerase chain reaction.

[0089] FIG. 9e shows a map of a spatial distribution of the ci values. Amplification can be observed in precisely the 12 micro-cavities containing stored primers and probes for the ABL target gene, with ci values in the range between 27.3 and 29.6.

[0090] FIG. 9f shows an associated graph with the normalized amplification curves of the twelve micro-cavities. In summary the measurement underscores the outstanding suitability of the receiving unit 105 for implementation of geometrically high multiplex sample analyses by means of molecular-diagnostics amplification methods.

[0091] FIG. 10 shows a flow diagram of a method 1000 for operating a receiving unit according to one exemplary embodiment. The method 1000 may be used, for example, for a receiving device as described in FIG. 1. This method 1000 comprises a step 1005 of the filling and sealing of the at least one micro-cavity with a fluid or with a second (sealing) fluid, which, for example, has only very little or no miscibility with the fluid; a step 1010 of the implementing of at least one possible reaction in the at least one micro-cavity to obtain a reaction result; and a step 1015 of the evaluating of the reaction result.

[0092] In other words the micro-cavities of the receiving unit are filled with the fluid. Subsequently the micro-cavities already filled beforehand with the fluid are sealed with a second (sealing) fluid, which has only very slight or no miscibility with the fluid. More particularly the second fluid, also referred to as sealing liquid, is a fluorinated hydrocarbon. Furthermore, according to this exemplary embodiment, reactions independent of one another are implemented, more particularly amplification reactions, such as polymerase chain reactions or isothermal amplification reactions, for example, for the detection of at least one target gene, for example, in the micro-cavities of the receiving unit. Reaction conditions suitable for these purposes are optionally produced by external action, such as introduction of heat or removal of heat, for example.

[0093] In one particularly preferred embodiment steps 1005, 1010, 1015 take place in automated form in a processing unit envisaged for the processing of the receiving device.

[0094] According to one exemplary embodiment, steps of the method 1000 may, in one advantageous version, be omitted and/or carried out in a changed sequence.

[0095] FIG. 11 shows a block diagram of an apparatus 1100 according to one exemplary embodiment. According to this exemplary embodiment the apparatus 1100 is designed for the implementation or activation of one of the methods described in FIG. 5 or 10. The apparatus 1100 is designed to read—in an input signal 1105 by means of a read—in unit 1110, for example, and to provide a control signal 1115 by means of a providing unit 1120. According to this exemplary embodiment, the apparatus optionally has an evaluating unit 1125 designed to evaluate information represented by the read—in signal 1105.

[0096] If an exemplary embodiment comprises an “and/or” conjunction between a first feature and a second feature, this should be read as meaning that according to one embodiment, the exemplary embodiment has both the first feature and the second feature, and, according to a further embodiment, it has either only the first feature or only the second feature.

[0097] Illustrative Specifications are Stated Below:

[0098] Thickness of the receiving element (125): 100 μm to 3000 μm, preferably 300 μm to 1000 μm

[0099] Lateral dimensions of the receiving element (125) or of the receiving face (130): 3 mm×3 mm to 30 mm×30 mm, preferably 5 mm×5 mm to 15 mm×15 mm

[0100] Number of the micro-cavity (135) and of the further micro-cavities (150):

[0101] 2 to 2000, preferably 50 to 500

[0102] Volume of the micro-cavity (135):

[0103] 1 nl to 100 nl, preferably 5 nl to 40 nl

[0104] Diameter of the micro-cavity (135):

[0105] 100 μm to 500 μm, preferably 250 μm to 400 μm

[0106] Depth of the micro-cavity (135):

[0107] 100 μm to 500 μm, preferably 200 μm to 300 μm

[0108] Aspect ratio (ratio of depth to diameter) of the micro-cavity (135):

[0109] 0.3 to 1.0, preferably 0.6 to 0.7

[0110] Distance of the edges of the micro-cavity (135) and at least one further micro-cavity (150) bordering the micro-cavity (135):

[0111] 70 μm to 300 μm, preferably 100 μm to 200 μm

[0112] Contact angle of water on the receiving face (130): <10° to 75°, preferably <10° to 40°

[0113] Reagents stored in the micro-cavities (135, 150): target-specific primers and probes, template DNA; additive: polyethylene glycol (PEG) with molecular weight of, for example, 6000 or 2000 and a concentration in the solution of 2-5% (w/v)

[0114] Fluid (Sample Liquid):

[0115] master mix for an amplification reaction such as a PCR or an isothermal amplification method, or constituents thereof, more particularly master mix without primers and/or probes which are present in the micro-cavities (135, 150)

[0116] Second Fluid (Sealing Liquid):

[0117] fluorinated hydrocarbon, such as 3M Fluorinert FC-40, Fluorinert FC-70, or Novec 7500

[0118] Flow rate for the filling and sealing of the micro-cavities (135, 150) of the receiving unit (105) in a receiving device (100) for micro-cavities (135, 150) having diameter of 350 μm and depth of 240 μm, where the chamber (115) has suitable dimensions such as 7 mm×7 mm×1 mm (volume˜50 μl): 5-10 μl/s