Laboratory-on-a-chip consumable with functionalizable reaction chamber and reaction chamber functionalization method

Abstract

Laboratory-on-a-chip consumable (10) provided with a reaction chamber (5) configured to contain a fluid sample, one or more direct access channels (2) for the functionalization of the reaction chamber (5), where the direct access channels (2) are through holes that extend from the surface of the consumable (10) to the reaction chamber (5), arranged to exclusively functionalize the surface of the reaction chamber (5). By virtue of this functionalization, the capacity to specifically bind the capturing agent in the reaction chamber without affecting the adjacent hydrophobic microfluidic channels is increased, thus reducing contamination and the presence of nonspecific signals generated by the binding of the antibodies to the base material.

Description

TECHNICAL SECTOR

[0001] The invention belongs to the sector of laboratories on a chip, more specifically it refers to a laboratory on a chip for carrying out ELISA tests.STATE OF THE ART

[0002] In ELISA (Enzyme-Linked Immunosorbent Assay) assays, functionalization of the reaction chamber surface is necessary for several reasons: Improvement of specific adhesion.

[0003] The ELISA method is based on the binding of antibodies or antigens to a solid surface, usually a polystyrene plate. However, proteins and other biomolecules do not easily adhere to these types of surfaces without proper treatment. By functionalizing the surface, reactive chemical groups are created that facilitate the adsorption or covalent binding of the biomolecules. This allows the antibodies or antigens to be efficiently immobilized on the surface. Appropriate targeting of biomarkers.

[0004] Antibodies have a specific orientation that must be respected so that they can correctly recognize and bind to their antigens. To perform an immunoassay, whether direct or indirect, the surface functionalization must be carried out in such a way that the antigen-antibody binding sites are available and exposed to the solution, improving the sensitivity and specificity of the assay. Avoid non-specific binding.

[0005] If the plate surface is not adequately treated, biomolecules may bind in an uncontrolled manner, potentially leading to increased background noise and false positives. Functionalization can help minimize nonspecific binding by creating an environment suitable for selective binding of the molecules of interest. Increase the retention capacity of biomolecules.

[0006] Functionalized surfaces can retain more biomolecules in a stable manner, which is important to ensure that the signal detected in the ELISA assay is strong and specific enough to obtain accurate and reproducible results. Common methods of functionalization include treatments with blocking proteins such as albumin or casein to cover undesired areas of the surface and reduce nonspecific binding. Covalent bonds via reactive chemical groups (such as amines, thiols or aldehydes) that react with biomolecules are also used. Another possibility is surface modification with polymers or active layers that increase the affinity for biomolecules.

[0007] In summary, functionalizing the surface of the reaction chamber ensures that the antibodies or antigens are well immobilized, correctly targeted and protected from interference, which improves both the accuracy and sensitivity of the ELISA assay. This functionalization process is one of the common steps within the field of immunoassays, and the presence of a functionalized surface is an essential requirement for carrying out this type of assay. To this end, the most common functionalization methods focus on two main strategies: 1) Functionalization of the reaction surface: mainly used in open systems or those with direct access to the reaction chamber. This method of functionalization is especially useful in hydrophilic materials with the capacity to bind biomolecules to their surface. In the case of closed devices, where access to the reaction chamber is difficult, this type of functionalization is uncommon due to the possible non-specificity derived from the functionalization of the entire fluidic path. In addition, the functionalization of the surface while it is not yet closed is seen to be of little use, since during the process of closing the system, said functionalized surface can be damaged when exposed to agents such as high temperatures or adhesive agents (organic solvents, adhesives, etc.). 2) Functionalization of external agents: such as functionalized particles or porous matrices, which are introduced into the reaction chamber for being contacted with the rest of the fluid reagents. This approach allows for the limitation of the functionalized space, but entails the problem of introducing external agents into a microfluidic system, which may lead to the generation of fluidic artifacts derived from this external body within the fluidic path. However, this is the most widely used functionalization method in microfluidic systems, as it ensures uniform and controlled functionalization within the system despite the problems derived from its use.

[0008] In reference to microfluidic systems, application WO2021233814 A1 describes a laboratory-on-a-chip in which it would be possible to perform different analytical tests by using a biocompatible microfluidic consumable driven by an external actuator system. This document refers to a multi-assay chip, capable of housing all the processes necessary for the creation of a complete analytical panel, including the possibility of integrating an immunoassay by functionalizing its internal surfaces. However, the configuration of fluidic inlets prevents the functionalization of a specific area, thus requiring the functionalization of the entire fluidic path. This process would not only result in a change in the behavior of the fluid within the designated channels, but would also increase the false positive rate by increasing the surface area on which nonspecific binding of biomolecules is possible. Likewise, application WO2021074310 A1 describes a propulsion system associated with a microfluidic consumable in which, using the same propulsion principle described in the present invention, it is possible to perform biological assays; however, the lack of accessibility to the reaction chamber by means other than through complex fluidic channels makes complicated the functionalization of the surface, made of hydrophobic material. The present invention therefore describes a method for, through the alternative configuration of the channels and inlets to the fluidic device, improving the functionalization of the reaction chamber through direct access thereto, thereby improving the specificity of the immunoassay and completing the multi-analytical capacity of the described device without altering the flow within the fluidic system.SUMMARY OF THE INVENTION

[0009] The present invention solves the technical problem outlined above by virtue of a laboratory-on-a-chip consumable provided with specific inlets to functionalize only the reaction chamber. More particularly, the invention relates to a laboratory-on-a-chip consumable, comprising a reaction chamber configured to contain a fluid sample, one or more direct access channels for functionalizing the reaction chamber, at least one reagent inlet hole for introducing reagents into the reaction chamber, a waste chamber for collecting waste fluids, a purge hole for evacuating waste fluids, fluidic channels that join the reagent inlet holes with the reaction chamber and the waste chamber, wherein the direct access channels are through holes that extend from the surface of the functional consumable to the reaction chamber, arranged to exclusively functionalize the surface of the reaction chamber. These direct accesses must be aligned with the reaction chamber, and have channels that communicate them with said chamber without affecting the rest of the fluidic path, avoiding retraction or pushing of other fluids contained in the rest of the channels of the design. Furthermore, these channels must allow direct evacuation of their contents in order to avoid contamination of the reaction chamber contents with possible residues from the functionalization process. Optionally, the consumable can be provided with metallizations to establish electrical connections, and electrical contacts for the interface with an electronic reading device. The consumable is preferably formed by joining several polymer layers, each with a thickness between 0.1 mm and 10 mm. Advantageously, the layers are formed using one or more of the following manufacturing methods: CNC machining, laser ablation, hot stamping or injection molding. Once the reaction chamber is functionalized, the direct access channels can be sealed using a biocompatible adhesive film, leaving them empty and sealed to prevent flow disruption during fluidic actuation of the remaining reagent introduction holes 1, 1'. The invention also comprises a method of functionalizing and manufacturing the chamber comprising: introducing a functionalizing agent into the reaction chamber through the direct access channels, incubating the functionalizing agent to react with the surface of the reaction chamber, washing the reaction chamber to remove the functionalizing agent through the direct access channels, introducing and incubating a capturing agent into the reaction chamber, washing the reaction chamber after the capturing agent has adhered, applying a blocking solution to the reaction chamber to prevent nonspecific binding of biological agents, and sealing the direct access channels after functionalization by a biocompatible adhesive film. The biocompatible adhesive film used to seal the direct access channels is advantageously an optically transparent adhesive film. The functionalizing agent may be a metal-ionic complex, such as a chromium ionic complex. The surface of the reaction chamber may also be functionalized by the addition of a capturing agent, such as an antibody or antigen, after functionalization with the metal-ionic complex.BRIEF DESCRIPTION OF THE FIGURES

[0010] In order to assist in a better understanding of the features of the invention and to complement this description, the following figure is included as an integral part thereof, the nature of which is illustrative and not limiting: Figure 1 shows the essential elements of the invention. Figure 2 shows the functional consumable of the invention from another perspective. Figure 3 includes a layer of biocompatible adhesive for closing the direct accesses to the chamber. DETAILED DESCRIPTION

[0011] Referring to Figure 1, the consumable 10 of the invention is part of a laboratory-on-a-chip composed of said consumable and a reader (not shown in the figures). The consumable is provided with a reaction chamber 5, the direct accesses for the functionalization of said chamber 2 and the rest of the elements of the functional consumable such as the reagent inlet holes 1, 1', the waste chamber 3, the purge hole for said waste 4, metallizations 6, electrical contacts with the electronic interface 7 and fluidic channels 8. The direct accesses 2 allow the functionalization of only the reaction chamber 5, thereby reducing the reactivity of the channels 8 in the event of the possible non-specific binding of biological agents along their path. These channels 8 connect the reagent inlet holes 1, 1' with the reaction chamber 5 and the waste chamber 3, thereby generating the fluidic path within the consumable 10.

[0012] The manufacture of this type of consumable 10 is based on joining different polymer layers that, by their combination, form channels for a complex fluidic path, also including electronic sensors and actuators that communicate with the reading device through an electronic interface (which is not part of the invention) that connects with the electrical contacts 7 of the consumable 10. For this purpose, these layers are made of a functionalizable material with a variable thickness between 0.1 mm and up to 10 mm, and can be shaped by different manufacturing methods (CNC machining, laser ablation, hot stamping or injection molding, among others). Materials suitable for this purpose are Polymethyl methacrylate (PMMA), Polyethylene terephthalate (PET), Polydimethylsiloxane (PDMS), Poly 3,4-ethylenedioxythiophene (PEDOT), Cyclic olefin copolymer (COC), Cycloolefin polymer (COP), Polyether ether ketone (PEEK) or Silicon.

[0013] In this process it is important to take into account the final arrangement of the reaction chamber 5, since the direct accesses 2 must be aligned with the inlet and outlet of the reaction chamber 5 once the consumable 10 has been assembled, being able to pass through different layers until reaching the reaction chamber 5. These direct accesses 2, therefore, consist of through holes that go from the surface of the consumable 10 to the reaction chamber 5, and can be subsequently closed once the functionalization process has finished by using a biocompatible adhesive film. Specifically, these accesses 2 must be shaped so that they are emptied directly and completely during the functionalization process to avoid contamination of the contents of the reaction chamber 5 in the successive steps, and they must also be included in the design so that they do not interrupt the flow from the reagent inlet holes 1, 1' and the reaction chamber 5 itself. In addition, a series of metallized layers must be added to this set of layers which, by means of a photolithography process, will form the electrical paths or metallizations 6 within the consumable 10, as well as the electrical contacts with the electronic interface 7.

[0014] As a functionalizable material for the fabrication of these layers, only polymers are chosen that, although hydrophobic by nature, are capable of being functionalized by exposure to functionalizing agents, with the use of metal-ion conjugates being the best method of functionalization for these surfaces. Being hydrophobic materials, they would also accept the use of other externally functionalized particles, acting in this case as a receiving chamber for them. The possibility of carrying out this functionalization in hydrophobic materials improves the sensitivity and specificity of the system by reducing the possibility of deposits of biomolecules by nonspecific binding, while allowing greater flexibility when choosing the base material on which to manufacture the device.

[0015] For the functionalization of the reaction chamber 5, the first step to be performed is the closing of the holes 1, which can be done by preloading reagents into said inlet holes 1 in order to close the confluent channels 8 in the reaction chamber 5. Once closed, the reaction chamber 5 is exposed to the functionalizing agent (for example, metal-ionic complex of chromium or another transition metal), introducing it through the direct accesses 2, and maintaining an incubation time that allows its reaction with the surface of the chamber. Once this incubation time has passed, the functionalizing agent is removed and washed through the direct accesses 2, then the capturing agent is added (antibody or antigen depending on the type of immunoassay to be performed) and the chamber is incubated for its correct adhesion to the free binding sites generated by the functionalizing agent. Once the capturing agent has been incubated, the chamber is washed through the direct accesses 2 and, as the last step of the functionalization process, a blocking solution is added which binds to the free binding sites to prevent the non-specific binding of other biomolecules in subsequent steps, incubating this solution for the time stipulated in its technical data sheet. Once this incubation time has elapsed, the reaction chamber 5 is emptied by aspiration of the contents through the direct accesses 2 and said direct accesses 2 are closed by using an insulating layer 9 (figure 3) in the form of a biocompatible adhesive transparent film - such as optical adhesive films, commonly used for sealing qPCR plates - for the correct use of the fluidic device in the following steps. From this point on, the direct accesses 2 are rendered useless and must be inert to the rest of the fluidic process carried out inside the consumable 10.

[0016] After completing the specific functionalization of the reaction chamber 5, it is possible to carry out the immunoassay, either immediately after the pretreatment of the surface, or after a time. To carry it out, the sample is introduced through the hole 1 designated for this purpose and the consumable 10 is introduced into a reading device in charge of controlling the fluidic advance within the consumable and the reading of both electrical and light signals emitted by the consumable 10. The reading device is provided with the mechanisms that facilitate the fluid impulses according to the stage of the immunoassay process in which it is, maintaining the incubation time of each step within the reaction chamber 5 and measuring the colorimetric signal generated in the last step of the immunoassay once a chromogen is added, such as 3,3',5,5'-Tetramethylbenzidine (TMB). In this way, the reading device performs the pulses of the reagents necessary for washing, the addition of the secondary antibody and the chromogen.

[0017] This immunoassay process is enhanced by the functionalization directed exclusively to the reaction chamber 5, showing the highest rate of colorimetric signal in reaction chamber 5 and only a negative-like background in channels 8 leading the chromogen into reaction chamber 5. This is due to the ability to specifically bind the capturing agent in the reaction chamber without affecting the adjacent hydrophobic microfluidic channels, thereby reducing the possibility of contamination and nonspecific signal generated by nonspecific binding of antibodies to the base material.

[0018] In view of this description and figures, the person skilled in the art may understand that the invention has been described according to some preferred embodiments thereof, but that multiple variations may be introduced in said preferred embodiments, without exceeding the object of the invention as claimed.

Claims

1. Lab-on-a-chip consumable (10), comprising: - a reaction chamber (5) configured to contain a fluid sample, - one or more direct access channels (2) for functionalizing the reaction chamber (5), - at least one reagent inlet hole (1) for introducing reagents into the reaction chamber (5), - a waste chamber (3) for collecting waste fluids, - a purge hole (4) for evacuating waste fluids, - fluidic channels (8) linking the reagent inlet holes (1) to the reaction chamber (5) and the waste chamber (3), wherein the direct access channels (2) are through holes extending from the surface of the consumable (10) to the reaction chamber (5), arranged to exclusively functionalize the surface of the reaction chamber (5).

2. Lab-on-a-chip consumable (10) according to claim 1, wherein the consumable contains metallizations (6) to establish electrical connections, and electrical contacts (7) for interfacing with an electronic reading device.

3. Lab-on-a-chip consumable (10) according to claim 1, wherein the consumable is formed by bonding several layers of polymer, each with a thickness between 0.1 mm and 10 mm,4. Consumable (10) according to claim 2, wherein the layers are formed using one or more of the following manufacturing methods: CNC machining, laser ablation, hot stamping or injection molding.

5. Consumable (10) according to any of claims 1 to 3, wherein the reaction chamber is functionalized and the direct access channels (2) sealed by means of a biocompatible adhesive film (9).

6. Method for functionalizing a reaction chamber (5) in a consumable (10) according to claims 1 to 4, characterized in that the functionalization comprises the following steps: - introducing a functionalizing agent into the reaction chamber (5) through the direct access channels (2), - incubating the functionalizing agent to react with the surface of the reaction chamber (5), - washing the reaction chamber (5) to remove the functionalizing agent through the direct access channels (2), - introducing and incubating a capturing agent in the reaction chamber (5), - washing the reaction chamber (5) after the capturing agent has adhered, - applying a blocking solution to the reaction chamber (5) to prevent nonspecific binding of biological agents, and - sealing the direct access channels (2) after functionalization using a biocompatible adhesive film.

7. Method according to claim 6, characterized in that the direct access channels (2) are sealed after the functionalization process by means of a biocompatible adhesive film.

8. Method according to claim 7, characterized in that the biocompatible adhesive film used to seal the direct access channels (2) is an optically transparent adhesive film.

9. Method according to any of claims 5-8, characterized in that the functionalizing agent is a metal-ionic complex, such as a chromium ionic complex.

10. Method according to any of claims 5-9, characterized in that the surface of the reaction chamber (5) is further functionalized by the addition of a capturing agent, such as an antibody or antigen, after functionalization with the metal-ionic complex.

11. Method according to any of claims 5-10, characterized in that a blocking solution is added to the reaction chamber (5) after the capturing agent has been immobilized, in order to block non-specific binding sites.

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