Coating for a cavity array for a microfluidic device in point-of-care diagnostics

Abstract

The invention relates to a coating (10) for a cavity array (110), wherein the coating (10) comprises a polymer matrix (11), wherein reagents (13) are embedded in the polymer matrix (11), in particular reagents (13) for carrying out a nucleic acid amplification, and wherein the coating (10) comprises a cover layer (12), wherein the cover layer (12) has a polymer, which is water-soluble when heated, for releasing the reagents (13), wherein the cover layer (12) is applied to the polymer matrix (11).

Description

[0001] Description

[0002] title

[0003] Coating for a cavity array for a microfluidic device in point-of-care diagnostics

[0004] State of the art

[0005] In molecular diagnostics, diseases can be detected based on nucleic acid biomarkers (DNA or RNA) of pathogens. Particularly in patient treatment, rapid pathogen detection can determine the preferred treatment options. In this case, clinical routine increasingly demands the simultaneous detection or screening of as many pathogens as possible. Typically, probe-based methods (qPCR, microarray, CRISPR / Cas) or sequencing (NGS) are used for detection. Since the relevant biomarkers / targets are often present only in very small quantities in the patient sample, detection usually takes place during or after nucleic acid amplification of the sample, especially via (quantitative) polymerase chain reaction (qPCR) or isothermal amplification methods.The use of such methods at the point of care and as immediate patient diagnostics can be implemented with microfluidic systems, whereby a patient sample is entered into a microfluidic cartridge and the cartridge is controlled in an analyzer to perform nucleic acid amplification inside the cartridge.

[0006] To detect or rule out multiple potential pathogens simultaneously, a large number of targets that could be responsible for a specific set of symptoms are screened in parallel, requiring the unambiguous detection of each individual target. This can be achieved, for example, by using different detection wavelengths of the specific probe used in qPCR, which is typically limited to a few targets. Another way to detect multiple targets simultaneously is to perform individual assays in multiple cavities of a cavity array in parallel. Such a

[0007] The method requires the placement of reagents in the corresponding cavities, which allow for specific target detection (for example, oligonucleotides for use as primers or probes). For instance, patent EP 3 993 905 B1 describes a microfluidic cartridge with such a cavity array (referred to there as a cavity array or chip) containing pre-filled reagents in the cavities. A challenge here is ensuring that, during sample filling, the specific reagents within the individual cavities are not carried out and into other cavities, as the cavities are interconnected via the liquid front during this phase. This would significantly complicate target detection, as the necessary reagents would no longer be sufficiently available, and could also lead to a false positive signal in another cavity.

[0008] Disclosure of the invention

[0009] Advantages of the invention

[0010] Against this background, the invention relates to a coating for a cavity array. The coating comprises a polymer matrix and a cover layer. Reagents are embedded in the polymer matrix, in particular reagents for carrying out nucleic acid amplification, for example oligonucleotides, primers and / or probes. The cover layer is applied to the polymer matrix and comprises a water-soluble polymer upon heating for the release of the reagents.

[0011] Furthermore, the invention relates to a cavity array for a microfluidic device and to a microfluidic device with such a cavity array, wherein at least one cavity, preferably several cavities, have such a coating. The microfluidic device can, in particular, be configured as a microfluidic cartridge for processing with an analytical instrument. The invention also relates to a method for coating one, preferably several, cavities of a cavity array. The cavity array can, in particular, be a substrate, especially a substrate comprising or consisting of silicon, with recesses on one side serving as cavities. The cavities can, for example, be at least partially cup-shaped, cylindrical, honeycomb-shaped, hemispherical, and / or rounded.Other cavity geometries with lateral recesses, for example one or more prongs, are also possible, wherein the recesses are preferably also filled with the coating and, in particular, covered by the coating's top layer. For example, the opening into the respective cavity can have one or more prongs. Such recesses facilitate filling the cavities, as capillary forces acting in the constrictions of the recesses allow liquid to enter the cavities more easily, and air can also escape more easily during filling. At least some of the cavities can have a volume between 10 picoliters (pl) and 10 microliters (pl), preferably 10 nanoliters (nl) to 300 nl.The invention advantageously significantly reduces the risk of carryover of specific reagents located upstream of cavities, particularly into other cavities, during the filling of the cavities (filling phase) with a liquid sample to be processed or analyzed. Due to the embedding of the reagents in a polymer matrix and subsequent layering with a polymer that is only soluble at higher temperatures, the release of these reagents can be selectively controlled, particularly when the reagents are used for a processing or detection reaction, which advantageously increases reaction efficiency. The polymer matrix contributes in particular to the delayed release of the embedded substances and thus reduces the risk of carryover into other cavities.The capping layer prevents carryover until a certain temperature is reached, as it is still sparingly soluble or insoluble at moderate temperatures, particularly a filling temperature between, for example, 40 and 45 °C. Specifically, the capping layer can only dissolve after the filling phase is complete and during the denaturation of nucleic acids introduced into the cavities with the sample, especially in the first cycle of a PCR or as preparation for isothermal amplification. This allows the release of reagents required for amplification, particularly primers and probes, from the polymer matrix. This ensures good availability of the specific reagents in time for the actual amplification, while effectively preventing reagent migration into neighboring cavities.Furthermore, the invention makes it possible to forego the otherwise usual covalent bonding of the reagents to the surface of the cavities and thus to enable better mobility of the reagents.

[0012] The polymer matrix may include or consist of one or more polysaccharides, polyamides, polyacrylamides, and / or polyvinyl alcohols, for example dextran as a polysaccharide or poly(2-ethyl-2-oxazoline) as a polyamide.

[0013] The polymer matrix can, in particular, be formed from a polymer solution, wherein the polymer solution comprises polyacrylamide with a molecular weight between 10,000 and 1,000,000, preferably between 40,000 and 150,000. According to a particular embodiment, the polymer solution comprises short-chain polyacrylamide with a molecular weight less than 50,000, preferably between 10,000 and 40,000, and / or medium- or long-chain polyacrylamide with a molecular weight greater than 100,000, preferably between 100,000 and 1,000,000. Preferably, the weight ratio between the short-chain and the medium- or long-chain polymers is between 8:1 and 12:1, preferably 10:1. Preferably, the concentration of the polyacrylamide does not exceed 1.0, and most preferably does not exceed 0.4 wt% in distilled water, in order to maintain sufficient dispensability of the polymer solution.If the reagents to be embedded include oligonucleotides, especially primers, the polyacrylamides can advantageously form hydrogen bonds with the oligonucleotides via the amide group, thus effectively delaying their release. Trehalose can be added to the polymer solution to stabilize the oligonucleotides.

[0014] The top layer preferably comprises or consists of a water-soluble polymer, in particular a polysaccharide and / or polyacrylamide, when heated. Due to its temperature-dependent solubility, the protective effect of the top layer can be selectively removed at a desired time, as described above. For example, the top layer comprises or consists of agarose, in particular agarose with a melting point below 55°C, which is also referred to as low-melt agarose.

[0015] An additional substance, such as a surfactant, can be added to the top layer to improve and ensure the filling of at least one cavity with the sample liquid during the filling phase. This can at least partially compensate for the hydrophobicity of the polysaccharide, particularly agarose, and improve the fillability for a subsequently added aqueous sample liquid.

[0016] The method for coating one, preferably several, cavities of a cavity array can comprise the following steps: i. Filling, in particular contactless dispensing, for example spotting or printing, the at least one cavity with a polymer matrix and with reagents to form the polymer matrix with the reagents embedded therein in the at least one cavity; ii. Filling, in particular contactless dispensing, for example spotting or printing, the at least one cavity with a cover layer, wherein the cover layer comprises a water-soluble polymer upon heating for the release of the reagents from the polymer matrix.

[0017] Before filling, the reagents to be embedded can be dissolved in the polymer matrix.

[0018] Before filling, at least one cavity can be at least partially or partially hydrophilized, for example via contactless dispensing, such as spotting or printing with a surfactant and / or trehalose.

[0019] After filling the cavity with the solution containing the polymer matrix and the embedded reagents, this solution is preferably dried before filling with the top layer. Drying can be achieved, for example, by waiting a predetermined time, such as 15 minutes, at room temperature, for example, 20 °C. Alternatively, the cavity or cavity array can be heated for drying, for example, to 40 °C.

[0020] Filling with the top layer may involve multiple filling processes, especially spot-filling with the polymer.

[0021] If the cavities have lateral recesses, as described above, these recesses are preferably also filled and thus coated.

[0022] Brief description of the drawings: Exemplary embodiments of the invention are shown schematically in the drawings and explained in more detail in the following description. The same reference numerals are used for the elements shown in the various figures that have a similar effect, thus avoiding a repeated description of the elements.

[0023] They show

[0024] Figure 1 shows an embodiment of a microfluidic device according to the invention with an embodiment of a cavity array according to the invention.

[0025] Figures 2a-c are schematic snapshots of an embodiment of the coating process and coating according to the invention.

[0026] Figure 3 shows a flowchart for the embodiment of the coating process according to the invention.

[0027] Embodiments of the invention

[0028] Figure 1 shows an embodiment of a microfluidic device according to the invention as a microfluidic cartridge 100, which includes an embodiment of the cavity array 110 according to the invention with an embodiment of the coating 10 according to the invention. The microfluidic cartridge 100 can, for example, be based on a microfluidic cartridge disclosed in German patent EP 3 993 905 B1. The microfluidic device, in particular the microfluidic cartridge 100, can be part of a microfluidic system for molecular diagnostics, especially for the detection of pathogens. For this purpose, the system can include an analyzer for receiving and processing the cartridge, as described, for example, in German patent documents DE 10 2016 222 075 A1 and DE 10 2016 222 072 A1.A biological sample, such as a swab, sputum, or blood, can be taken into a receiving chamber 101 of the cartridge 100 and transferred to a processing chamber 102. In the processing chamber 102, nucleic acids contained in the sample can be purified, for example, by binding the nucleic acids to a filter (not shown) and subsequently removing sample residues using a wash buffer located upstream in the cartridge, for example, in a reagent chamber 104. This wash buffer is then passed through the processing chamber 102 into a waste chamber 105. If the nucleic acids are still contained within cells in the sample, the cells can be lysed to release the nucleic acids, for example, by mixing the sample with a lysis buffer located upstream in a further reagent chamber 103 before purification.The lysis buffer can also be a combined lysis and binding buffer, which, in addition to lysis, also establishes favorable chemical conditions for the binding of nucleic acids to the filter. After purification, the nucleic acids can be released from the filter again, for example with the aid of an eluate medium, and transferred to the amplification chamber 106, in which the cavity array 110, for example in the form of a silicon substrate, is located (schematically shown from above in Figure 1). The cavity array 110 comprises several cavities 111 – in Figure 1, 16 cavities 111 with circular boundaries are shown as an example – in which the amplification reactions, for example, PCR, can take place in parallel. For example, the cavities 111 are formed as pot-shaped, hemispherical or cylindrical recesses in the top surface of the silicon substrate and have, for example, a volume between 2 and 80 nanoliters (nl).In the cavities 111, reagents required for PCR and optical luminescence detection, in particular freeze-dried reagents, such as polymerases, nucleotides, or optical probes, can be pre-positioned. At least one, preferably several or all, cavities 111 have a coating according to the invention, in which at least some of these reagents 13 are contained. In particular, the coating can contain different primers and / or probes for the respective nucleic acid segments to be amplified, depending on the cavity. Figures 2a, 2b, and 2c show a cross-section of a single cavity 111 of the cavity array 110 and sketch three snapshots of an embodiment of the coating process 500 according to the invention in this cavity 111. Figure 3 shows the steps of this coating process 500 as a flowchart 500.

[0029] In a first step 501 of the process 500, the reagents 13 to be incorporated into the coating, in particular primers (oligonucleotides) and probes for pathogen-specific detection, can be dissolved in an aqueous polymer matrix. The polymer matrix can contain polysaccharides, for example dextran dissolved at a concentration of more than 2 wt%, polyamides, for example poly(2-ethyl-2-oxazoline), polyacrylamides, or polyalcohols, in particular polyvinyl alcohols. Before the mixture 11 of polymer matrix and reagents 13 to be incorporated therein is introduced into the cavity 111 in a third step 503, as shown in Figure 2b, the cavity 111 can be at least partially hydrophilized in a second step 502 of the process 500, for example by spotting with a surfactant or trehalose, for example aqueous Tween® 20 with a proportion of 0.01 to 0.1 wt% or a 1.5% trehalose solution.As indicated in Figure 2a, hydrophilization can be carried out by dispensing or spotting with droplets 9 in the picoliter range, for example, 300 to 400 picoliters per drop, from a commercially available dispenser 200, such as the sciFLEXARRAYER S12 from SCIENION GmbH, and alternatively also before the first step 501. Due to the fillability of the cavities 111 in the nanoliter range, certain polymers as polymer matrix and certain concentrations of the polymer matrix solution are advantageous. For example, the cavity 111 has a volume between 2 and 80 nanoliters (nLI), so that, for example, between 2 and 80 nanoliters (nLI) of polymer matrix solution can be introduced into the cavity 111. For example, the polymer matrix solution can comprise polyacrylamides with a molecular weight between 10,000 and 1,000,000, preferably between 40,000 and 150,000, the concentration of which does not exceed 0.4 wt% in distilled water for sufficient dispensability.The polyacrylamides can form hydrogen bonds with the oligonucleotides via their amide groups, thereby delaying their release when dissolved in an aqueous solution. Trehalose, for example approximately 1 wt%, can be added to the solution to stabilize the primers.

[0030] As shown in Figure 2c, in a fourth step 504, preferably after drying the mixture 11 of polymer matrix and reagents 13 located in the cavity 111 by heating to 40 °C, a protective coating 12 is applied to the cavity 111 and to the introduced polymer matrix 11 together with the reagents (13) embedded therein, in particular by spotting. A water-soluble polysaccharide that dissolves when heated above a certain temperature can be used as the coating. For example, so-called low-melt agarose with a melting point below 55 °C can be used, for example, in concentrations up to 0.4 wt%. A small amount of surfactant can be added to this solution to facilitate filling the cavity 111, for example, Tween® 20 at a concentration of 0.1 wt%.Instead of agarose, another polysaccharide or a polyacrylamide can also be introduced, preferably repeatedly, as a cover layer 12, in particular by spot application. For the above-mentioned exemplary volume of cavity 111, between 2 and 80 nanoliters (nLI) of cover layer volume can be introduced into the cavity 111.

[0031] As already explained above, the cover layer 12 has the advantage that when the cavity 111 is filled below the melting temperature of the cover layer material, for example at a temperature between 40 and 45 °C in the case of low-melt agarose, the coating 10 does not dissolve, whereas when heated to temperatures above 50 to 60 °C, for example above 52 °C in the case of low-melt agarose, particularly in the denaturation step of the first cycle of a PCR, which usually involves heating to over 90 °C, the cover layer 12 dissolves and the reagents 13 are preferably released from the polymer matrix 11 in a delayed manner.

Claims

Claims 1. Coating (10) for a cavity array (110), wherein the coating (10) comprises a polymer matrix (11) wherein reagents (13) are embedded in the polymer matrix (11), in particular reagents (13) for carrying out nucleic acid amplification, and wherein the coating (10) comprises a cover layer (12) wherein the cover layer (12) has a water-soluble polymer upon heating for the release of the reagents (13), wherein the cover layer (12) is applied to the polymer matrix (11).

2. Coating (10) according to claim 1, wherein the reagents (13) comprise oligonucleotides, in particular primers for nucleic acid amplification.

3. Coating (10) according to claim 1 or 2, wherein the polymer matrix (11) comprises one or more polysaccharides, polyamides, polyacrylamides and / or polyvinyl alcohols.

4. Coating (10) according to one of the preceding claims, wherein the polymer matrix (11) is formed from a polymer solution, the polymer solution comprising polyacrylamides with a molecular weight between 10,000 and 1,000,000, preferably between 40,000 and 150,000.

5. Coating (10) according to one of the preceding claims, wherein trehalose is added to the polymer matrix (11), in particular to the polymer solution for the formation of the polymer matrix (11), especially for stabilizing oligonucleotides in the reagents (13) to be embedded.

6. Coating (10) according to any of the preceding claims, wherein the polymer comprises one or more polysaccharides, for example agarose, and / or one or more polyacrylamides.

7. Coating (10) according to claim 6, wherein the top layer (12) comprises agarose having a melting point of less than 55°C.

8. Coating (10) according to one of the preceding claims, wherein a substance, in particular a surfactant, is added to the top layer (12) for easier filling of the at least one cavity.

9. Cavity array (110) for a microfluidic device (100), wherein at least one cavity has a coating (10) according to one of the preceding claims.

10. Cavity array (110) according to claim 9, wherein the polymer matrix (11) in the at least one cavity is completely covered with the cover layer (12).

11. Microfluidic device (100), in particular a microfluidic cartridge for processing with an analyzer, for example for the detection of pathogens, comprising a cavity array (110) according to one of claims 9 or 10.

12. Method (500) for coating one, preferably several, cavities of a cavity array (110), comprising the steps: • Filling (503) the at least one cavity with a polymer matrix (11) and with reagents (13) in order to form the polymer matrix (11) with the embedded reagents (13) in the at least one cavity • Filling (504) comprising at least one cavity with a cover layer (12), wherein the cover layer (12) comprises a water-soluble polymer upon heating for the release of the reagents (13) from the polymer matrix (11).

13. Method (500) according to claim 12, wherein the reagents (13) are dissolved in the polymer matrix (11) before the at least one cavity is filled with the polymer matrix (11).

14. Method (500) according to claim 12 or 13, wherein the at least one cavity is at least partially or regionarily hydrophilized, for example by means of a Spotting with a surfactant and / or trehalose before filling.

15. Method (500) according to any one of claims 12 to 14, wherein the filling with the top layer (12) comprises multiple filling, in particular spotting, with the polymer.

16. Method (500) according to any one of claims 12 to 15, wherein a surfactant is added to the cover layer (12) for easier filling of the cavities.

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