Reactor for the generation of concentration gradients of fluids

Abstract

The invention relates to a gradient reactor for generating a concentration gradient, comprising: a) a first layer comprising i) at least one fluid inlet for an injection of at least one fluid, ii) at least one dilutant inlet for an injection of a dilutant, and iii) at least one outlet for an ejection of the at least one fluid, b) a second layer, c) a third layer suitable for culturing microorganisms or cells, wherein a concentration gradient is formable in the second layer, and the concentration gradient is transferable from the second layer to the third layer. The invention further relates to a system comprising i) the gradient reactor according to any one of the preceding claims, wherein the gradient reactor optionally further comprises pumps, preferably micropumps, for automated injection of the at least one fluid and the dilutant, ii) a camera system, and iii) a computer program comprising instructions, optionally wherein the instructions, when the program is executed by a computer, cause the computer to analyze the growth, proliferation and / or survival of the microorganisms or cells. The invention also relates to the use of the gradient reactor according to any one of the preceding claims, for the generation of overlapping stationary or dynamic concentration gradients, for analyzing the efficacy or effect of different biologically active compounds, for assessing possible combinatory therapies, for assessing the probability of future resistance to the different biologically active compounds, or for analyzing pharmacokinetics. The invention also relates to a method for generating one or more concentration gradients, comprising: i) providing a gradient reactor according to the invention, or a system according to the invention, ii) simultaneously injecting the dilutant and the at least one fluid, into the at least one dilutant inlet and the at least one fluid inlet, respectively, iii) generating one or more concentration gradients in the second layer, iv) wherein the one or more concentration gradients transfer from the second layer to the third layer, wherein the third layer is suitable for culturing microorganisms or cells.

Description

[0001] REACTOR FOR THE GENERATION OF CONCENTRATION GRADIENTS OF FLUIDS

[0002] DESCRIPTION

[0003] The invention is in the field of reactors used for the generation of (overlapping) concentration gradients of fluids comprising biologically active compounds, and for analyzing the efficacy and / or effect of said biologically active compounds on microorganisms, particularly pathogenic microorganisms, or cells, e.g. tumor cells.

[0004] BACKGROUND OF THE INVENTION

[0005] The significant increase of antibiotic-resistant bacteria represents one of the greatest medical challenges worldwide.

[0006] Several different methods for Antimicrobial Susceptibility Testing (AST) are known in the art. Conventional, phenotypical, (proliferation-based) AST approaches are performed on pure, cultured isolates. The gold standard are dilution approaches in liquid media and agar. Further frequently used methods are the Kirby-Bauer (KB) disk diffusion test and concentration gradient test strips such as the Epsilometertest (HATTAB, S. er al. Rapid Phenotypic and Genotypic Antimicrobial Susceptibility Testing Approaches for Use in the Clinical Laboratory. Antibiotics (Basel). 2024 Aug 22;13(8):786).

[0007] For the KB disk diffusion test, platelets soaked with antibiotics are placed onto inoculated agar plates and the susceptibility is determined based on the size of the inhibition zone around the platelets.

[0008] Epsilometer tests function similarly. Here, strips with predefined gradients of an antimicrobial substance are placed onto inoculated agar plates. Based on the resulting inhibition zone, the minimal inhibitory concentration (MIC) can then be read from a scale on the strip.

[0009] For the aforementioned test systems, commercial solutions exist already, some of which can also be carried out automatically (VAN BELKUM, A. et al. Innovative and rapid antimicrobial susceptibility testing systems. Nat Rev Microbiol. 2020 May;18(5):299-311).

[0010] US 2013 / 0068310 A1 discloses a microfluidic device comprising (a) a porous membrane, (b) a gradient layer defining a plurality of gradient micro-channels, where the gradient layer is coupled to a top surface of the membrane, (c) a distributor layer defining a plurality of distributor microchannels, where the distributor micro-channels are coupled to the plurality of gradient microchannels, where the distributor layer defines at least one inlet opening and at least one outlet opening, each inlet opening and outlet opening are coupled to the plurality of distributor microchannels, and (d) self-supporting means coupled to one or more of the porous membrane, the gradient layer and the distributor layer.US 2020 / 0048680 A1 discloses a fluidic device that has a culture chamber configured to house a 3D culture matrix comprising a culture of microorganisms. A concentration gradient of a test substance is established over the 3D culture matrix by providing respective fluid flows at different end portions of the culture chamber and comprising different concentrations of the test substance. The response of the microorganisms to the test substance is determined based an the position of a border zone in the 3D culture matrix.

[0011] Nevertheless, the required work effort and cost expenditure and the time span until the result is available are still critical parameters, such that the focus also lies on the development of faster methods (“Rapid AST”) since some time. Besides phenotypic approaches, genotypic approaches, which are based on detection of specific genes that convey resistance, play an important role. Several “Rapid AST”-based methods reached already commercial use and are approved for use in clinical diagnostics; further approaches are in the development pipeline of different companies (HATTAB, S. Rapid Phenotypic and Genotypic Antimicrobial Susceptibility Testing Approaches for Use in the Clinical Laboratory. Antibiotics (Basel). 2024 Aug 22;13(8):786; VAN BELKUM, A. Innovative and rapid antimicrobial susceptibility testing systems. Nat Rev Microbiol. 2020 May;18(5):299-311 ; NEEDS, S.H. et al. Challenges in Microfluidic and Point-of-Care Phenotypic Antimicrobial Resistance Tests. Front. Meeh. Eng., 15 September 2020, Sec. Micro- and Nanoelectromechanical Systems, Volume 6 - 2020).

[0012] Due to the increasing problem of antimicrobial resistance, improved systems for susceptibility testing address a major medical need and have a high market potential.

[0013] Current systems often require 5 to 7 days to select an appropriate drug, e.g. an antifungal drug. Hence, therapies often can only slowly be initiated or changed, which contributes to high mortality rates.

[0014] Therefore, further improved systems are needed, which can quickly generate and adapt concentration gradients.

[0015] This would be particularly advantageous for rapid susceptibility and combination therapy testing, as well as to predict resistance development in order to improve diagnostics and therapy guidance so that medicaments, such as antibiotics, can be employed in a more targeted and economic way.

[0016] SUMMARY OF THE INVENTION

[0017] In light of the prior art, the technical problem underlying the present invention is to provide alternative and / or improved means for generating a concentration gradient.

[0018] This problem is solved by the features of the independent claims. Preferred embodiments of the present invention are provided by dependent claims.

[0019] The present invention addresses the above-mentioned need by providing a reactor that can generate one or more compound concentration gradients within, for example, several minutes toa few, e.g. 2, or within 24, hours. The concentration gradients can be stationary or dynamic and it can be changed from a stationary to a dynamic concentration gradient generation and vice versa. Moreover, due to the way the concentration gradients are generated, a plurality of different concentrations and concentration combinations of different compounds can be tested simultaneously. The present invention enables the continuous observation of the evolutive adaptation of microorganisms and cells. Thus, a fast prediction of the current and future effectiveness of the respective (e.g. antibiotic, antifungal, etc.) therapy can be determined.

[0020] The invention relates to a gradient reactor for generating a concentration gradient, comprising:

[0021] a first layer comprising

[0022] i) at least one fluid inlet for an injection of at least one fluid,

[0023] ii) at least one dilutant inlet for an injection of a dilutant, and

[0024] iii) at least one outlet for an ejection of the at least one fluid,

[0025] a second layer,

[0026] a third layer suitable for culturing microorganisms or cells,

[0027] wherein a concentration gradient is formable in the second layer, and the concentration gradient is transferable from the second layer to the third layer.

[0028] In embodiments, the concentration gradient is formable and / or formed in the second layer. In embodiments, the concentration gradient is primarily formed in the second layer and preferably subsequently transferred from the second layer to the third layer.

[0029] In embodiments, the concentration gradient is formed in the second layer, and the concentration gradient is transferred from the second layer to the third layer.

[0030] In embodiments, the second layer comprises a material selected from the group consisting of i) a porous material, such as a glass filter, ii) stainless steel, iii) 3D-printed material, iv) a material comprising microchannels, and v) combinations thereof.

[0031] In embodiments, the second layer comprises or consists of a material selected from the group consisting of i) a porous material, such as a glass filter, ii) stainless steel, iii) 3D-printed material, iv) a material comprising microchannels, v) one or more polymers, and vi) combinations thereof. In embodiments, the second layer comprises or consists of a porous material selected from the group consisting of i) a glass filter, ii) stainless steel, iii) 3D-printed material, iv) a material comprising microchannels, and v) combinations thereof.

[0032] In embodiments, the 3D printed material comprises or consists of one or more polymers, UV-resins, metals (alloys), ceramics, silicium, carbon, cellulose, composite materials, or combinations thereof.

[0033] In embodiments, the second layer comprises or consists of one or more polymers and is preferably porous.The aforementioned materials, such as the porous glass filter, are particularly well suited to enable convection. Convection is beneficial, since it enables a fast, stable, and uniform mixing of liquids and thus a well-defined and reproducible gradient formation.

[0034] In embodiments, the second layer comprises or consists of a material enabling convection, such as a porous material, preferably wherein the material prevents cells or microorganisms from passing into the first layer while allowing compounds, e.g. biologically active compounds, and liquid, such as fluids and dilutants, to pass from the first layer to the second layer.

[0035] The skilled person knows how to test whether a concentration gradient is formable in the second layer and / or whether the concentration gradient is transferable from the second layer to the third layer and the result can be directly and positively verified by routine tests or procedures known to the skilled person and which do not require undue experimentation. The skilled person similarly knows how to select suitable materials for the second and third layer in order to achieve said functions. For example, suitable materials that enable convection and / or diffusion are known. Suitable tests thereto may include, for example, tests to visualize liquid flow / motion and / or density changes, such as a test in which the one or more liquids are stained with a colorant. Additional tests may include, for example, a measurement of a change in pH, and / or the use of fluorescently or otherwise labelled compounds, the motion of which may be traced with suitable cameras, for instance.

[0036] In embodiments, the third layer has a thickness of 0.05-10 mm, preferably 0.1-5 mm.

[0037] Shear forces on the cells or microorganisms due to liquid flow should preferably generally be avoided, since they may lead to, e.g., changes in growth behaviour, changes in motility, a “washing away” of the microorganisms or cells, membrane ruptures, extensional stress, elongational stress, and / or difficulties in performing long-term tests. The arrangement of the gradient reactor advantageously avoids or reduces (excessive) shear forces onto the microorganisms or cells, preferably due to the separation of the third layer from the first layer via the second layer and preferably due to the properties of the second layer to form the gradient, which is then transferred into the third layer, thereby preferably reducing liquid flow speed of the liquid that may come into contact with the microorganisms or cells. In embodiments, the liquid flow has no or only a negligible effect on microorganism or cell growth in the third layer.

[0038] In embodiments, each of the at least one fluids comprises a different biologically active compound, wherein the different biologically active compounds influence or are suspected to influence growth, proliferation and / or survival of microorganisms or cells, preferably wherein the third layer comprises the microorganisms or cells.

[0039] In embodiments, the different biologically active compounds are selected from the group consisting of

[0040] i) antimicrobial agents, such as antibiotic, antiviral, antifungal, or antiparasitic agents, ii) biocides,

[0041] iii) stressors, such as salt,and

[0042] iv) drugs, such as chemotherapeutic agents.

[0043] In embodiments, the concentration gradient is transferable from the second layer to the third layer by diffusion.

[0044] In embodiments, the dilutant is selected from the group consisting of water and medium.

[0045] In embodiments, at least a portion of the at least one fluid inlet is arranged substantially perpendicularly to a surface of the second layer and towards a center of the surface of the second layer.

[0046] In embodiments, the at least one fluid inlet is four fluid inlets, wherein the ends of said fluid inlets, through which the fluids are ultimately injected into the reactor, are arranged to form the corners of a square.

[0047] In embodiments, at least a portion of one of the at least one dilutant inlets is arranged between the end of the at least one fluid inlet through which the fluid is ultimately injected into the reactor, and the at least one outlet.

[0048] In embodiments, the concentration gradient is a stationary or dynamic concentration gradient. In embodiments, the first, second and third layer are parts of the gradient reactor.

[0049] The invention further relates to a system comprising

[0050] the gradient reactor according to the invention, wherein the gradient reactor optionally further comprises pumps, preferably micropumps, for automated injection of the at least one fluid and the dilutant,

[0051] a camera system, and

[0052] a computer program comprising instructions, optionally wherein the instructions, when the program is executed by a computer, cause the computer to analyze the growth, proliferation and / or survival of the microorganisms or cells.

[0053] In embodiments, the camera system enables the taking of one or more images of a region of interest within the third layer and / or the microorganisms or cells.

[0054] In embodiments, the system may comprise an integrated scale for displaying the effective concentration of the compound(s) and mixtures of compounds. In embodiments, said integrated scale replaces the camera (system).

[0055] In embodiments, the analysis of the growth, proliferation and / or survival of the microorganisms or cells comprises the computation of the effective concentration of the different biologically active compounds, preferably the computation of the MIC and / or one or more microorganism or cell growth border zones.The invention further relates to a use of the gradient reactor, system or methods according to the invention, for the generation of overlapping stationary or dynamic concentration gradients, for analyzing the efficacy or effect of different biologically active compounds, for assessing possible combinatory therapies, for assessing the probability of future resistance to the different biologically active compounds, or for analyzing pharmacokinetics.

[0056] In some embodiments, the use may be for generating microorganisms, which have a high salt resistance, and which can, for instance, be used for biogas production from algae.

[0057] The invention further relates to a method for generating one or more concentration gradients, comprising:

[0058] providing a gradient reactor according to the invention, or a system according to the invention,

[0059] simultaneously injecting the dilutant and the at least one fluid, into the at least one dilutant inlet and the at least one fluid inlet, respectively,

[0060] generating one or more concentration gradients in the second layer,

[0061] wherein the one or more concentration gradients transfer from the second layer to the third layer, wherein the third layer is suitable for culturing microorganisms or cells.

[0062] In embodiments, the one or more concentration gradients transfer from the second layer to the third layer, wherein microorganisms or cells are cultured on or in the third layer.

[0063] In embodiments, the concentration gradient is formed by convection in the second layer, and optionally diffusion into the third layer.

[0064] In embodiments, the concentration gradient is formed by mixing of the at least one fluid, preferably at least two fluids or dilutions or mixtures thereof, and optionally the dilutant, in the second layer, preferably by convection, and optionally by a diffusion of the concentration gradient into the third layer.

[0065] DETAILED DESCRIPTION OF THE INVENTION

[0066] The invention relates to a gradient reactor for generating a concentration gradient, comprising:

[0067] a first layer comprising

[0068] i) at least one fluid inlet for an injection of at least one fluid,

[0069] ii) at least one dilutant inlet for an injection of a dilutant, and

[0070] iii) at least one outlet for an ejection of the at least one fluid,

[0071] a second layer,

[0072] a third layer suitable for culturing microorganisms or cells,wherein a concentration gradient is formable in the second layer, and the concentration gradient is transferable from the second layer to the third layer.

[0073] In embodiments, the concentration gradient is formed in the second layer, and the concentration gradient is transferred from the second layer to the third layer.

[0074] In embodiments, not only one concentration gradient, but at least one, e.g. one or two or three or four concentration gradients are formable or formed in the second layer and transferable or transferred from the second layer to the third layer.

[0075] First layer

[0076] The first layer comprises at least one fluid inlet into which a fluid, which preferably comprises a biologically active compound of interest, can be injected.

[0077] In preferred embodiments, at least a portion of said at least one fluid inlet is arranged substantially perpendicularly to a surface of the second layer and towards a center of the surface of the second layer.

[0078] In preferred embodiments, the reactor comprises four fluid inlets.

[0079] In embodiments, the fluid inlets and / or dilutant inlets according to the invention are tubes or channels or similar structures connectable or connected to injection means, which may comprise, but are not limited to, pumps, such as micropumps, and fluid containers, such as syringes, adapted for fluid and / or dilutant injection.

[0080] The injection means according to the invention can be any means, which can accommodate a fluid according to the invention and can inject it into the reactor according to the invention.

[0081] Hence, multiple injection means are required, at least one for each fluid and at least one for the dilutant.

[0082] In embodiments, the fluid(s) and dilutant are directly injected from the inlets into the second layer. In embodiments, the gradient is generated by microfluidics.

[0083] In preferred embodiments, the at least one fluid inlet is four fluid inlets, wherein the ends of said fluid inlets, through which the fluids are ultimately injected into the reactor, are arranged to form the corners of a square.

[0084] In said arrangement, a first fluid comprising a first compound according to the invention can be injected into a first of said fluid inlets, a second fluid comprising a second compound according to the invention can be injected into a second of said fluid inlets, which is arranged opposite of the first fluid inlet in said “square”, and the same or two different dilutions or mixtures of the first and second fluid comprising the first and second compound, respectively, can be injected into the other two of said fluid inlets. One, two, three, or four of said fluids can be injected into one, two, three, or four of said fluid inlets, respectively. The aforementioned “dilution or mixture” shall mean a fluid which comprises a combination of the first and the second compound. The concentration of the first and the second compound is predetermined, but can be adjusted, depending on which concentrations are of interest to be tested. For example, the first compound may be mixed withthe second compound in such a way that the mixture comprises 10% of the undiluted first compound and 90% of the undiluted second compound. Alternatively, they could be mixed to result in an equimolar mixture, or they are further diluted to a desired degree by using a dilutant. That way, any desired concentration of the first and second compound can be achieved. The skilled person knows howto mix and optionally dilute the fluids comprising the compounds in order to achieve the desired biologically effective end concentrations that shall be tested.

[0085] In said arrangement, the ends of a first dilutant inlet, through which the dilutant ultimately is injected into the reactor, may be arranged as a first torus-shaped structure, which may, in embodiments, be arranged substantially parallel to a surface of the second layer. In embodiments, the ends of the first dilutant inlet may additionally comprise one or two spotshaped structures in said first torus-shaped structure to facilitate injection of the dilutant into the first torus-shaped structure from said spot-shaped structures.

[0086] In said arrangement, the ends of a first outlet, through which the fluid(s) ultimately is / are ejected from ordrain off the reactor, may, in embodiments, be arranged as a second torus-shaped structure, which encircles the first torus-shaped structure, and which may, in embodiments, be arranged substantially parallel to a surface of the second layer. In certain embodiments, the ends of the first outlet may additionally comprise one or two spot-shaped structures in said second torus-shaped structure to facilitate the ejection ordrainage of the fluid(s).

[0087] In said arrangement, the ends of a second dilutant inlet, through which the dilutant ultimately is injected into the reactor, may be arranged as a third torus-shaped structure, which encircles the second torus-shaped structure, and which may, in embodiments, be arranged substantially parallel to a surface of the second layer. In embodiments, the ends of the second dilutant inlet may additionally comprise one or two spot-shaped structures in said third torus-shaped structure to facilitate injection of the dilutant into the third torus-shaped structure from said spot-shaped structures.

[0088] In alternative embodiments, all torus-shaped structures can be replaced by tube-shaped structures extending from the bottom of the first layer towards the second layer, thus ending in the aforementioned spot-shaped structures (top view).

[0089] In embodiments, at least a portion of one or more, preferably all, of the at least one fluid inlets, the at least one dilutant inlets, and / or the at least one outlet is arranged substantially perpendicularly to a surface of the second layer, preferably wherein said surface of the second layer is opposite to a surface that faces the third layer.

[0090] In alternative embodiments, at least a portion of one or more, preferably all, of the at least one fluid inlets, the at least one dilutant inlets, and / or the at least one outlet is arranged in the same plane as a surface of the second layer, preferably wherein said surface of the second layer is opposite to a surface that faces the third layer.

[0091] In embodiments, the at least one fluid inlet is one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, or more fluid inlets.

[0092] SecondIn the second layer, the fluids (if more than one is used) and / or, optionally, their dilutions and / or mixtures, are mixed. In embodiments, the fluids are at least two fluids, wherein each fluid comprises at least one biologically active compound, wherein said fluids are mixed in the second layer, preferably by convection. In alternative embodiments, a fluid and a dilutant are mixed in the second layer, preferably by convection. In embodiments, the structure of the second layer enables an active mixing of the fluids (if more than one is used) and / or, optionally, their dilutions and / or mixtures. In preferred embodiments, the mixing is done by convection. In embodiments, two or more fluids and / or, optionally, their dilutions and / or mixtures, are mixed within the second layer, preferably by convection. In embodiments, the mixing enables the gradient formation. In embodiments, the second layer comprises a material selected from the group consisting of i) a porous material, such as a glass filter or a porous filter made of another material, ii) stainless steel, iii) 3D-printed material, iv) a material comprising microchannels, v) a mesh structure, and vi) combinations thereof.

[0093] In embodiments, the second layer comprises a porous material. In embodiments, said porous material enables convection while preferably preventing direct contact between the first and the third layer.

[0094] In embodiments, the second layer has a thickness of 0.05-10 mm, preferably 0.1-9 mm, more preferably 0.2-8 mm, even more preferably 0.3-7 mm.

[0095] Third layer

[0096] The third layer according to the invention is suitable for culturing microorganisms or cells. In embodiments, the third layer comprises culture or growth medium for microorganisms or cells. In embodiments, the culture medium or growth medium comprises a solid, liquid, or semi-solid medium configured to support the growth and / or proliferation of a population of microorganisms or cells. In preferred embodiments, the culture medium or growth medium comprises a solid medium, preferably wherein the medium is configured to support the growth and / or proliferation of a population of microorganisms or cells. Each microorganism or cell may require a different type of culture or growth medium. Fungi, for instance, are often cultured in agar and cellulose. Hence, in embodiments, the third layer comprises or consists of agar and / or cellulose. The use of a liquid culture or growth medium is preferably used in combination with a well-damped system, so that the liquid cannot be affected, impaired or intermixed by external influences during or after the gradient formation.

[0097] In embodiments, the third layer is or comprises a growth medium for the microorganisms or cells. In embodiments, the third layer is or comprises as a diffusion medium for the concentration gradient.

[0098] Advantageously, the third layer may serve as a growth medium for the microorganisms or cells and as a diffusion medium for the concentration gradient.

[0099] In embodiments, the third layer comprises microorganisms or cells. In embodiments, the microorganisms or cells are bacteria, viruses, fungi, oomycetes, plasmodium, parasites,mammalian cells, such as murine and / or human cells, cells forming a biological tissue and / or organoid, and / or combinations thereof. In embodiments, the microorganisms or cells can be obtained from a sample of a subject. Said sample may be a tissue, blood, plasma, serum, urine, cerebrospinal fluid, lung, skin, gastrointestinal, stool sample or any other sample obtained from a subject. In embodiments, the sample comprises pathogenic bacteria, viruses, fungi, oomycetes, plasmodium, parasites, and / or combinations thereof. Alternatively or additionally, the sample comprises non-pathogenic bacteria, viruses, fungi, oomycetes, plasmodium, parasites, and / or combinations thereof.

[0100] In embodiments, the microorganisms or cells are arranged on and / or within the third layer. In embodiments, the third layer comprises or consists of a single, continuous culturing area. Hence, preferably the microorganisms or cells are cultured on a single continuous layer.

[0101] In embodiments, the third layer has a thickness of 0.05-10 mm, preferably 0.05-8 mm, more preferably 0.05-7 mm, even more preferably 0.1-5 mm, even more preferably 0.3-3 mm, even more preferably 0.5-1.5 mm, even more preferably 1 mm ±20%.

[0102] In embodiments, the third layer has a thickness of 0.05-10 mm, preferably 0.05-8 mm, more preferably 0.05-7 mm, even more preferably 0.1-5 mm, even more preferably 0.3-5 mm, even more preferably 1-3.5 mm, even more preferably 1 mm ±20% or 3.5 mm ±20%.

[0103] In embodiments, the third layer is an inlay, such as a well.

[0104] In embodiments, the inlets and outlets are physically separated from and thus not adjacent to the third layer.

[0105] In embodiments, the microorganisms or cells are inoculated on or are cultured in the culture or growth medium. The microorganisms or cells grow against the concentration gradients of the biologically active compounds and their combinations in the overlapping area. In embodiments, the bacteria can be tracked in real-time. In some embodiments, the microorganisms or cells comprise detectable labels, such as fluorescent markers or proteins. In additional or alternative embodiments, areas free or substantially free of, or comprising detectably less microorganisms or cells can be detected, for example, visually by eyesight or by a camera. Only resistant microorganisms or cells will migrate towards the sources with highest concentrations of the biologically active compounds. Hence, in embodiments, the spatial distribution of the microorganisms or cells enables the analysis and identification of optimal concentrations, concentration ratios, the minimum inhibitory concentration, and / or the speed of resistance evolution with respect to the (different) biologically active compound(s) or combinations of different biologically active compounds.

[0106] Fourth layer

[0107] In optional embodiments, the reactor further comprises a fourth layer between the second layer and the third layer, preferably configured to avoid contamination of the reactor with the microorganisms or cells, or with metabolic products from the microorganisms or cells, or with the culture and / or growth medium of the microorganisms or cells, or combinations thereof.In embodiments, said fourth layer is or comprises a semi-permeable membrane, for example a removable insert comprising a semi-permeable membrane. Said semi-permeable membrane is configured to enable transfer of the concentration gradient (and thus of the fluid(s)) from the second layer to the third layer, while preventing the transfer of microorganisms or cells, or metabolic products from the microorganisms or cells, or the culture and / or growth medium of the microorganisms or cells, or combinations thereof, to the second layer.

[0108] In embodiments, the fourth layer is an inlay, such as a well.

[0109] Further embodiments and aspects

[0110] In embodiments, microorganisms or cells with known treatment and / or resistance history, which were obtained from a subject, are compared to the predictions of resistance evolution, efficacy or effect results of the (different) biologically active compound(s) and / or the pharmacokinetics of the present invention.

[0111] In embodiments, appropriate and likely effective treatment can be initiated, or treatment can be adjusted, based on the results of the analysis. Hence, the present invention enables a personalized and fast treatment guidance.

[0112] Fungal infections, for example, are often neglected, although about 6.5 million acute, lifethreatening fungal infections exist every year worldwide with about 3.8 million deaths. Since the present invention enables fast (within 24 hours) and personalized treatment guidance, the present invention may advantageously help to decrease mortality. It is additionally advantageous, that the reactor according to the invention can be fully automated and / or standardized.

[0113] In embodiments, the therapy guidance may comprise information on effective concentrations of a biologically active compound, or of combinations of biologically active compounds in order to provide an optimal therapy.

[0114] In embodiments, the therapy guidance may comprise an initiation or adjustment of a therapy based on the effective concentrations of a biologically active compound, or of combinations of biologically active compounds.

[0115] In embodiments, the second layer is sandwiched by the first layer and the third layer. In embodiments, the first layer is below the second layer. In embodiments, the third layer is above the second layer. In embodiments, the first layer is below the second layer and the third layer is above the second layer. In embodiments, the layers are arranged vertically, e.g. as a vertical stack. In embodiments, the first layer is in direct contact with the second layer, and preferably the third layer is in direct contact with the second layer or with the fourth layer if it is present. Hence, in embodiments, all layers are preferably in direct contact with the adjacent layer. In embodiments, the second layer is arranged on the opposite side of the side of the first layer that harbours the inlets, preferably wherein the second layer is in direct contact with the first layer, and optionally, the third layer is arranged on the opposite side of the second layer that is arranged on the opposite side of the side of the first layer that harbours the inlets, wherein preferably, the third layer is in direct contact with the second layer.Such aforementioned arrangements of the first and second, and optionally the third layer, is advantageous, since gravity may thus be employed to support the ejection from or draining off of the fluid(s) and dilutant. The aforementioned layer arrangement of the gradient reactor also has the advantage that the large contact area between the layers enables or supports diffusive mass transport.

[0116] In some embodiments, the third layer is optional (see also Fig. 1C). In such an embodiment, the gradient is formed in the second layer, e.g. by convection, but no microorganisms or cells are tested and no diffusion into a third layer occurs.

[0117] Hence, the invention also relates to a gradient reactor for generating a concentration gradient, comprising:

[0118] a first layer comprising

[0119] i) at least one fluid inlet for an injection of at least one fluid,

[0120] ii) at least one dilutant inlet for an injection of a dilutant, and

[0121] iii) at least one outlet for an ejection of the at least one fluid,

[0122] a second layer,

[0123] optionally a third layer suitable for culturing microorganisms or cells,

[0124] wherein a concentration gradient is formable in the second layer, and optionally wherein the concentration gradient is transferable from the second layer to the third layer.

[0125] The invention also relates to a gradient reactor for generating a concentration gradient, comprising:

[0126] a first layer comprising

[0127] i) at least one fluid inlet for an injection of at least one fluid,

[0128] ii) at least one dilutant inlet for an injection of a dilutant, and

[0129] iii) at least one outlet for an ejection of the at least one fluid,

[0130] a second layer,

[0131] optionally a third layer suitable for culturing microorganisms or cells,

[0132] wherein preferably a concentration gradient is formable in the second layer, and optionally wherein the concentration gradient is transferable from the second layer to the third layer.

[0133] In embodiments, the reactor further comprises a lid and a glass plate on top of the third, or, if present, the fourth layer.

[0134] In embodiments, the reactor further comprises a gas inlet and / or outlet for regulation of O2 and / or CO2 concentrations.In embodiments, the reactor further comprises an integrated temperature control module.

[0135] In embodiments, the gradient reactor further comprises pumps, preferably micropumps, for automated injection of the at least one fluid and the dilutant, and optionally for ejection of the at least one fluid and the dilutant.

[0136] In embodiments, the reactor can be used for simultaneous testing of the efficacy of at least two antifungals in a patient sample in a single process step.

[0137] In embodiments, dynamically overlapping drug or compound gradients are generated within two hours.

[0138] The invention also relates to a gradient reactor for generating a concentration gradient, comprising:

[0139] a first layer comprising

[0140] i) four fluid inlets for an injection of at least one fluid, preferably wherein two of said fluid inlets are for injection of fluids comprising two different biologically active compounds and the two other fluid inlets are for injection of two different fluids comprising both of the two different biologically active compounds, preferably wherein the two different biologically active compounds are present in different dilutions or amounts within the two different fluids comprising both of the two different biologically active compounds,

[0141] ii) two, preferably opposing, inner dilutant inlets for an injection of a first and / or second dilutant,

[0142] iii) two, preferably opposing, outlets for an ejection of the at least one fluid,

[0143] iv) two, preferably opposing, outer dilutant inlets for an injection of a third and / or fourth dilutant, wherein the first and / or second dilutants may be the same or different than the third and / or fourth dilutant,

[0144] wherein the two outlets are arranged such that they are sandwiched by the two inner dilutant inlets and the two outer dilutant inlets,

[0145] a second layer,

[0146] optionally a third layer suitable for culturing microorganisms or cells,

[0147] wherein preferably a concentration gradient is formable in the second layer, and optionally wherein the concentration gradient is transferable from the second layer to the third layer.

[0148] The invention also relates to a gradient reactor for generating a concentration gradient, comprising:

[0149] a first layer comprising

[0150] i) one fluid inlet for an injection of a fluid,ii) two, preferably opposing, inner dilutant inlets for an injection of a first and / or second dilutant,

[0151] iii) two, preferably opposing, outlets for an ejection of the fluid,

[0152] iv) two, preferably opposing, outer dilutant inlets for an injection of a third and / or fourth dilutant, wherein the first and / or second dilutants may be the same or different than the third and / or fourth dilutant,

[0153] wherein the two outlets are arranged such that they are sandwiched by the two inner dilutant inlets and the two outer dilutant inlets,

[0154] a second layer,

[0155] optionally a third layer suitable for culturing microorganisms or cells,

[0156] wherein preferably a concentration gradient is formable in the second layer, and optionally wherein the concentration gradient is transferable from the second layer to the third layer. The invention also relates to a gradient reactor for generating a concentration gradient, comprising:

[0157] a first layer comprising

[0158] i) a fluid inlet for an injection of a fluid,

[0159] ii) two, preferably opposing, dilutant inlets for an injection of a first and / or second dilutant, iii) two, preferably opposing, outlets for an ejection of the fluid,

[0160] wherein the two outlets are arranged such that they are further away from the fluid inlet than the two dilutant inlets,

[0161] a second layer,

[0162] optionally a third layer suitable for culturing microorganisms or cells,

[0163] wherein preferably a concentration gradient is formable in the second layer, and optionally wherein the concentration gradient is transferable from the second layer to the third layer. The invention further relates to a method for generating one or more concentration gradients, comprising:

[0164] providing a gradient reactor according to the invention, or a system according to the invention,

[0165] simultaneously injecting the dilutant and the at least one fluid, into the at least one dilutant inlet and the at least one fluid inlet, respectively,

[0166] generating one or more concentration gradients in the second layer,optionally, wherein if a third layer is present, the one or more concentration gradients transfer from the second layer to the third layer, wherein the third layer is suitable for culturing microorganisms or cells.

[0167] The invention further relates to a method for generating one or more concentration gradients, comprising:

[0168] providing a gradient reactor according to the invention, or a system according to the invention,

[0169] simultaneously injecting the dilutant and the at least one fluid, into the at least one dilutant inlet and the at least one fluid inlet, respectively,

[0170] generating one or more concentration gradients in the second layer,

[0171] wherein the one or more concentration gradients transfer from the second layer to the third layer, wherein the third layer is suitable for culturing microorganisms or cells.

[0172] In embodiments, the one or more concentration gradients transfer from the second layer to the third layer, wherein the third layer is not only suitable for culturing microorganisms or cells, but actually comprises microorganisms or cells.

[0173] In embodiments, the at least one fluid can be one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, or more fluids.

[0174] In embodiments, each of the at least one fluids comprises one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, or more biologically active compounds.

[0175] In embodiments, each fluid or combination or mixture is injected into another fluid inlet.

[0176] The principle of the concentration gradient generation is that one or more fluids, each comprising a biologically active compound, or mixtures of two or more biologically active compounds, can be injected into the reactor. The fluids (if more than one is used) mix in the second layer, preferably by convection. In embodiments, these fluids are at least two fluids, wherein each fluid comprises at least one biologically active compound. However, the same principle also applies to a fluid comprising at least one biologically active compound and a dilutant, such that, in embodiments, a fluid and a dilutant may also be mixed in the second layer, preferably by convection. In embodiments, the fluid(s) and the dilutant are injected simultaneously. In alternative embodiments, the fluid(s) and the dilutant are injected sequentially, wherein either the fluid(s) are injected first or the dilutant is injected first. In embodiments, the dilutant “guides” or “pushes” the fluids towards the third layer and dilutes the fluids. This function is fulfilled by the dilutant streaming in from the inner dilutant inlet(s). In embodiments, the dilutant streaming in from the outer dilutant inlet(s) limits the space to which the fluids extend and may also, in embodiments, dilute the fluids, so that the gradient generation is largely limited to the area within and restricted by the outlets (see also Fig. 6). Outside of the area restricted by the outlets, microorganisms and cells may grow without the presence of compound(s), which may, in embodiments, represent a (growth / proliferation starting) zone in which the microorganisms or cells can grow and / orproliferate without being restricted by the biologically active compound. If only one fluid is injected, only the dilutant inlets and the outlet on the side of the used fluid inlet may be needed in the methods according to the invention (see also Fig. 1C).

[0177] When the concentration gradient is established, or while it is formed, the concentration gradient can, in embodiments, transfer from the second layer to the third layer, preferably by diffusion. Since preferably, diffusion only or mainly occurs within the third layer, the gradient formation is very fast. In preferred embodiments, the third layer is so thin (e.g. only about 0.05-10 mm, preferably only about 0.1-5 mm, more preferably only about 1 mm ±20%) that the concentration gradient transfers without loss, or without substantial loss, of compound concentration into the direction of the diffusion. This is an advantage over systems that are based on fluid streambased gradient generation in which fluids are lead past a culture medium.

[0178] In embodiments, the time needed to build the concentration gradient(s) ranges from 10 min to 24 hours, preferably from 12 min to 20 hours, more preferably from 15 min to 12 hours, even more preferably from 17 min to 8 hours, even more preferably from 20 min to 2.5 hours.

[0179] In embodiments, the gradient is formed in the second layer, and optionally the third layer, within 10 min to 48 hours, preferably within 10 min to 24 hours, more preferably within 12 min to 20 hours, even more preferably within 15 min to 12 hours, even more preferably within 17 min to 8 hours, even more preferably within 20 min to 5 hours, even more preferably within 20 min to 4 hours, even more preferably within 20 min to 2.5 hours.

[0180] In embodiments, the gradient is formed in the second layer, and optionally the third layer, within 2 hours, preferably within 1.5 hours, more preferably within 1 hour, even more preferably within 50 minutes, even more preferably within 45 minutes, even more preferably within 40 minutes, even more preferably within 35 minutes, even more preferably within 30 minutes, even more preferably within 25 minutes, even more preferably within 20 minutes, even more preferably within 15 minutes, even more preferably within 10 minutes.

[0181] Typically, the time to build the concentration gradient depends on the diffusion properties of the fluid(s) comprising the biologically active compound and the diffusion properties of the dilutant. In embodiments, the time needed to build the concentration gradient(s) in the third layer ranges from 10 min to 2 hours, or from 20 min to 1.5 hours, or from 30 min to 1 hour, per mm of the third layer.

[0182] In embodiments, the time needed to build the concentration gradient(s) in the third layer ranges from 10 min to 1 hours, or from 20 min to 1 hours, or from 30 min to 1 hour, per mm of the third layer.

[0183] In embodiments, the time needed to build the concentration gradient(s) in the third layer is 10 min ±20%, or 15 min ±20%, or 20 min ±20%, or 25 min ±20%, or 30 min ±20%, or 35 min ±20%, or 40 min ±20%, or 45 min ±20%, or 50 min ±20%, or 55 min ±20%, or 60 min ±20%, or 65 min ±20%, or 70 min ±20%, or 75 min ±20%, or 80 min ±20%, or 85 min ±20%, or 90 min ±20%, or95 min ±20%, or 100 min ±20%, or 105 min ±20%, or 110 min ±20%, or 115 min ±20%, or 120 min ±20%, per mm of the third layer.

[0184] In embodiments, the growth, proliferation and / or survival of microorganisms or cells is tracked for up to 48 h, preferably for up to 24h, more preferably for up to 20 hours, even more preferably for up to 15 hours, even more preferably for up to 13 hours, even more preferably for up to 10 hours. In embodiments, the growth, proliferation and / or survival of microorganisms or cells is tracked for 1-48 h, preferably 2-24h, more preferably 3-6h.

[0185] The invention further relates to a method for detecting an influence of at least one biologically active compound on the growth, proliferation and / or survival of microorganisms or cells, comprising:

[0186] providing a gradient reactor according to the invention, or a system according to the invention,

[0187] simultaneously injecting the dilutant and the at least one fluid, into the at least one dilutant inlet and the at least one fluid inlet, respectively, wherein each of the at least one fluids comprises a different biologically active compound, wherein the different biologically active compounds influence or are suspected to influence growth, proliferation and / or survival of microorganisms or cells,

[0188] generating one or more concentration gradients for the different biologically active compounds in the second layer,

[0189] wherein the one or more concentration gradients transfer from the second layer to the third layer, wherein the third layer comprises the microorganisms or cells,

[0190] wherein an influence on the growth, proliferation and / or survival of the microorganisms or cells is analyzed.

[0191] In embodiments, the diffusion constant is calculated or known for the tested compounds, wherein preferably, said calculated diffusion constant enables the numerical pre-simulation of the gradient propagation / formation such that every desired gradient can automatically be generated by the reactor.

[0192] In embodiments, the gradient is quantified using a photometric measurement.

[0193] In embodiments, the gradient is quantified using fluorescent biologically active compounds. Such fluorescence may be inherent to the biologically active compound and / or may stem from a label attached to the biologically active compound.

[0194] In embodiments, the biologically active compounds are labelled with a marker that can be detected visually or biochemically, such as a fluorescent label or a tag, such as a streptavidin, avidin, or biotin tag.In embodiments, the reactor, system and methods of the invention can be used for diagnostic purposes (e.g. susceptibility testing or identification of resistant microorganisms or cells), environmental monitoring, or pharmaceutical development of compounds.

[0195] In embodiments, the reactor and system and methods of the invention provide a patient-specific point-of-care system for rapid combination therapy of infections, e.g., bacterial, viral or fungal infections.

[0196] In embodiments, the reactor, system and methods of the invention can be used to experimentally test antibiotic resistance evolution for new treatment regimens and new drugs before application. Thus, antibiotics, for example, can be used sustainably, the spread of antimicrobial resistance (AMR) genes and organisms can be reduced, and persistent infections can be treated more efficiently.

[0197] It is an advantage of the reactor, system, and methods according to the invention, that different compounds, dilutions and / or mixtures thereof can be tested at once, and due to the resulting overlapping concentration gradients, a large amount of different concentrations and combinatorial combinations (with a certain resolution, (almost) all concentration combination gradations) can be tested. In embodiments, a dilution of one or more biologically active compounds or a combination of two or more different biologically active compounds is tested. In embodiments, a, preferably uniform, dilution of one or more biologically active compounds and a combination of two or more different biologically active compounds is tested simultaneously. In embodiments, the dilution of two fluids, or of a fluid to a dilutant, is 1 :2, 1 :3, 1 :4, 1 :5, 1 :6, 1 :7, 1:8, 1:9, 1:10, 1:20, 1:30, 1:40, 1:50, 1:60, 1:70, 1:80, 1:90, 1:100, 1:150, 1:200, 1:250, 1:300, 1:500, 1:750, 1:1000, or more, each of which may be ±20%.

[0198] It is further an advantage of the reactor, system, and methods according to the invention, that i) the desired concentration gradients can be generated quickly, for example, within 2 hours, in the third layer, ii) the gradient is dynamically adaptable, iii) they can be automated, and iv) the desired concentration gradient can be simulated.

[0199] In embodiments, the biologically active compound, such as an antibiotic, is isolated from the third layer prior to its quantification.

[0200] In embodiments, the gradient is quantified by isolating the biologically active compound, such as an antibiotic, from the third layer, followed by quantification of its concentration, preferably using a photometric measurement.

[0201] In embodiments, the minimum inhibitory concentration is determined.

[0202] In embodiments, sterility is achieved, preferably for up to 10 days, more preferably up to 8 days, more preferably up to 6 days, more preferably up to 4 days, more preferably up to 2 days.

[0203] In embodiments, microorganism or cell growth across the formed gradient is observed and measured, preferably for up to 10 days, more preferably up to 8 days, more preferably up to 6 days, more preferably up to 4 days, more preferably up to 2 days.The reactor, system, and methods according to the invention advantageously enable a testing of a large number of different compounds in different concentrations and / or dilutions, preferably simultaneously.

[0204] It is a further advantage of the reactor, system, and methods according to the invention, that convection (e.g. in the second layer) and diffusion (e.g. and the third layer) can both be employed, thus enabling a (fast) generation of stationary concentration gradients, while also enabling a quick switch between different compounds to be tested and / or their concentrations due to dynamic concentration gradient generation. For the stationary gradient generation, a constant supply of the fluids and dilutant is provided, at least until the gradient is fully formed. If the gradient shall be changed or if other compounds or combinations shall be tested, i.e. if a dynamic gradient is required, the fluid supply can be stopped and dilutant can be injected into the fluid inlets. Due to the low (or lack of) compound concentration in the dilutant, the compounds will diffuse from the third layer to the second layer and will be ejected or will drain from the outlets. Subsequently, a new concentration gradient can be generated. In alternative embodiments, a dynamic gradient is produced by varying the concentration of the injected fluid(s). This results in a shift of the concentration distribution and a change of the concentration gradient at the surface of and / or in the third layer, so that, in embodiments, different and / or changing compound concentrations affect the microorganisms or cells. Hence, in embodiments, the reactor and methods according to the invention can be used for stationary and / or dynamic concentration gradient generation. Gradients can thus not only be generated localized, but also temporarily. In embodiments, the different concentration gradients overlap to some degree. Based on the knowledge of the concentration of the compounds in the different fluids and the flow behavior of the fluids and the dilutant, the concentration of each compound on each point of the third layer can be computed. Based on this information, conclusions on the minimum inhibitory concentration, pharmacokinetics etc. can be drawn.

[0205] Definitions

[0206] As used herein, the term “reactor” and “gradient reactor” are used interchangeably.

[0207] As used herein, the term “gradient” and “concentration gradient” are used interchangeably.

[0208] As used herein, the term “compound” and “biologically active compound” are used interchangeably.

[0209] A “fluid”, as used herein, preferably refers to fluids comprising a biologically active compound. “Convection”, as used herein, preferably refers to a liquid flow that that is due to the combined effects of liquid and including component property heterogeneity and body forces on said liquid. “Diffusion”, as used herein, preferably refers to the net movement of e.g. molecules, such as biologically active compounds, preferably from a region of higher concentration to a region of lower concentration.

[0210] A “torus-shaped structure” means a structure having the shape of a torus or donut, but may also refer to shapes in which the surface of the “torus” has corners, wherein the corners, for example,have about 90°, and optionally wherein the surface comprises one or more substantially planar surfaces.

[0211] “The minimum inhibitory concentration” or “MIC” as used herein is the lowest concentration of a chemical, usually a drug, which prevents visible in vitro growth of bacteria or fungi.

[0212] “Biocides” as used herein refers to a diverse group of poisonous substances including preservatives, insecticides, disinfectants, and pesticides used for the control of organisms that are harmful to human or animal health or that cause damage to natural or manufactured products. Examples include pesticides, such as fungicides; herbicides; insecticides; algicides; molluscicides; miticides; piscicides; rodenticides; and slimicides, and antimicrobials, such as germicides; antibiotics; antibacterials; antivirals; antifungals; antiprotozoals; and antiparasitics, and spermicides.

[0213] “Stressors” as used herein refers to a chemical or biological agent causing stress to an organism. Examples include disinfectants, salt, alcohol, drugs.

[0214] In general, the features disclosed in the context of the gradient reactor, or the system, ora method, or the use of the reactor or system of the invention also relate to and are herewith disclosed in the context of any of the other ones of the gradient reactor, or the system, or a method, or the use of the reactor or system of the present invention and vice versa.

[0215] FIGURES

[0216] The invention is demonstrated byway of example through the figures disclosed herein. The figures provided represent particular, non-limiting embodiments and are not intended to limit the scope of the invention.

[0217] Figure 1:

[0218] A) Schematic cross-sectional view of the gradient reactor according to the invention.

[0219] B) Schematic cross-sectional view of the gradient reactor according to the invention showing two centrally located inlets (in this case serving as inlets for exemplary antibiotic A and B, respectively) and one further centrally located inlet through which a mixture of antibiotic A and B can be injected. A further centrally located inlet, which is not visible as it is located behind the other three centrally located inlets, can also be used for injection of a mixture of antibiotic A and B. The two dilutant inlets next to the centrally located inlets are connected via a torus-shaped structure as indicated in subfigure A. Similarly, the outer two dilutant inlets and the two outlets are connected via torus-shaped structures, respectively.

[0220] C) Sectional view of the numerical simulation of the combination approach of diffusion and convection at different time points of the gradient formation. Different coloring of the concentration ratio of one of the substances.Figure 2: Structure of the assembled (top panels) and disassembled (bottom panels) gradient reactor according to the invention.

[0221] Figure 3: Structure of the system according to the invention. In this exemplary assembly, the reactor is connected to syringes used for fluid injection and ejection.

[0222] Figure 4: Concentration gradient formation in an embodiment of the reactor without a third layer, wherein the concentration gradient is only formed in the second layer (in this example, the second layer is a glass filter). Upper panel: a schematic representation of microorganism growth in particular regions of the growth medium in dependence of the concentration of the biologically active compounds. Bottom left: the picture was taken a few seconds after injection of the fluids.

[0223] Bottom right: the picture was taken one minute after injection of fluids.

[0224] Figure 5: Workflow of an embodiment of the method of the invention. A patient sample is obtained and the microorganism(s) comprised therein are inoculated and incubated on a suitable growth medium in the reactor according to the invention. Concentration gradients for two different biologically active compounds are generated and the effect on the microorganisms is detected and analyzed. A prognosis of the efficacy and of the resistance can be made based on the results of the analysis.

[0225] Figure 6: Sectional view of the reactor depicting an exemplary simulation of the combination approach of convection (in second layer, here: “porous media”) and diffusion (into the third layer, here: “growth media”) at different time points of the gradient formation (A-L). The inlet in the center, which is sandwiched by the inlets through which two different biologically active compound-comprising fluids are injected, comprises a mixture of the two different exemplary biologically active compound-comprising fluids.

[0226] Figure 7: Ternary phase diagram to illustrate the expected concentration and combination gradations in the reactor. Comparison of 100 direct combinations by pipetting mixtures in a microbouillon dilution and the coverage of the concentration range in the nutrient medium layer of the reactor. The data points were taken from the numerical simulation.

[0227] Figure 8: Optical density of E.coli MG1655 and a sample from an area with a high antibiotic concentration during inoculation with 4 pg / ml Ampicillin for 16 hours.

[0228] Figure 9: Schematic view of an embodiment of the first layer of the gradient reactor (A) with one central fluid inlet, two inner dilutant inlets, two outlets, and two outer dilutant inlets. A fluorescence image of a fluorescein gradient after 6h of gradient formation is shown in (B) and four independent fluorescence intensity plots of a fluorescein gradient from different experiments and different indicated time points after gradient formation, and optionally different flow parameters, are depicted in (C-F).

[0229] Figure 10: Schematic top view on the first layer of two embodiments of the gradient reactor according to the invention.A) One embodiment of the gradient reactor with one central inlet for a biologically active compound (e.g. an antibiotic) (A), two inner dilutant inlets (B), two outlets (C) and two outer dilutant inlets (B).

[0230] B) One embodiment of the gradient reactor with two central inlets for two different biologically active compounds (e.g. antibiotics) (A and B), two central inlets fora mixture of the two different biologically active compounds (2x AB), two inner dilutant inlets (C), two outlets (D) and two outer dilutant inlets (C).

[0231] Figure 11: Schematic view (side (A), cross-sectional (B), partial (D)) of an embodiment of an assembled gradient reactor (e.g. as in Figure 10A) including one view on the first layer (C), and a schematic view of a simulated gradient of Fluorescein (E) formed in the reactor embodiment of (A)-(D).

[0232] Figure 12: Evolution (resistance development) in bacteria grown in the reactor after challenge with Levofloxacin. The maximum concentration of Levofloxacin used was 2 pg / ml. The experiment duration was 6 days. The bacterium was Escherichia Coli CC 25922 (DSM 1103), grown on 0.5% agarose.

[0233] EXAMPLES

[0234] The invention is demonstrated through the examples disclosed herein. The examples provided represent particular embodiments and are not intended to limit the scope of the invention. The examples are to be considered as providing a non-limiting illustration and technical support for carrying out the invention.

[0235] Example 1:

[0236] The present invention allows statements about the effectiveness of a particular compound concentration and an assessment of the probability of future resistances. Experiments were able to demonstrate resistance evolution of E.coli MG1655 against Ampicillin within the reactor. The detected MIC of the sample had a rise in MIC from 2 pg / ml to 4 pg / ml of Ampicillin. See Fig. 8. Example 2: Quantification of a fluorescent dye gradient

[0237] As a proof-of-principle experiment, a fluid comprising fluorescein (a fluorescent dye) was injected into the reactor and a stationary gradient was formed in the second layer and transferred to the third layer (0.5% agarose gel). 4-24 hours after injection (Figure 9 shows plots for 3h, 4h, 5.5.h, and 6h), fluorescence imaging was performed and intensity measured and quantified (Figure 9). Example 3: Quantification of a fluorescent antibiotic agent gradient via photometric measurements

[0238] A stationary Levofloxacin (Levo) gradient was generated using the gradient reactor according to the invention. Samples were punched out of the agar after gradient formation and Levo was washed out of the agar. Subsequently, Levo concentration was photometrically measured. The experiment was performed twice and shows that the gradient is measurable.Example 4: Evolution experiments with bacteria

[0239] It could be shown that by using an antibiotic gradient that was generated by the gradient reactor according to the invention, sterility could be achieved for 6 days. Furthermore, two experiments showed that the minimum inhibitory concentration (MIC) was increased and bacteria grew against the gradient.

[0240] Figure 12 shows the results of one of such experiment. 6 days after the start of the gradient formation, agarose samples were taken from the third layer and divided into 40 samples. Only samples with an OD600 > 0.005 were analyzed (here: 13 out of N=40 samples). In sampling zone A (outermost zone), the rise in MIC is less than in zones B, C, and D. This indicates that resistance towards Levofloxacin was generated, which was particularly strong in the bacteria that had contact to a higher concentration of Levofloxacin.

[0241] As used throughout herein, “MIC” is the lowest concentration of a biologically active compound that prevents visible growth of the microorganisms or cells. An increase in the MIC therefore indicates acquired resistance of the microorganisms or cells towards the tested biologically active compound. A bacterial growth against the gradient is also an indicator of acquired resistance.

Claims

CLAIMS1. A gradient reactor for generating a concentration gradient, comprising:a first layer comprisingi) at least one fluid inlet for an injection of at least one fluid,ii) at least one dilutant inlet for an injection of a dilutant, andiii) at least one outlet for an ejection of the at least one fluid,a second layer,a third layer suitable for culturing microorganisms or cells,wherein a concentration gradient is formable in the second layer, and the concentration gradient is transferable from the second layer to the third layer.

2. The gradient reactor according to claim 1 , wherein the second layer comprises a material selected from the group consisting of i) a porous material, such as a glass filter, ii) stainless steel, iii) 3D-printed material, iv) a material comprising microchannels, and v) combinations thereof.

3. The gradient reactor according to any one of the preceding claims, wherein the third layer has a thickness of 0.05-10 mm, preferably 0.1-5 mm.

4. The gradient reactor according to any one of the preceding claims, wherein each of the at least one fluids comprises a different biologically active compound, wherein the different biologically active compounds influence or are suspected to influence growth, proliferation and / or survival of microorganisms or cells, preferably wherein the third layer comprises the microorganisms or cells.

5. The gradient reactor according to claim 4, wherein the different biologically active compounds are selected from the group consisting ofi) antimicrobial agents, such as antibiotic, antiviral, antifungal, or antiparasitic agents,ii) biocides,iii) stressors,andiv) drugs, such as chemotherapeutic agents.

6. The gradient reactor according to any one of the preceding claims, wherein the concentration gradient is transferable from the second layer to the third layer by diffusion.

7. The gradient reactor according to any one of the preceding claims, wherein the dilutant is selected from the group consisting of water and medium.

8. The gradient reactor according to any one of the preceding claims, wherein at least a portion of the at least one fluid inlet is arranged substantially perpendicularly to a surface of the second layer and towards a center of the surface of the second layer.

9. The gradient reactor according to any one of the preceding claims, wherein the at least one fluid inlet is four fluid inlets arranged to form the corners of a square.

10. The gradient reactor according to claim 8 or 9, wherein one of the at least one dilutant inlets is arranged between the at least one fluid inlet and the at least one outlet.

11. The gradient reactor according to any one of the preceding claims, wherein the concentration gradient is a stationary or dynamic concentration gradient.

12. The gradient reactor according to any one of the preceding claims, wherein the first layer is below the second layer and the third layer is above the second layer.

13. The gradient reactor according to any one of the preceding claims, wherein at least a portion of one or more, preferably all, of the at least one fluid inlets, the at least one dilutant inlets, and / or the at least one outlet is arranged substantially perpendicularly to a surface of the second layer.

14. The gradient reactor according to any one of the preceding claims, wherein two or more fluids and / or, optionally, their dilutions and / or mixtures, are mixed within the second layer, preferably by convection.

15. A system comprisingthe gradient reactor according to any one of the preceding claims, wherein the gradient reactor optionally further comprises pumps, preferably micropumps, for automated injection of the at least one fluid and the dilutant,a camera system, anda computer program comprising instructions, optionally wherein the instructions, when the program is executed by a computer, cause the computer to analyze the growth, proliferation and / or survival of the microorganisms or cells.

16. The system according to claim 15, wherein the analysis of the growth, proliferation and / or survival of the microorganisms or cells comprises the computation of the effective concentration of the different biologically active compounds.

17. Use of the gradient reactor according to any one of the preceding claims, for the generation of overlapping stationary or dynamic concentration gradients, for analyzing the efficacy or effect of different biologically active compounds, for assessing possible combinatory therapies, for assessing the probability of future resistance to the different biologically active compounds, or for analyzing pharmacokinetics.

18. A method for generating one or more concentration gradients, comprising:providing a gradient reactor according to any one of claims 1 to 14, or a system according to any one of claims 15 to 16,simultaneously injecting the dilutant and the at least one fluid, into the at least one dilutant inlet and the at least one fluid inlet, respectively,- generating one or more concentration gradients in the second layer,wherein the one or more concentration gradients transfer from the second layer to the third layer, wherein the third layer is suitable for culturing microorganisms or cells.

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