DE102024203913B3 - Coating for a cavity array for a microfluidic device in point-of-care diagnostics - Google Patents

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

State of the art

In molecular diagnostics, diseases can be detected based on nucleic acid biomarkers (DNA or RNA) of the pathogens. Particularly in patient treatment, rapid detection of pathogens can determine the preferred treatment options. In this case, clinical routine increasingly requires the simultaneous detection and interrogation of as many pathogens as possible. Probe-based methods (qPCR, microarray, CRISPR/Cas) or sequencing (NGS) are typically used for detection. Since the relevant biomarkers/targets are often present only in very small quantities in the patient material taken, detection is usually carried out during or after nucleic acid amplification of the material to be examined, particularly using (quantitative) polymerase chain reaction ((q)PCR) or isothermal amplification methods. The use of such procedures at the point of care and as near-patient immediate diagnostics can be implemented using microfluidic systems, whereby a patient sample is inserted into a microfluidic cartridge and the cartridge is controlled in an analysis device to carry out nucleic acid amplification inside the cartridge.

In order to simultaneously detect or exclude multiple potential pathogens, a large number of targets are queried in parallel that could be responsible for a specific symptom, which requires 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 possibility for detecting multiple targets simultaneously is to perform individual detections in multiple wells of a well array in parallel. Such a method requires the presence of reagents in the corresponding wells that allow specific target detection (for example, oligonucleotides for use as primers or probes). For example, the patent specification EP 3 993 905 B1 a microfluidic cartridge with such a cavity array (referred to there as a cavity array or chip) with reagents stored in the cavities. One challenge here is to ensure that when the cavities are filled with the sample, the specific reagents within the individual cavities are not carried out and carried into other cavities, since during this phase the cavities are connected to each other via the liquid front. This would significantly complicate target detection, as the required reagents would no longer be available in sufficient quantities and could also lead to a false positive signal in another cavity.

Disclosure of the invention

Advantages of the invention

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

The invention further relates to a cavity array for a microfluidic device and to a microfluidic device having such a cavity array, wherein at least one cavity, preferably a plurality of cavities, has such a coating. The microfluidic device can be designed in particular as a microfluidic cartridge for processing with an analytical device. The invention also relates to a method for coating one, preferably a plurality of cavities of a cavity array. The cavity array can in particular be a substrate, in particular a substrate comprising or consisting of silicon, with recesses on one side as cavities. The cavities can, for example, be at least partially pot-shaped, cylindrical, honeycomb-shaped, hemispherical, and/or rounded. Other geometries of the cavities 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 cover layer of the coating. For example, the opening into the respective cavity can have one or more prongs. Such recesses facilitate filling of the cavities, as capillary forces acting in the narrow spaces 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 (µl), preferably 10 nanoliters (nl) and 300 nl.

The invention advantageously reduces the risk of carryover of specific reagents stored in cavities, in particular into other cavities, during filling of the cavities (filling phase) with a substance to be processed. The release of the reagents can be controlled specifically, particularly when the reagents are used for a processing or detection reaction, which advantageously increases reaction efficiency. The polymer matrix contributes in particular to a delayed release of the embedded substances and thus reduces the risk of carryover to other cavities. The cover layer prevents carryover up to a certain temperature, as it is still sparingly soluble or insoluble at moderate temperatures, particularly a filling temperature between 40 and 45 °C, for example. In particular, the cover layer can be dissolved only after completion of the filling phase and during a denaturation of nucleic acids introduced into the wells with the sample, particularly during the first cycle of a PCR or in preparation for isothermal amplification, in order to release reagents required for amplification, in particular primers and probes, from the polymer matrix. This ensures good availability of the specific reagents in time for the actual amplification, while effectively largely preventing the reagents from being leached into neighboring wells. Furthermore, the invention makes it possible to dispense with the otherwise usual covalent bonding of the reagents to the surface of the wells, thus enabling better mobility of the reagents.

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

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 of 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. The weight ratio between the short-chain and the medium- or long-chain polymers is preferably between 8:1 and 12:1, more preferably 10:1. The concentration of the polyacrylamide preferably does not exceed 1.0, most preferably not more than 0.4% by weight in distilled water to maintain sufficient dispensability of the polymer solution. If the reagents to be embedded comprise oligonucleotides, in particular primers, the polyacrylamides can advantageously form hydrogen bonds with the oligonucleotides via the amide group, thus effectively delaying release. Trehalose can be added to the polymer solution to stabilize the oligonucleotides.

The cover layer preferably comprises or consists of a polymer that is water-soluble upon heating, in particular polysaccharide and/or polyacrylamide. Due to the temperature-dependent solubility, as described above, the protective effect of the cover layer can be selectively removed at a desired time. For example, the cover 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.

A further substance can be added to the cover layer to improve and ensure the filling of at least one cavity with the sample liquid to be analyzed during the filling phase, for example, a surfactant. This can at least partially compensate for the hydrophobicity of the polysaccharide, particularly the agarose, and improve the fillability for a later-added aqueous sample liquid.

1. i. Befüllen, insbesondere kontaktloses Dispensen, zum Beispiel Spotten oder Drucken, der zumindest einen Kavität mit einer Polymermatrix und mit Reagenzien, um in der zumindest einen Kavität die Polymermatrix mit den darin eingebetteten Reagenzien auszubilden
2. ii. Befüllen, insbesondere kontaktloses Dispensen zum Beispiel Spotten oder Drucken , der zumindest einen Kavität mit einer Deckschicht, wobei die Deckschicht ein bei Erwärmung wasserlösliches Polymer für eine Freisetzung der Reagenzien aus der Polymermatrix umfasst.

The method for coating one, preferably several, cavities of a cavity array may comprise the following steps:

1. i. Filling, in particular contactless dispensing, for example spotting or printing, the at least one cavity with a polymer matrix and with reagents in order to form the polymer matrix with the reagents embedded therein in the at least one cavity
2. ii. Filling, in particular contactless dispensing, for example spotting or printing, of at least one cavity with a cover layer, wherein the cover layer comprises a polymer which is water-soluble when heated for releasing the reagents from the polymer matrix.

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

Before filling, the at least one cavity can be hydrophilized at least partially or in regions, for example by contactless dispensing, for example spotting or printing with a surfactant and/or a trehalose.

After filling the cavity with the solution comprising the polymer matrix with the reagents embedded therein, this solution is preferably dried before filling with the The top layer begins to dry. Drying can be achieved, for example, by waiting for a specified time, for example, 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.

Filling with the cover layer may involve multiple filling, in particular spotting with the polymer.

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

Short description of the drawings

Embodiments of the invention are schematically illustrated 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 and having a similar effect, and a repeated description of the elements is omitted.

• 1 ein Ausführungsbeispiel einer erfindungsgemäßen mikrofluidischen Vorrichtung mit einem Ausführungsbeispiel eines erfindungsgemäßen Kavitätenarrays,
• 2a-c schematische Momentaufnahmen zu einem Ausführungsbeispiel des erfindungsgemäßen Beschichtungsverfahren und der erfindungsgemäßen Beschichtung sowie
• 3 ein Flussdiagramm zu dem Ausführungsbeispiel des erfindungsgemäßen Beschichtungsverfahren.

It shows

• 1 an embodiment of a microfluidic device according to the invention with an embodiment of a cavity array according to the invention,
• 2a -c schematic snapshots of an embodiment of the coating method according to the invention and the coating according to the invention and
• 3 a flow chart for the embodiment of the coating method according to the invention.

Embodiments of the invention

1 shows an embodiment of a microfluidic device according to the invention as a microfluidic cartridge 100, which has 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 coating described in the patent specification EP 3 993 905 B1 The microfluidic device, in particular the microfluidic cartridge 100, can be part of a microfluidic system for molecular diagnostics, in particular for the detection of pathogens. For this purpose, the system can comprise an analysis device for receiving and processing the cartridge, as described, for example, in the documents DE 10 2016 222 075 A1 and DE 10 2016 222 072 A1 described.

A biological sample, such as a smear, sputum, or blood, can be collected in a receiving chamber 101 of the cartridge 100 and passed on 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 stored upstream in the cartridge, for example in a reagent chamber 104, which is passed through the processing chamber 102 into a waste chamber 105. If the nucleic acids are still present in cells in the sample, the cells can be lysed to release the nucleic acids, for example by mixing the sample with a lysis buffer stored upstream in another reagent chamber 103 before purification. The lysis buffer can also be a combined lysis and binding buffer, which, in addition to lysis, also creates favorable chemical conditions for binding of the nucleic acids to the filter. After purification, the nucleic acids can be detached from the filter, for example, using an eluate medium, and transported to the amplification chamber 106, in which the cavity array 110, for example in the form of a silicon substrate, is located (in 1 shown schematically from above). The cavity array 110 comprises several cavities 111 - in 1 16 cavities 111 with circular boundaries are shown, by way of example, in which the amplification reactions, for example a PCR each, can proceed in parallel. For example, the cavities 111 are formed as pot-shaped, hemispherical, or cylindrical recesses in the upper side of the silicon substrate and have, for example, a volume between 2 and 80 nanoliters (nl). Reagents required for the PCR and the optical luminescence readout can be stored in the cavities 111, in particular in (freeze-)dried form, for example polymerases, nucleotides, or optical probes. At least one, preferably several, or all of the 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.

The 2a , 2b and 2c show a single cavity 111 of the cavity array 110 in cross section and outline three snapshots of an embodiment of the coating method 500 according to the invention in this cavity 111. 3 shows the steps of this coating process 500 as a flow chart 500.

In a first step 501 of the method 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 comprise polysaccharides, for example dextran, dissolved for example at a concentration of more than 2% by weight, polyamides, for example poly(2-ethyl-2-oxazoline), polyacrylamides, or polyalcohols, in particular polyvinyl alcohols. Before in a third step 503, as in 2b shown, the mixture 11 of polymer matrix and reagents 13 to be accommodated therein is introduced into the cavity 111, the cavity 111 can be at least partially hydrophilized in a second step 502 of the method 500, for example by spotting a surfactant or a trehalose, for example aqueous Tween® 20 with a proportion of 0.01 to 0.1% by weight or a 1.5% trehalose solution. As in 2a As indicated, hydrophilization can be performed by dispensing or spotting drops 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 regime, certain polymers as the polymer matrix and certain concentrations of the polymer matrix solution are advantageous. For example, cavity 111 has a volume between 2 and 80 nanoliters (nLl), so that, for example, between 2 and 80 nanoliters (nLl) of polymer matrix solution are introduced into 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, whose concentration does not exceed 0.4% by weight in distilled water for sufficient dispensability. The polyacrylamides can form hydrogen bonds with the oligonucleotides via the amide group, thus delaying their release when dissolved in an aqueous solution. To stabilize the primers, trehalose, for example, approximately 1% by weight, can be added to the solution.

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

As already explained above, the cover layer 12 has the advantage that when the cavity 111 is filled below the melting temperature of the material of the cover layer, 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, in particular in the denaturation step of the first cycle of a PCR, which usually entails heating to above 90 °C, the cover layer 12 dissolves and the reagents 13 are released from the polymer matrix 11, preferably with a delay.

Claims (16)

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 that is water-soluble when heated for releasing the reagents (13), wherein the cover layer (12) is applied to the polymer matrix (11). Coating (10) according to Claim 1 , wherein the reagents (13) comprise oligonucleotides, in particular primers for nucleic acid amplification. Coating (10) according to Claim 1 or 2 , wherein the polymer matrix (11) comprises one or more polysaccharides, polyamides, polyacrylamides and/or polyvinyl alcohols. Coating (10) according to one of the preceding claims, wherein the polymer matrix (11) is formed from a polymer solution, wherein the polymer solution comprises polyacrylamides having a molecular Weight between 10,000 - 1,000,000, preferably between 40,000 and 150,000. 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 forming the polymer matrix (11), in particular for stabilizing oligonucleotides in the reagents (13) to be embedded. Coating (10) according to one of the preceding claims, wherein the polymer comprises one or more polysaccharides, for example agarose, and/or one or more polyacrylamides. Coating (10) according to Claim 6 , wherein the cover layer (12) comprises agarose having a melting point lower than 55°C. Coating (10) according to one of the preceding claims, wherein a substance, in particular a surfactant, is added to the cover layer (12) for easier filling of the at least one cavity. Cavity array (110) for a microfluidic device (100), wherein at least one cavity has a coating (10) according to one of the preceding claims. 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). Microfluidic device (100), in particular a microfluidic cartridge for processing with an analysis device, for example for detecting pathogens, comprising a cavity array (110) according to one of the Claims 9 or 10 . A method (500) for coating one, preferably multiple, cavities of a cavity array (110), comprising the steps of: • 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 reagents (13) embedded therein in the at least one cavity. • Filling (504) the at least one cavity with a cover layer (12), wherein the cover layer (12) comprises a polymer that is water-soluble upon heating for releasing the reagents (13) from the polymer matrix (11). Procedure (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). Procedure (500) according to Claim 12 or 13 , wherein the at least one cavity is at least partially or regionally hydrophilized, for example by spotting with a surfactant and/or a trehalose, before filling takes place. Method (500) according to one of the Claims 12 until 14 , wherein the filling with the cover layer (12) comprises multiple filling, in particular spotting, with the polymer. Method (500) according to one of the Claims 12 until 15 , wherein a surfactant is added to the cover layer (12) to facilitate filling of the cavities.

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