ANALYZING DEVICE HAVING FUNCTIONALIZED CRYOGELS

Abstract

A device having a channel with a sequence of compartments having molecules having specific binding sites, this sequence of compartments being suitable for the specific binding of analytes, characterized in that the molecules having specific binding sites are bound to porous cryogels as carriers and the cryogels are chemically bonded to the wall of the channel, a method for producing the device and the use of the device in analytics.

Claims

1. A device comprising a channel including a wall and having a sequence of compartments containing molecules having specific binding sites, said sequence of compartments is adapted for specific binding of analytes, porous cryogels chemically bonded to the wall, and the molecules having specific binding sites are bound to the porous cryogels as a support.

2. The device as claimed in claim 1, wherein the channel comprises a microchannel.

3. The device as claimed in claim 1, further comprising a microfluidic chip on which the channel is located.

4. The device as claimed in claim 1, wherein the channel comprises microfluidic elements.

5. The device as claimed in claim 4, wherein each said microfluidic element has multiple compartments, each comprising the molecules having specific binding sites, said molecules are bound to the porous cryogels in each case and are different for each compartment, and further comprising compartments without cryogels as separation regions.

6. The device as claimed in claim 1, further comprising means for generating pressure differences comprising at least one of a centrifuge, a centrifugal force-generating device, an external upstream or downstream pump system, an absorption material which supply or discharge liquids in a reproducible manner, or a microfluidic pump device.

7. The device as claimed in claim 1, wherein the channel comprises microfluidic channels configured for a flow generated solely or at least in part by capillary forces acting in the microfluidic channels.

8. The device as claimed in claim 1, wherein the cryogels comprise copolymers with functional groups including at least one of vinyl, epoxy or aldehyde groups or benzophenone groups including at least one of, polyurethane, epoxides reacted with polyamines or polyols, amino group-containing molecules bound by dialdehydes, prepolymers, polymers obtainable from ethylenically unsaturated molecules by free-radical chain reaction, azido group-containing monomers or polymers, anthraquinone-, or psoralen (derivative)-consisting or obtainable materials; or copolymers comprising benzophenone groups from/of a copolymer of the simplified formula ##STR00002## where “stat” stands for randomly arranged segments and means a random arrangement of the groups shown.

9. The device as claimed in claim 4, wherein the microfluidic elements are formed of glass or plastic which is transparent for visible or UV light.

10. The device as claimed in claim 1, wherein the channel is a microchannel having a smallest diameter transverse to a longitudinal direction across parts or an entire length of the microchannel of 1000 μm.

11. A process for producing a device as claimed in claim 1, comprising alternately, supplying volumes of initially charged solutions containing, firstly, precursor molecules of the porous cryogels as support and, secondly, the molecules having specific binding sites for the specific binding of analytes that are to be immobilized and are different for each volume, or the precursor molecules thereof, in sequence to the channel; cooling the filled device in order to freeze the solutions contained therein; and then carrying out reactions to develop a formation of the cryogels, a binding thereof to the wall of the channel and a binding of the molecules having specific binding sites that are to be immobilized or the precursor molecules thereof.

12. The process as claimed in claim 11, further comprising triggering the reactions by electromagnetic radiation.

13. The process of claim 11, wherein the channel comprises a microfluidic element.

14. A method of performing an assay using the device of claim 1, comprising conducting a liquid or gaseous sample containing sample molecules through the device and identifying bound molecules.

15. The method as claimed in claim 14, further comprising initially charging detection molecules at a start of the channel which comprises a microfluidic channel, and subsequently carrying out a test in one step, with mass transfer being passively.

16. The method of claim 15, wherein the passive mass transfer includes capillary filling followed by continuous mass transfer by coupling to superabsorbent polymers.

17. The device as claimed in claim 7, further comprising an absorbent material downstream of the microfluidic channels or placed at ends thereof.

18. The process of claim 11, further comprising, supplying, between the volumes containing the cryogel precursors and the molecules having specific binding sites that are to be immobilized or the precursor molecules thereof, precursor molecules of the cryogels without molecules to be immobilized or other liquid or gaseous separation substances in order to form a separation of the cryogels containing different ones of the molecules as the specific binding sites.

19. The process of claim 11, further comprising thawing and rinsing the cryogel.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0050] Particularly preferred embodiments of the invention are also found in the description, the abstract and the claims, which are all incorporated here by reference into the description.

[0051] The figures show:

[0052] FIG. 1: Schematic representation of a capillary during the production process for a capillary-based device according to the invention. The upper part shows a capillary after it has been filled with different polymer solutions, which are separated from one another by air pockets. The lower picture shows the same capillary after freezing and exposure to UV radiation and washing, as described in the example.

[0053] FIG. 2: Schematic representation of a test setup for carrying out enrichment and binding of sample molecules when using a device according to the invention.

[0054] FIG. 3: Schematic representation of a selected capillary as microfluidic element according to the sandwich immunoassay carried out as per the example for the detection of analytes, together with (here exemplarily) fluorescence reader.

[0055] FIG. 4: Measured fluorescence intensities (=average gray value T−average gray value NC) for the IL-6 concentrations used in the example. For each concentration, two data points (two capillaries) are plotted. The blank measurement was carried out five times to ascertain the detection limit (LOD signal=Blank+3σ) via the calibration curves (dotted line, R.sup.2=0.997). LOD=26 pg/mL.

DETAILED DESCRIPTION

[0056] The following example serves to illustrate the invention without limiting its scope, but also represents special embodiments of the invention.

[0057] The relevant test setup is demonstrated purely exemplarily by the figures described in detail, including with respect to the reference signs, hereinbelow (the relevant reference signs can also be used in the more general description and the claims in the case of the corresponding features):

[0058] To demonstrate both the production process for and the bioanalytical applicability of the device according to the invention, commercial glass capillaries as an example of microfluidic elements 1 (Minicaps 5 μL, Hirschmann Laborgeräte GmbH & Co. KG, Hauptstraße 7-15, 74246 Eberstadt, Germany) having a diameter of 450 μm were first treated with a benzophenone-containing silane (triethoxy benzophenone silane, see O. Prucker, C. A. Naumann, J. Rühe, W. Knoll and C. W. Frank, J. Am. Chem. Soc., 1999, 121, 8766-8770) and subsequently filled, with the aid of a peristaltic pump (IPC ISMATEC from Cole-Parmer GmbH, Futtererstr. 16, 97877 Wertheim, Germany), with three different liquid compartments 2, 3 and 4, which were separated from one another in each case by an air pocket as separation substance 5 (see FIG. 1). The liquid compartments were:

[0059] 1. 60 mg/L of a benzophenone-containing copolymer (PDMAA-5% MABP-2.5% SSNa, see M. Rendl et al., Langmuir, 2011, 27, 6116) dissolved in PBS (=NC) (liquid compartment 2 in FIG. 1)

[0060] 2. NC+0.05 mg/mL of an antibody against human interleukin 6 (IL-6) (=T) (MAB206-100, R&D Systems, Bio-Techne GmbH, Borsigstraße 7a, 65205 Wiesbaden-Nordenstadt, Germany) (liquid compartment 3 in FIG. 1)

[0061] 3. NC+0.01 mg/L biotinylated BSA (=PC) (A8549, Sigma-Aldrich Chemie GmbH, Munich, Germany) (liquid compartment 4 in FIG. 1).

[0062] In each case, 0.45 μL of liquid (A, B or C) or air as separation substance 5 were filled in.

[0063] (Note: The formula of the polymer used under 1. can be depicted as follows:

##STR00001##

[0064] The distribution of the individual monomers in the polymer is random.)

[0065] The filled liquid compartments A, B, C were subsequently frozen by cooling the capillaries to −25° C. After freezing, the capillaries were exposed to UV light (365 nm) for 15 min (corresponding to an energy dose of around 30 J/cm.sup.2) (VL-UVA 135.M, 365 nm, 28 mW/cm.sup.2, Vilber Lourmat, Germany) in order to photochemically excite the benzophenone groups. As a result, three processes take place simultaneously through nonspecific, free-radical reactions (C-H insertion reactions): [0066] crosslinking of the polymer strands (development of the cryogel matrix) [0067] attachment of the sample molecules (antibody and BSA) to the cryogel matrix [0068] attachment of the cryogel matrix to the silanized glass capillary wall (spatial fixation of the constituents of the liquid compartments as compartments).

[0069] Cryogels produced by this method had pore sizes in the low μm range (typically 5-25 μm).

[0070] Resultant microfluidic elements 1 are depicted in FIG. 1, bottom. In said figure, 6 refers to the regions without molecules having a specific binding site (regions of the separation substance 5 in FIG. 1, top), containing in this case the wash buffer used for washing. 7, 8 and 9 refer to cryogel sections bound to the fluidic element 1, corresponding to the liquid compartments 2, 3, 4 stated above under A, B and C.

[0071] FIG. 2, besides the cryogel sections 7, 8 and 9, shows exemplarily part of the test setup, wherein further besides already described features from FIG. 1. A gaseous or, in this case, liquid sample containing analytes 11 from a microtiter plate 10 is drawn through the microfluidic element 1—in this case, by means of a pump device 13. In parallel, further (not depicted) such microfluidic elements 1 are fed from different wells having different liquid samples containing analytes 11—each microfluidic element 1 can, then, be connected to a, for example, peristaltic multichannel pump as pump device 13. Thereafter, the assay components, which are initially charged in a multi-well plate (microtiter plate), are drawn successively through the capillaries. (12 shows—unlike in the following example—a possible site for an alternatively possible initial charging of detection molecules).

[0072] In the example, the procedure is as follows: after the exposure, the capillaries (as microfluidic elements 1) were thawed and connected to a peristaltic 12-channel pump as pump device 13 (IPC ISMATEC from Cole-Parmer GmbH, Futtererstr. 16, 97877 Wertheim, Germany). The capillaries were washed for approx. 2 hours with PBS/0.1% BSA at an average flow rate of 2 μL/min. Thereafter, a classic sandwich immunoassay (with optical detection of a fluorescently labeled detection antibody) was carried out using various IL-6 standards (0 to 1000 pg/mL in PBS/0.1% BSA). To this end, the following liquid samples containing analytes 11 and reagents/solutions were pumped in steps through the capillaries for the indicated time:

[0073] 1. IL-6 standards for 90 min

[0074] 2. PBS/0.1% Tween for 10 min

[0075] 3. Biotinylated detection antibody BAF206, R&D Systems, Bio-Techne GmbH, Borsigstraße 7a, 65205 Wiesbaden-Nordenstadt, Germany) (1 μg/mL in PBS/0.1% BSA) for 40 min

[0076] 4. PBS/0.1% Tween for 10 min

[0077] 5. Streptavidin-Cy5 (1 μg/mL in PBS/0.1% Tween) for 20 min (PA45001, GE Healthcare Europe GmbH, Oskar-Schlemmer-Str. 11, 80807 Munich, Germany)

[0078] 6. PBS/0.1% Tween for 15 min.

[0079] The sections 14, 15 and 16 in FIG. 3 that follow from the cryogel sections 7, 8, 9 shown in FIG. 1 and FIG. 2 have the correspondingly specifically bound sample molecules, to which biotinylated detection antibody and streptavidin-Cy5 are bound. Fluorescence is excited by a light source 17 and detected by an optical detector 18 (which can be arranged at any site, for example at the two positions shown).

[0080] In the specific example, the capillaries were analyzed in a commercial fluorescence reader (Fluorescent Array Imaging Reader, Sensovation AG, Markthallenstraße 5, 78315 Radolfzell, Germany).

[0081] The results present with different amounts of IL-6 are shown in FIG. 4. The linearity allows a calibration, by which IL6 samples can then be quantified.