Surface-functionalized substrate, and method for producing same

Abstract

The invention relates to a surface-functionalized substrate (20), in particular for a microfluidic cartridge, having oligonucleotide chains (10), each of which is covalently bonded to the surface of the substrate (20). The substrate is produced by providing functionalized oligonucleotide chains (10), providing a substrate (20) with a surface which is functionalized, and bonding the oligonucleotide chains (10) to the substrate by means of a reaction between a functionalization of the oligonucleotide chains (10) and a functionalization of the surface.

Description

[0001] Description

[0002] title

[0003] Surface-functionalized substrate and process for its production

[0004] The present invention relates to a surface-functionalized substrate. Furthermore, the present invention relates to a process for producing the surface-functionalized substrate.

[0005] State of the art

[0006] In molecular diagnostics, diseases can be detected based on nucleic acid biomarkers (DNA or RNA). To detect a target, oligonucleotides are reacted with the nucleic acid biomarkers as primers or probes. If several potential pathogens are to be detected or excluded simultaneously, this can be achieved by performing individual detections with different oligonucleotides in parallel. In a microfluidic device (lab-on-chip), a microcavity array can be provided for this purpose, in which an oligonucleotide is located in each cavity. Alternatively or in addition to separation via individual cavities, multiplexing can be achieved by using different fluorophores within the same cavity. These fluorophores can each be coupled to specific probes.

[0007] WO 2007 / 139723 A1 describes chemically modified oligonucleotide primers for the amplification of nucleic acids that can function as probes. These primers have thermally labile side chains as a chemical modification. Only at the high temperatures of an amplification reaction are the thermally labile side chains cleaved. This can be used to increase the specificity of a detection reaction. Disclosure of the Invention

[0008] The surface-functionalized substrate comprises oligonucleotide chains, each covalently bound to a surface of a substrate, particularly a silicon substrate. This has the advantage of preventing carryover of the oligonucleotide chains.

[0009] This is particularly advantageous when the substrate is a microcavity array and the surface is a surface cavity of the microcavity array. Filling these cavities with a sample liquid to be analyzed is typically done by flushing the microcavity array with this sample liquid. However, this poses the risk that oligonucleotides stored in the cavities may be ejected from them or even carried over to other cavities. This significantly complicates target detection, as the required oligonucleotides are no longer available in sufficient quantities. In the case of significant carryover, this can lead to a false-positive signal in another cavity.

[0010] In particular, the microcavity array is arranged in a microfluidic cartridge, in particular in a chamber of the microfluidic cartridge. The invention therefore also relates to a microfluidic cartridge comprising such a microcavity array. Such cartridges are used as disposable inserts in microfluidic analysis systems. A microfluidic cartridge has a channel system through which a sample liquid can be transported to the microcavity array. Furthermore, reagents can be stored upstream in the microfluidic cartridge, with which the sample is reacted before it is directed onto the microcavity array. Such reagents can, for example, serve for pre-amplification of the sample, since the relevant targets are often present only in very small quantities in patient material.

[0011] In one embodiment of the surface-functionalized substrate, some or all of the oligonucleotide chains are bound to a surface of the substrate via at least one thermolabile functionalization. While thermolabile functionalizations of oligonucleotide chains are known, these traditionally only serve to prevent chemical reactions of the oligonucleotide chains below an amplification temperature. However, such functionalized oligonucleotide chains can be washed out of the cavities of a microcavity array just as easily as unfunctionalized oligonucleotide chains.Attaching oligonucleotide chains to the surface of cavities by other means, for example, using hydrophilic polymers, has the disadvantage that such hydrophilic polymers dissolve more slowly in water than oligonucleotides, resulting in slow release of the oligonucleotides and often delaying amplification reactions in the cavities. It is also possible that the oligonucleotides escape from the polymer matrix too quickly. The use of hydrophobic polymers also makes it difficult to fill the cavities with sample fluid. Overall, therefore, it can be said that the release of oligonucleotides is difficult to predict and control.In contrast, covalent attachment to the surface of the substrate via a thermolabile functionalization enables, on the one hand, the reliable prevention of discharge of the oligonucleotides when filling the cavities and, on the other hand, a rapid release of the oligonucleotide chains when the denaturation temperature is reached.

[0012] In various embodiments of the surface-functionalized substrate, the thermolabile functionalization can, in particular, be either a side chain linked to a phosphate group in the oligonucleotide chain or a terminal functionalization located at a phosphate group at the 5' end of the oligonucleotide chain. If the thermolabile functionalization is a side chain, it can be provided that several phosphate groups of the oligonucleotide, starting from its 3' end, each have a thermolabile functionalization through which they are bound to the substrate. By attaching the oligonucleotide chain to the substrate multiple times, high stringency is achieved.

[0013] In another embodiment of the surface-functionalized substrate, thermolabile functionalization is omitted for at least some of the oligonucleotide chains. Instead, oligonucleotide chains covalently bound to the substrate surface are each bound to a complementary oligonucleotide chain via base pairing. In this case, the oligonucleotide chain covalently bound to the surface does not act as the reaction partner in an amplification reaction; rather, this role is fulfilled by the complementary oligonucleotide chain. This is released from the covalently bound oligonucleotide chain at a typical amplification temperature, as this temperature is sufficient to break the base pairing.

[0014] To avoid unwanted enzyme reactions on the oligonucleotide chain covalently bound to the surface, a preferred embodiment comprises natural and unnatural nucleic bases. Natural nucleic bases are understood to mean adenine, cytosine, guanine, thymine, and uracil. Unnatural nucleic bases are understood to mean all other nucleic bases. Sequences of the natural nucleic bases can form base pairs with the complementary oligonucleotide chain, while the unnatural nucleic bases prevent enzyme reactions. To create multiple attachment options for the complementary oligonucleotide chain to the covalently bound oligonucleotide chain, the covalently bound oligonucleotide chain can preferably comprise multiple sequence repeats of natural nucleic bases, each of which is separated from the other by unnatural nucleic bases.

[0015] In another preferred embodiment of the invention, the oligonucleotide chain is chemically modified. It is particularly preferably embodied as a peptide nucleic acid (PNA). This means that its sugar-phosphate backbone is replaced by pseudopeptides. Such chemically modified oligonucleotide chains also cannot be amplified or elongated enzymatically.

[0016] In the process for producing the surface-functionalized substrate, functionalized oligonucleotide chains are provided. Furthermore, a substrate with a functionalized surface is provided. Finally, the oligonucleotide chains are bound to the substrate by a reaction between the functionalization of the oligonucleotide chains and the functionalization of the surface.

[0017] The reaction is, in particular, an addition reaction or a condensation reaction with elimination of water. The addition reaction can, for example, be an azide-alkyne cycloaddition, as used in click chemistry.

[0018] The functionalization of the oligonucleotide chains preferably includes a thiol group, a primary amine group, or an aldehyde group. Such groups are well suited for the formation of covalent bonds and the reaction conditions that can occur in the presence of an oligonucleotide chain. Furthermore, such reactions can occur below the temperature of amplification reactions. This is advantageous if the functionalization should remain thermolabile so that it can be cleaved from the oligonucleotide chain at a later time during an amplification reaction.

[0019] The surface functionalization preferably comprises a chemical group selected from the group consisting of a thiol group, a carboxylic acid group, an aldehyde group, a sulfonic acid group, an epoxy group, and an isothiocyanate group. Such groups can be easily attached to a substrate surface, such as a silicon surface, as terminal functionalizations of hydrocarbon spacers and also react below conventional amplification temperatures to form covalent bonds.

[0020] One possible method of attachment is to react the functionalized oligonucleotide chains with a bismaleimide in a Michel addition reaction, followed by a further Michel addition reaction of the surface functionalizations with the bismaleimide. This can be achieved by first adding the functionalized oligonucleotide chains to the bismaleimide, followed by a further addition reaction between the bismaleimide and the surface functionalization. Alternatively, the bismaleimide can be first added to the surface functionalization and then reacted with the functionalized oligonucleotide chains. This creates a favorable bond between the oligonucleotide chains and the surface. The bismaleimide can, in particular, be phenylbismaleimide.

[0021] If the functionalization of the oligonucleotide chains is not thermolabile, it is preferred that complementary oligonucleotide chains are bound to the oligonucleotide chains via base pairing in a further reaction step. This is done, in particular, by adding the complementary oligonucleotide chains to the substrate functionalized with the covalently bound oligonucleotide chains and heating the resulting reaction mixture to a temperature of preferably at least 90°C, particularly preferably at least 95°C.

[0022] Short description of the drawings

[0023] Embodiments of the invention are illustrated in the drawings and are explained in more detail in the following description.

[0024] Figure 1 shows the chemical structure of a functionalized oligonucleotide chain in one embodiment of the invention.

[0025] Figure 2 shows the chemical structure of a functionalization of the oligonucleotide chain according to Figure 1.

[0026] Figure 3 shows a reaction scheme for the production of a surface-functionalized substrate according to an embodiment of the invention.

[0027] Figure 4 shows a reaction scheme for the thermal cleavage of an oligonucleotide chain from a surface-functionalized substrate according to one embodiment of the invention. Figure 5 schematically shows a substrate whose surface has multiple functionalizations used in different embodiments of the method according to the invention.

[0028] Figure 6 shows the chemical structure of a functionalized oligonucleotide chain in another embodiment of the invention.

[0029] Embodiments of the invention

[0030] Figure 1 shows an oligonucleotide chain 10 containing several nucleic bases Bi to Bs. A phosphate bridge of the oligonucleotide chain has a functionalization X. This is shown in Figure 2. This is a 14-mercapto-4-oxo-tetradecyl group. This is attached to the phosphate bridge at its 1-position, which is located on the right side in Figure 2. The mercapto group is located at the 14-position on the left side in Figure 2, but is not shown there.

[0031] A complete schematic representation of the functionalized oligonucleotide chain is shown in Figure 3. To produce a surface-functionalized substrate, in a first embodiment of the process according to the invention, it is combined with phenylbismaleimide as bismaleimide 30. In a first Michel addition 41, the mercapto group is added to the carbon-carbon double bond of a maleimide group of the bismaleimide 30. The oligonucleotide chain 10, further functionalized in this way, is then filled into a cavity of a substrate 20, which in the present embodiment is designed as a silicon microcavity array. This substrate is functionalized on its surface with mercaptoethyl groups, which have thiol groups 21 as reactive terminal groups.In a second Michel addition 42, the previously unreacted carbon-carbon double bond of the second maleimide group of bismaleimide 30 reacts with one of these thiol groups 21. Finally, hydrolysis 43 of the two now thio-substituted maleimide groups of bismaleimide 30 occurs. This yields a substrate 20 that is stable at room temperature and whose surface is modified by the oligonucleotide chain 10. However, this modification is thermolabile in the region of the 4-oxotetradecyl group.

[0032] The microcavity array is surface-modified in each of its cavities in the manner described above, for example, with three different types of oligonucleotide chains for the detection of a different target. One type of oligonucleotide chain 10 acts as a forward primer, one type of oligonucleotide chain 10 as a reverse primer, and one type of oligonucleotide chain 10 as a probe. All three oligonucleotide chains 10 are specific for a particular nucleic acid target. The microcavity array is then installed in a microfluidic cartridge (not shown).

[0033] The microfluidic cartridge can be used to analyze a biological sample. After the sample has been pre-amplified in a fluidic layer of the cartridge, the microcavity array is flushed with the resulting sample solution to fill all cavities with the solution. Amplification reagents are added to the sample solution, which enter the cavities together with the pre-amplified sample. Using a heater, the microcavity array is then heated to an initial denaturation temperature. Figure 4 shows that this heating leads to the cleavage of the thermolabile functionality X from the oligonucleotide chain 10. The oligonucleotide chain 10 is thus released from the surface of the substrate 20 and can undergo an amplification reaction together with the sample and the amplification reagents after the reaction mixture has been cooled to an amplification temperature.By using different oligonucleotide chains 10 in the different cavities as probes for different targets, the surface-functionalized substrate 20 enables the detection of a large number of potential pathogens alongside each other.

[0034] In several further embodiments of the method according to the invention, different functional groups can be provided on the oligonucleotide chains 10 and on the substrate 10 for linking the oligonucleotide chains 10 to the substrate 20 than in the first embodiment of the method. Thus, the mercapto group on the functionalization of the oligonucleotide chain 10 can be replaced by a primary amine group. A 2-(N-formyl-N-methyl)aminoethyl group can also function as a thermolabile functionalization of the oligonucleotide chain, with its formyl group, as an aldehyde functionality, effecting the linking of the oligonucleotide chains 10 to the substrate 20. Instead of its mercapto group 21, the substrate 20 can have various other reactive groups 22 to 26, which are shown in Figure 5. Thus, a carboxylic acid group 22, an aldehyde group 23, a sulfonic acid group 24, an epoxy group 25 or an isothiocyanate group 26 may be provided.

[0035] In a further embodiment of the method, the thermally labile 4-oxo-tetradedecyl group X between the oligonucleotide chain 10 and its reactive mercapto group or primary amine group is omitted. Instead, an ethylene group, for example, is arranged between this reactive group and the phosphate group. In this embodiment, the oligonucleotide chain 10 is covalently bound to the substrate 20 without being thermally cleaved from it. It does not function as a probe itself, but rather has at least one base sequence that is complementary to an oligonucleotide acting as a probe. This oligonucleotide is introduced into the cavity of the microcavity array after the oligonucleotide chain 10 has been bound to the substrate 20.By heating to a temperature of 95°C and subsequent cooling to a temperature below 50°C, base pairing is created between the free oligonucleotide chain acting as a probe or primer and the surface-bound oligonucleotide chain. When the sample liquid is introduced into the wells of the microwell array and heated to the denaturation temperature, denaturation occurs, breaking the base pairing and releasing the oligonucleotide chain acting as a probe. Some of the nucleic bases Bi to Bs of the oligonucleotide chain 10 bound to the surface of the substrate 20, which are not complementary to the oligonucleotide chain acting as a probe, are unnatural nucleic bases.This prevents enzymes from binding to this oligonucleotide chain 10 during the amplification reaction taking place in the cavities, or from preventing a reaction, elongation, or amplification if binding occurs. They are not accepted by the enzyme as a template. In yet another embodiment of the method, the backbone of the oligonucleotide chain 10 consists of aminoethylglycine units that are linked to one another via neutral amide bonds instead of the charged phosphodiester bonds according to the previous embodiments. This is shown in Figure 6. The oligonucleotide chain 10 does not have a thermally labile group X, but merely a reactive group R, by means of which the oligonucleotide chain 10 is covalently bound to the substrate 20 without it being able to be thermally cleaved from the substrate.As in the previous embodiment, the oligonucleotide chain 10 does not function as a probe itself, but rather has at least one base sequence that is complementary to an oligonucleotide acting as a probe. This oligonucleotide is introduced into the cavity of the microcavity array after the oligonucleotide chain 10 is bound to the substrate 20. All nucleic bases Bi to Bs are natural nucleic bases. The use of non-natural nucleic bases is not permitted in this embodiment.

[0036] This embodiment is not required because the oligonucleotide chain 10 is a PNA chain that cannot be enzymatically amplified or elongated.

Claims

Claims 1 . Surface-functionalized substrate (20), in particular for a microfluidic cartridge, comprising oligonucleotide chains (10) which are each covalently bound to a surface of the substrate (20).

2. Surface-functionalized substrate (20) according to claim 1, characterized in that the substrate (20) is a microcavity array and the surface is a surface in cavities of the microcavity array.

3. Surface-functionalized substrate (20) according to one of claims 1 to 2, characterized in that at least some of the oligonucleotide chains (10) are each bound to a surface of the substrate (20) via a thermolabile functionalization (X).

4. Surface-functionalized substrate (20) according to claim 3, characterized in that the thermolabile functionalization (X) is a side chain.

5. Surface-functionalized substrate (20) according to claim 3, characterized in that the thermolabile functionalization (X) is a terminal functionalization.

6. Surface-functionalized substrate (20) according to one of the preceding claims, characterized in that a complementary oligonucleotide chain is bound to the oligonucleotide chains (10) via base pairing.

7. Surface-functionalized substrate (20) according to claim 6, characterized in that the oligonucleotide chains (10) comprise natural and unnatural nucleic bases (Bi - Bs).

8. Surface-functionalized substrate (20) according to claim 6 or 7, characterized in that the oligonucleotide chains (10) are peptide nucleic acids.

9. Microfluidic cartridge comprising a surface-functionalized substrate (20) according to one of the preceding claims.

10. A method for producing a surface-functionalized substrate (20) according to one of claims 1 to 8, comprising the following steps: Providing functionalized oligonucleotide chains (10), providing a substrate (20) whose surface is functionalized, and binding the oligonucleotide chains (10) to the substrate by means of a reaction between a functionalization of the oligonucleotide chains (10) and a functionalization of the surface.

11. The method according to claim 10, characterized in that the reaction (42, 43) is an addition reaction or a condensation reaction.

12. The method according to claim 10 or 11, characterized in that the functionalization of the oligonucleotide chains (10) comprises a chemical group selected from the group consisting of a thiol group, a primary amine group and an aldehyde group.

13. Method according to one of claims 10 to 12, characterized in that the functionalization of the surface comprises a chemical group selected from the group consisting of a thiol group (21), a carboxylic acid group (22), an aldehyde group (23), a sulfonic acid group (24), an epoxy group (25) and an isothiocyanate group (26).

14. Method according to one of claims 10 to 13, characterized in that the functionalized oligonucleotide chains (10) are reacted with a bismaleimide (30) in a Michel addition (41) and the functionalizations of the surface are reacted with the bismaleimide (30) in a further Michel addition (42).

15. Method according to one of claims 10 to 14, characterized in that complementary oligonucleotide chains are bound to the oligonucleotide chains (10) via base pairing.

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