EP4247556A1

EP4247556A1 - Microfluidic flow cell and system for analyzing or diagnosing biofilms and cell cultures, and the use thereof

Info

Publication number: EP4247556A1
Application number: EP21815510.9A
Authority: EP
Inventors: Ralf Heermann; Athanasios Gazanis
Original assignee: Johannes Gutenberg Universitaet Mainz
Current assignee: Johannes Gutenberg Universitaet Mainz
Priority date: 2020-11-23
Filing date: 2021-11-22
Publication date: 2023-09-27
Also published as: US20240002764A1; DE102020130870B3; CA3200697A1; WO2022106673A1

Abstract

The present invention relates to microfluidic flow cells for analyzing or diagnosing biofilms and cell cultures, comprising a carrier plate with a specimen chamber formed therein, the latter being peripherally bounded by chamber walls and a base, a cover plate which is fluid-tightly connectable to the carrier plate, an inflow with an inflow channel integrated therein, the latter opening into the specimen chamber via an opening, and an outflow with an outflow channel integrated therein. Holding elements for fastening the carrier plate to an object stage of a microscope or a holding device are formed on the end sides of the carrier plate. The invention further relates to systems and their use for analyzing or diagnosing biofilms and cell cultures using such microfluidic flow cells.

Description

Microfluidic flow cell and system for analyzing or diagnosing biofilms and cell cultures and their use

TECHNICAL AREA

The present invention relates to microfluidic flow cells and systems for analyzing or diagnosing biofilms and cell cultures. The invention also relates to the use of such microfluidic flow cells or systems for analyzing or diagnosing biofilms or cell cultures.

STATE OF THE ART

Microfluidic systems and methods are basically known from the prior art, because devices and methods based on microfluidic principles have become widely established in the laboratory area. Its advantages include, among other things, a lower consumption of reagents and sample materials per test run while at the same time increasing the number of test runs that can be carried out simultaneously. A distinction is made between covered or closed microfluidic systems, in which the carrier plate, the cover plate and wall structures provided between them are connected to one another permanently and not just temporarily by clipping or jamming. The wall areas of the support plate, together with the cover plate and the support plate, form a channel system which is delimited by corresponding inner surfaces.

Such microfluidic systems can include channels and cavities whose dimensions are comparable to the dimensions of biological cells and tissue structures. It is thus possible to cultivate cells under in vivo-like conditions, for example by setting a defined perfusion. Under such conditions, it is possible for the cells to retain their phenotype, which is relevant for diagnostics, among other things, so that the results are obtained under physiological conditions as far as possible. Such complex cell arrangements can be used, for example, to determine the toxicity, metabolism and mechanisms of action of active ingredients in the pharmaceutical industry.

A previously new field for the use of microfluidic flow cells is the analysis and diagnostics of biofilms, i.e. colonization of microorganisms on surfaces, for example on the surfaces of drinking water systems or pipelines in the food industry or the pharmaceutical industry. Surfaces can be colonized either by one type of bacteria or by different types of microorganisms. In In a biofilm, the microorganisms are exposed to completely different environmental conditions compared to their free-living conspecifics (Hall-Stoodley et al., 2004; Fleming et al., 2016). In the network of a biofilm, microorganisms are far more resistant than free-swimming, individual cells. In the medical field, far higher doses of antibiotics must therefore be used to combat biofilms of clinically relevant bacteria, since the microorganisms in the biofilm are enveloped in a slimy matrix and are thus protected from the effects of the antibiotics (Bryers et al., 2008; Fleming et al., 2017). However, biofilms not only cause massive problems in medicine and hygiene, but also affect other branches of industry, such as drinking water systems or the marine sector, such as the shipping industry, where biofilms form on the hulls of ships and thus increase the fuel consumption of the ships exponentially increase (Little et al., 2008; Niaounakis et al., 2017; de Carvalho et al., 2018). With the help of microfluidic flow cells, it is possible to understand and analyze such biofilms in their different occurrences, since the conditions in the flow cell can be realistically mapped. For example, it is possible to test the effect of drugs or antimicrobial substances, which are applied to surfaces and materials with paint or in drinking water hoses, on the effect of living cells in a flow under different environmental conditions in a closed system.

Although microfluidic cells are known for the analysis of biological systems, they have various disadvantages, such as a lack of compatibility with different microscope types or microscope variants, flexibility in the choice of surfaces, pressure build-up or air bubble entry. For example, 3D-printed microscopy chambers for multidimensional imaging are known (Alessandri, K., et al.: All-in-one 3D printed microscopy chamber for multidimensional imaging, the UniverSlide. Sei. Rep. (2017) 7:42378). Another system is a membrane-integrated microfluidic flow cell equipped with cell chambers and coverslip (Epshteyn, AA, et al.: Membrane-integrated microfluidic device for high-resolution live cell imaging. Biomicrofluidics (2011) 5(4): 046501-046501 -6).

Flow cells, as they are available on the market today and used in various laboratories worldwide, always have to meet minimum criteria in order to ensure clear, realistic and reproducible results in analysis or diagnostics. For example, the supply of the microorganisms with fresh nutrient medium or test solutions (eg blood serum) and the drainage of used medium must be guaranteed. For an optimized analysis it is also necessary that the flow cell is adaptable in terms of flow velocities, because microorganisms or cell cultures must be able to be cultivated in the microfluidic flow cell in such a way that the cells adhering to the bottom in the biofilm are not washed off by excessively high flow velocities. Nevertheless, an optimal supply of fresh nutrients must be guaranteed (Gale et al., 2018). If the flow rate in the microfluidic flow cell is too low, this often leads to overgrowth due to the strong development of biomass, which can lead to a blockage of the microfluidic.

Also, known microfluidic flow cells are not compatible with different microscopes (Paie et al., 2018; AbuZineh et al., 2018; Babic et al., 2018; Sakai et al., 2019; www.biofilms.biz; www.ibisci.com ). Microscopes differ in their structure and function, so that the use of the flow cells available on the market is often limited to certain types of microscopes, for example flow cells that are only transparent on one side. For example, such flow cells are only compatible with a CLSM (confocal laser scanning microscope) microscope, where fluorescent samples are excited from one side of the flow cell and microscopic observation of the samples can only be performed on the same side. If the laser and detector are on different sides of the flow cell for fluorescence analysis of biological samples, such a microfluidic flow cell is not suitable for use with a CLSM. Conversely, microscopic flow cells designed for CLSM are not compatible with conventional light or fluorescence microscopes because the light source or laser and detector are on different sides. In addition to these limitations, most conventional microfluidic flow cells are only suitable for a few conventional microscopes due to their design, i.e. for those in which the lens is located above the object stage. Due to their design, such cells cannot be used in inverted microscopes, since the cell already rests on the lens without being securely held by a holder.

Furthermore, conventional microfluidic flow cells can only be used to a limited extent for the analysis or diagnostics of biofilms, since their flow rate is too low and increased pressure is built up in the sample chamber of the microfluidic flow cell, which can damage the growing biofilm. Such cells also give the microorganisms very little space for biofilm formation, which is why mature biofilms can hardly be analyzed later microscopically or macroscopically (www.ibisci.com, www.biofilms.biz, www.ibidi.com). Another disadvantage of existing microfluidic flow cells is that they can only be operated in combination with an air bubble trap ("bubble trap").

Otherwise there is a risk that an air bubble that has penetrated the closed system will destroy the entire biofilm, since there is no space for the air bubble to escape. The river can also stall, making accurate analysis impossible (Babic et al., 2018). The lack of such an air bubble trap often leads to the failure of an entire test series, since the inflow or outflow of the medium in the microfluid is disrupted or prevented. In addition, such air bubble traps are very expensive to purchase and can only be used once.

Another problem with known microfluidic flow cells is that it is not possible to exchange surfaces for testing other surface materials. The base of the available cells is usually limited to glass or polystyrene, so that the available flow cells already contain the base for the biofilm colonization. However, such bases do not necessarily represent realistic conditions of the biological systems to be examined, so that other surfaces have to be used for the experiments or diagnostics (Pinto et al., 2019). These, in turn, cannot be analyzed with conventional microfluidic flow cells. In addition, a previously fixed base surface has the difficulty that it is not flexible and cannot be further processed as a result of experiments.

Conventional cells, which are not intended for microscopy analysis, therefore have the disadvantage that only prefabricated surfaces can be used due to their design and geometry (e.g. www.biofilms.biz). It is therefore also not possible to use materials of different geometries flexibly. A material to be tested would therefore have to be adapted to a specific cell geometry for analysis or diagnostics, otherwise one would be forced to use a prefabricated material which is only suitable for a specific flow cell. If a user is not able to adapt his material to the corresponding conditions or even uses a material that is not prefabricated by the supplier, analysis of biofilms in the microfluidic flow cell can only be carried out under suboptimal conditions.

Conventional microfluidic flow cells essentially consist of polycarbonate (PC) or polymethyl methacrylate (PMMA) and are accordingly expensive (Wolfaardt et al., 1994; Tolker-Nielsen et al., 2014; www.plasticker.de). In addition, both types of material are very difficult to process, which is why additional costs arise during processing. PRESENTATION OF THE INVENTION

Against this background, it is the object of the present invention to provide a microfluidic flow cell in which the above-mentioned disadvantages existing in the prior art are eliminated. This object is achieved by a flow cell having the features of claim 1. Preferred variants are found in the dependent claims.

The flow cell according to the invention addresses the disadvantages of known flow cells in the prior art and provides solutions, in particular with regard to the compatibility of microscopes, the flexibility in the choice of surfaces, the pressure build-up and the introduction of air bubbles. This makes the flow cell according to the invention more flexible in its application and also more cost-effective than conventional microfluidic flow cells when using inexpensive materials in production. In addition, the flow cell according to the invention can be reused several times, which again reduces costs and is environmentally friendly.

Like the known flow cells, the microfluidic flow cell according to the invention comprises a carrier plate with a sample chamber formed therein. The sample chamber is delimited on the circumference by chamber walls and, depending on the embodiment variant, can have a permanently integrated or removable base. On the opposite side, i.e. on the upper side, the sample chamber is delimited by a cover plate which is connected to the carrier plate in a fluid-tight manner. Depending on the design variant, the cover plate can either be firmly connected to the carrier plate or can also be flexibly removed using appropriate fastening means. The cover panel is preferably held by a cover frame. As a result, one or more surfaces of the cover plate or the cover plate itself can be easily replaced. This makes the flow cell according to the invention flexibly usable for different applications, for example due to the choice of materials for the surfaces or their equipment. In a preferred variant, the cover plate is transparent; alternatively, depending on the area of application, it can also be opaque.

The bottom of the flow cell is either firmly connected to the support plate or, in the case of the removable variant, is held by a bottom frame. This means that the surfaces of the floor can also be flexibly exchanged. Depending on the design, the floor can be transparent or opaque. For the media inflow is also an inlet with a provided therein integrated inlet channel, which is preferably located on the front side of the support plate. The inlet channel opens into the sample chamber via an opening. A drain with a drain channel integrated in it is provided for media drainage.

According to the invention, holding elements for attaching the support plate to an object table of a microscope or a holding device are now provided on the end faces of the support plate, which makes the flow cell compatible for a large number of different microscopes. The holding elements are preferably designed in such a way that they can be inserted into a holding device which can be pivoted between 0° and 180° via a pivoting device. This also enables vertical installation compared to conventional microfluidic flow cells, which can usually only be installed horizontally. The flow cell according to the invention, including its holding elements, preferably has the dimensions of a conventional slide, so that the flow cell according to the invention can be used for both normal and inverted microscopes. This feature eliminates the need for a so-called air bubble trap. Due to the vertical installation (i.e. in an arrangement offset by 90° compared to the horizontal installation), any air bubbles that are present migrate upwards and can be removed, for example by means of a pump, without coming into contact with the sample to be analyzed. Furthermore, the two holding elements formed on the end faces enable simple, secure, inexpensive and quick attachment, for example by means of a rubber fastener, latching lugs, clamping elements or by means of longitudinal joints.

Previous designs of microfluidic flow cells did not allow an analysis of biofilms or diagnostics of cell cultures at all or only to a limited extent, partly because the known systems build up too high a flow pressure in the sample chamber, which damages the biofilm. In order to reduce the flow pressure in the sample chamber, the flow cell according to the invention therefore provides for the diameter of the inflow channel of the inflow and of the outflow channel of the outflow to be greater than 1 mm, preferably between 1 mm and 3 mm, preferably between 1.5 mm and 2. is 5 mm. Cells with a diameter of less than 1 mm for the inflow channel and the outflow channel offer little space for microorganisms to form biofilms, which is why mature biofilms can hardly be analyzed microscopically and macroscopically later on.

Provision is preferably made for the surfaces, the cover plate and/or the base to be removable from the carrier plate. In alternative variants, the surfaces on which the biofilm is cultivated can be removed or exchanged. The surfaces are preferably supported by a base frame or cover frame held. The surfaces are preferably made of materials that promote biofilm formation. After the exchange, the surfaces can be placed back on the flow cell. The cell is closed with the cover plate or base plate. The design according to the invention makes it possible to cultivate biofilms between 0° and 180°, preferably between 0 and 90°. With an offset option between 0° and 180°, biofilms can be cultivated on both sides of the surfaces. Of course, all angle ranges and angle values within the range between 0° and 180° are also covered by the invention.

In a preferred variant, it is provided that the sample chamber is also significantly larger than existing solutions, ie has a larger volume, with volumes of 3.5 cm ³ to 8 cm ³ being preferred and a volume of between 5 and 5.5 cm ³ is particularly preferred. The height of the support plate is between 6 and 12 mm, preferably about 8 mm. The large-volume design of the sample chamber contributes to reduced pressure and thus gentle treatment of the biofilm. Air bubbles that may have been drawn in also stay away from the biofilm, since the chamber is large enough for the air bubbles to escape. The height of the chamber also enables operation in the vertical (ie offset by 90° compared to the horizontal) process, which allows colonization of both surfaces by microorganisms and thus the build-up of a biofilm. The large volume of the sample chamber enables the analysis and diagnostics of both small-scale (mm) and large-scale (cm) samples. The samples can be attached to the bottom of the sample chamber, which prevents the material from blurring in the test medium.

The sample chamber of the carrier plate is delimited on the underside by a chamber floor. The cover plate and/or the base are preferably fastened to the carrier plate in a detachable manner, ie the cover plate and/or the base can be completely removed from the sample chamber. Due to the fact that the cover plate and/or the base can be removed, the sample chamber in the microfluidic flow cell according to the invention is freely accessible, so that cleaning and thus reuse of the cell is also easily possible. In addition, the flow cell according to the invention can be quickly treated with a new medium or a washing solution. This also makes it possible to freely select the material and the material geometry of the cover plate and the base to precisely fit the carrier plate. The sample chamber, the cover plate and/or the floor are variable in height and can be individually dimensioned depending on the requirement profile or application. The cover plate and/or the base can be attached using one of the usual types of attachment, for example by latching, screwing, clamping, Gluing or using a click system. In order to make a large number of microscopes compatible, a preferred variant provides that the length ratio of the end face to the long side including the holding elements of the carrier plate is between 1:2.5 and 1:3.5, preferably about 1:3.

In order to achieve the high flow rates in the sample chamber that are required for cleaning or rinsing the cell, significantly higher flow rates are selected than in an operation for cultivating biofilms, in which a flow rate of approx. 5 ml/hour is sufficient. In a preferred variant it is therefore provided that the flow cell is equipped with a pump which is fluidically connected to the inlet and outlet and supplies a flow rate of at least 4 mm/sec in the sample chamber. Such high velocities and the high flow volumes of the flow cell allow the sample chamber to be efficiently rinsed after operation, e.g. after performing an experiment, or to be thoroughly cleaned for another test run, e.g. to remove adherent bacteria. In addition, in the flow cell according to the invention, not only were the diameters of the inflow and outflow increased in comparison to existing solutions, but the length of the inflow and outflow to the sample chamber was also increased. The length of the inlet or outlet is preferably at least 3 mm, preferably between 8 mm and 10 mm, preferably about 5 mm. The extended inlets and outlets prevent the connecting hoses from slipping off and are also suitable for very large flow rates (e.g. 100 ml/min).

In a preferred variant, in which the cover plate can be removed, it is provided that a groove for receiving a seal is formed on the upper side of the carrier plate. In the alternative embodiment where the top and bottom panels are removable, a groove with gasket is formed on both the top and bottom to seal the cell. The seal is preferably a silicone seal, for example a silicone cushion, which seals off the sample chamber from the outside. The seal introduced into the groove is preferably exchangeable and can be renewed if necessary. Alternatively, an O-ring can also be used.

In a preferred variant, fastening holes are provided on the upper side of the carrier plate, for example in order to attach a separate viewing window. This allows the analyzes or experiments to be carried out and the corresponding parameters, such as the flow rate in the sample chamber, to be monitored. In preferred embodiment variants of the microfluidic flow cell according to the invention, several flow cells are connected to one another. This is preferably done via latching lugs, which are located on the longitudinal sides of the carrier plate and cooperate with corresponding receptacles for the latching lugs of adjacent flow cells. In this way, several flow cells can be arranged side by side in a modular fashion and fastened to one another. In a further developed embodiment variant, several flow cells are held by a frame, which preferably consists of several frame elements, in which at least one frame element can be plugged into another frame element. The frame is opened by removing a frame element and the flow cells can be pushed into the frame via a groove. Provision is preferably made for the individual flow cells to be connected to one another via corresponding connecting devices, for example the aforementioned latching lugs or latching receptacles. The groove of the frame cooperates with holding elements of the flow cell formed on the end face of the carrier plate.

The invention also relates to a system for analyzing or diagnosing biofilms and cell cultures, comprising one or more microfluidic flow cells, as described here, and a holding device for accommodating one or more flow cells. The holding device is preferably designed in such a way that the one or more flow cells can be pivoted within an angular range of 0 to 180°, preferably 0 to 90°. The system further includes a pump fluidly connected to the inlet and outlet. The pump is preferably designed in such a way that the desired flow rate of at least 4 mm/sec is achieved in the sample chamber when required.

In a preferred variant it is provided that the holding device is a frame with several frame elements. The frame preferably has a groove for accommodating one or more flow cells, which groove cooperates with the retaining elements of the support plate of a flow cell.

In a preferred embodiment variant, the system comprises a stand with a holding device (preferably in the form of a frame) for accommodating one or more flow cells. The holding device is preferably pivotably mounted via swivel joints, so that the flow cell or the frame with a plurality of flow cells can be pivoted over an angular range of 0° to 180°. In a preferred variant, the rotary joints can be pivoted by a motor. Alternatively, instead of a rotary joint, a stepper motor can also be used, which can make the angle adjustments electronically. The control of the stepper motor is preferably software-based, for example via a mobile communication device such as a smartphone. Support elements are preferably provided for mounting the rotary joints attached on both sides of the holding device, in order to pivot the flow cell(s) or the holding device within the angle range of 0° to 180°. In addition, a pump is provided which is fluidly connected to the inlet and outlet. The holding device is preferably mounted in a stand with brackets connected to the pivots.

In order to keep the costs for the production of a flow cell according to the invention as low as possible and also because of the advantageous suitability as a sample surface, at least the carrier plate is made entirely of polyethylene (PE). In an alternative variant, the use of polypropylene (PP) is also possible, as this material can be autoclaved. This choice of material allows optional processing of the cell to adapt it to the specific experimental situation. The use of flexible surfaces in the sample chamber makes the microfluidic flow cells according to the invention even more flexible in use. The fact that the cells can be reused and that they can be manufactured cheaply are further important advantages over conventional solutions.

In a further developed embodiment of the invention, the microfluidic flow cell includes control elements in order to be able to operate the microfluidic flow cell semi-automatically or fully automatically. The aim is to control the cultivation of the biofilms or cell cultures on the corresponding surfaces and to carry out the incubation according to a program sequence. The central component here is a microcontroller, with which one or more motor drivers can be driven, which in turn are responsible for the actuation of drive units (e.g. stepper motors). A first stepping motor assumes the function of a pump in order to pump the incubation medium into the sample chamber via the inlet. A further drive unit can be pivoted in an angular range of 0 to 180°, preferably 0 to 90°, for the rotation of the flow cells. It is also possible via the microcontroller to carry out a time-dependent gradual swiveling of the flow cell over the angle range. For example, it can be provided that an angle of 0° is selected during the adhesion phase. The plate can then be rotated step by step up to an angle of 90° via a time gradient. This also allows air bubbles to escape, for example, so that they do not interfere with the formation of the biofilms or cell cultures. The controller can also ensure that the medium trickles down onto the biofilms or cell cultures as gently as possible over a preset angle. The microcontroller also receives sensor-transmitted data, which it can use to carry out appropriate control. So is For example, a flow sensor is responsible for determining the flow rate, which in turn can be set via the system control. A temperature sensor can ensure that a heating element is switched on as soon as the temperature in the bottom chamber falls below a predefined value. Conversely, a fan can ensure that the temperature is turned down when it reaches a predetermined threshold. The combination of heating element and fan thus enables ideal incubation conditions in the sample chamber. A timer or time control also ensures when and for how long certain process steps are carried out, for example.

The microfluidic flow cells according to the invention can be used for the analysis or diagnostics of biological samples or biofilms. They enable microscopic and macroscopic analyzes of biological samples and facilitate the analysis of biofilms in drinking water and supply lines as well as in pipelines in the food industry or the pharmaceutical industry.

The invention is explained in more detail in the following drawings. However, the invention is by no means restricted to the embodiment variants described therein. Rather, combinations of individual features of embodiment variants are also possible and are included in the basic idea of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

1 shows a side view of a first embodiment variant of a microfluidic flow cell according to the invention.

FIG. 2 shows the embodiment variant shown in FIG. 1 in plan view.

3 shows a further variant in a side view.

FIG. 4 shows the embodiment variant of FIG. 3 in plan view.

Fig. 5 shows the two variants of Fig.1 / 2 and Fig. 3/4 from the front.

6 shows an exploded view of a variant of a flow cell with a cover plate and base.

7 shows an embodiment variant with a removable cover plate and a fixed base. 8 shows an embodiment variant with a removable cover plate and removable base.

9 shows an arrangement of several support plates connected to one another in a row.

10 shows a holding device (frame) with two flow cells integrated therein.

11 shows individual components of a holding device in the form of a frame.

12 shows a system for analyzing or diagnosing biofilms and cell cultures, consisting of a stand, holding device and swivel joints for pivoting one or more flow cells.

13 shows a further developed embodiment with control elements.

14 shows a block diagram of an apparatus controlled via a microcontroller.

WAYS TO CARRY OUT THE INVENTION

1 shows a first embodiment variant of a microfluidic flow cell according to the invention. This comprises a carrier plate 10 with a sample chamber 20 formed therein, which is delimited on the peripheral side by four chamber walls 24 . An inlet 16 with an inlet channel 17 integrated therein and an outlet 18 with an outlet channel 19 integrated therein can be seen on the end faces 11 of the carrier plate 10 . The inlet channel 17 or the outlet channel 19 opens into the sample chamber 20 via corresponding openings 26 in the chamber wall 24 . The recess facilitates the connection and retention of tubing to the inlet 16 or outlet 18 without disturbing the external geometry of the cell.

To ensure compatibility for normal and inverted microscopes, holding elements 12 are formed on the end faces 11 of the support plate 10 in order to attach the support plate 10 to an object table of a microscope or alternatively to a holding device (e.g. for vertical operation in a 90° position). Such a holding device preferably enables adjustment in an angle range of 0 to 180°, which enables colonization of the surfaces with biofilm on both sides. In the variant shown, the retaining elements 12 are in the form of strips, ie are designed and have a rectangular web preferably an aspect ratio between long side and wide side of 3: 1. The width of the holding elements 12 preferably corresponds to the width of the carrier element 10. In the variant shown, the long side of the holding elements 12 (ie the end face 11 of the carrier plate 10) is 24 mm long, while the holding elements 12 have a width of 8 mm. The total length of the support plate 10 including the two holding elements 12 is 75 mm with a width of about 24 to 25 mm. The flow cell according to the invention thus differs only insignificantly from the dimensions of a conventional slide for microscopy, which makes it compatible with a large number of microscopes. With the two holding elements 12, the cell can be attached to the objective table of a microscope in a simple manner. The depth of the sample chamber is preferably between 7 and 8 mm, so that the cell is suitable for both normal and inverted microscopes. In the preferred variant, the sample chamber 20 itself is 40 mm long and 16 mm wide.

In order to reduce the pressure in the sample chamber 20, the diameters of the inlet channel 17 of the inlet 16 and the outlet channel 19 of the outlet 18 are increased according to the invention, namely to a diameter of greater than 1 mm, preferably greater than 1.5 mm.

The variant shown is designed without a floor 22 (not shown), but can be equipped with a removable or permanently installed floor 22 if required. A cover plate 40 is placed on top of the carrier plate 10 and connected to the carrier plate 10 . A closed sample chamber 20 is thus present. If the cover plate 40 is transparent, the test procedure and the individual parameters can be easily observed. The length of the inlet 16 and the outlet 18 is approximately 5 mm in order to channel large flow quantities into the enlarged sample chamber 20 and to prevent connecting hoses from slipping off the inlet 16 and the outlet 18 . This enables large flow rates of up to 100 ml/min.

In Fig. 2, the variant shown in Fig. 1 is shown in plan view. You can see the proportions of the carrier plate 10, the holding elements 12 and the sample chamber 20.

In all variants, the length ratio of the front side 11 to the long side 13 including the holding elements 12 is approximately 1:3. Lengths of the inlets 16 and the outlets 18 of between 5 mm and 10 mm are preferred. Preferred diameters of the inlet channel 17 of the inlets 16 and the outlet channel 19 of the outlets 18 are between 1.5 mm and 2.5 mm.

FIG. 3 shows a further variant in which the sample chamber 20 can be equipped either with or without a base 22 . The special thing about this one A variant is a groove 30 for a seal 32 running around the top of the support plate 10. In the variant in which the base plate is designed to be removable, there is also a groove 30 on the underside of the support plate 10 for liquid-tight sealing of the cell. The base 22 is either fixed to the support plate 10 or, depending on the variant, designed as a removable base plate. The sample chamber 20 is sealed off from the outside by the seal 32 . Fastening openings 34 can also be seen on the upper side of the carrier plate 10, via which a separate see-through window can be attached. The variant shown can be seen again in FIG. 4 in a plan view. The cover plate 40 is preferably designed to be transparent. The choice of materials for the cover plate 40 or the base 22 allows ideal test conditions to be created in order to analyze biofilms or cell cultures, in particular in order to carry out macroscopic analyzes of biological samples.

Since the height of the sample chamber 20 is preferably between 8 and 10 mm, a significantly reduced pressure can be used, which protects the biofilms to be examined and also deflects any air bubbles that may be present. In addition, the height of the sample chamber 20 allows the operation to be performed in the vertical mode so as to culture the biofilm on both the top plate 40 and the bottom 22 surfaces. In contrast to conventional solutions, no air bubble trap is required. The retaining elements 12 formed on the end faces 11 of the carrier plate 10 enable alternating operation between a horizontal, vertical or stepless position in an angular range between 0 and 180°. A further advantage of the geometry according to the invention can be seen in the fact that several flow cells can be pushed into one another in a modular manner, i.e. a space-saving parallel connection is possible.

FIG. 5 shows a side view of the end face 11 of the carrier plate 10 from the two embodiment variants described above. You can see the U-shaped recess 14 with the inlet 16 or outlet 18 enclosed therein and the inlet channel 17 or outlet channel 19 integrated therein.

A variant of the flow cell according to the invention is shown in FIG. 6 in which the cover plate 40 is held by a cover frame 43 . The floor 22 in turn is held by a floor frame 23 . In this variant, both surfaces, ie the cover plate 40 and the base 22, are interchangeable. In this way, different surfaces can be colonized with a flow cell with biofilms or cell cultures. The biological samples can be used for further experiments outside of the flow cell. This enables, for example, the use of special antibodies Fluorescent markers or DNA probes for analysis or diagnostics, where the smallest amounts of the substances have to be applied to the surface with pinpoint accuracy. At least one latching lug 42 is also formed on the longitudinal side 13 of the support plate 10 and cooperates with a corresponding latching receptacle 44 of an adjacent flow cell. In the variant shown, there is a latching lug 42 and a latching receptacle 44 on the longitudinal side 13 of the carrier plate 10. Corresponding features can also be found on the opposite sides, but offset, so that when individual flow cells are connected in series, the adjacent carrier plates 10 can be connected to one another in a modular manner are.

FIG. 7 shows the variant in which only the cover plate 40 but not the base 22 can be removed. The locking lugs 42 or locking receptacles 44 can also be seen here. 7A illustrates that the top panel 40 is removable while the bottom 22 is fixed. 7B shows the assembled configuration. This variant is suitable for applications that do not involve microscopy. With this variant, for example, macroscopic analyzes of biological samples can be carried out. The experiments can be viewed live through a transparent cover plate 40 . The samples can be removed from the sample chamber 20 via the cover plate 40 for subsequent experiments.

Fig. 8 shows the variant with a removable cover plate 40 and base 22 (not shown). The latching lugs 42 and the latching receptacles 44 can also be seen here. You can also see the removable cover frame 43 for holding the cover plate 40 and the base frame 23 for receiving the base 22. The cover frame 43 and the base frame 23 are designed in such a way that the surfaces to be colonized with the biofilm or the cell cultures, i.e. the Top plate 40 and the bottom 22 can accommodate.

9 shows the modular arrangement of three flow cells. The individual carrier plates 10 are connected to one another via the latching lugs 42 or latching receptacles 44 formed on the longitudinal sides 13 .

A holding device 50 according to the invention can be seen in FIG. 10, which consists of a frame with a plurality of frame elements. In the variant shown, a frame element 52 is slipped onto another frame element of the frame and the frame is thus closed around the circumference. The support plates 10 of the two flow cells shown are guided in the groove 54 of the frame via the holding elements 12 formed on the end faces 11 and are thereby held in the holding device 50 . In Fig. 11, the individual frame elements are shown again in detail. The frame element 52 (FIG. 11B) is pushed onto the frame of the holding device 50 (FIG. 11A) via a corresponding nose 56, the nose 56 of one frame element (52) cooperating with the groove 54 of the frame.

12 shows a system for analyzing or diagnosing biofilms and cell cultures, consisting of a stand 60, a stand plate 62, support elements 64 and swivel joints 66, between which a holding device 50 according to the invention with the support plates 10 arranged therein is pivotably mounted. With this system, multiple flow cells can be pivoted together because the frame of the holding device 50 can accommodate multiple flow cells. The swivel joints 66 allow the entire holding device 50 with the flow cells accommodated therein to be pivoted over an angular range of preferably 180°. Alternatively, a stepper motor can also be used (not shown). The flow cell is connected to a pump via appropriate lines and connections so that the inlet 16 and outlet 18 communicate with one another fluidically.

FIG. 13 shows a system for analyzing or diagnosing biofilms and cell cultures, which is equipped with one (several) microfluidic flow cells. A holding device 50 accommodates a carrier plate 10 with a sample chamber 20 formed therein. The carrier plate 10 can be rotated in an angular range between 0 and 180° via rotary joints 66, with a working range between 0 and 90° preferably being selected. The holding device 50 is connected to the system via carrier elements 64 . The system includes a microcontroller (not shown) and is equipped with appropriate sensors to receive parameters required for analysis or diagnostics and perform appropriate control. The system thus comprises at least one flow sensor and one temperature sensor in order to determine the flow rate and the temperature in the sample chamber. The microcontroller then ensures that the aforementioned parameters are achieved via appropriate control devices. Thus, the system includes a heating element and a fan to create predetermined incubation conditions. Furthermore, the microcontroller also drives one or more drive units, for example stepper motors, in order to guide an incubation medium into the sample chamber and to pivot the carrier plate 10 in a predetermined angular range. On the front there is a display with a touchscreen 70 for monitoring and control, via which programming can be carried out. The control and regulation units of the system according to the invention are explained in more detail in FIG. FIG. 14 shows a block diagram of a microcontroller-controlled system for analyzing or diagnosing biofilms and cell cultures. The core is a microcontroller, which takes on various tasks. A power supply unit and a voltage regulator as well as corresponding switching elements are required to operate the system. A real-time clock (RTC) takes over the scheduling and sequence of individual process steps, ie the RTC is used to determine when and for how long certain process steps are carried out. In this case, programming can take place in accordance with previously defined parameters. A temperature sensor monitors the temperature and transmits the data to the microcontroller. If the temperature falls below a certain, predetermined threshold value, the microcontroller activates a heating element so that the temperature is increased. Conversely, the microcontroller controls a fan if the temperature in the sample chamber or the temperature of the incubation medium is above a certain threshold value. The microcontroller also controls the drive units, ie the motor drivers and the stepper motors. Motor driver I and stepping motor I ensure that the incubation medium flows into the sample chamber. The Motor Driver II and Stepper Motor II cause the platen to rotate within a predetermined angular range or to a specified angle. The microcontroller can be programmed in such a way that the angle changes gradually over a period of time. For example, the angle in the sticking phase can be 0°, with the plate being continuously pivoted upwards to 90° over the course of time. In this range of angles, air bubbles will escape upwards, leaving the surface of the slide free of air bubbles. For connectivity, the system is also equipped with a USB socket and an Internet adapter, for example. A display with a touchscreen ensures that the user can operate it accordingly.

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Claims

Patent Claims:

1 . Microfluidic flow cell for analyzing or diagnosing biofilms and cell cultures, comprising:

- a carrier plate (10) with a sample chamber (20) formed therein, which is delimited on the peripheral side by chamber walls (24) and a floor (22),

- A cover plate (40) which can be connected in a fluid-tight manner to the carrier plate (10), an inlet (16) with an inlet channel (17) integrated therein, which opens into the sample chamber (20) via an opening (26), an outlet ( 18) with a drainage channel (19) integrated therein, characterized by the following features:

- On the end faces (11) of the carrier plate (10) are holding elements (12) for fastening the carrier plate (10) to an object table of a microscope or a holding device (50).

2. Microfluidic flow cell according to claim 1, characterized in that the cover plate (40) and / or the bottom (22) is removably attached to the carrier plate (10).

3. Microfluidic flow cell according to claim 1 or 2, characterized in that the retaining elements (12) formed on the end faces (11) of the carrier plate (10) of the flow cell are designed in the form of strips, the aspect ratio between the longitudinal side and the broad side preferably being 3:1.

4. Microfluidic flow cell according to one of Claims 1 to 3, characterized in that a latching lug (42) and a latching receptacle (44) are formed on at least one longitudinal side (13) of the carrier plate, which engage with the corresponding latching receptacles (44) or Latches (42) of adjacent flow cells cooperate.

5. Microfluidic flow cell according to one of claims 1 to 4, characterized in that the diameter of the inlet channel (17) of the inlet (16) and the outlet channel (19) of the outlet (18) are> 1 mm.

6. Microfluidic flow cell according to any one of claims 1 to 5, characterized in that on the top or bottom of the carrier plate T (10). Groove (30) for receiving a seal (32) is formed.

7. Microfluidic flow cell according to one of claims 1 to 6, characterized in that the cover plate (40) is accommodated in a cover frame (43) and/or the base (22) in a base frame (23).

8. Microfluidic flow cell according to any one of claims 1 to 7, characterized in that at least the support plate (10) is made entirely of polyethylene (PE) or polypropylene (PP).

9. System for analyzing or diagnosing biofilms and cell cultures, comprising

- one or more microfluidic flow cells according to any one of claims 1 to 8,

- a holding device (50) for receiving one or more flow cells, wherein the holding device (50) is designed such that the one or more flow cells can be pivoted within an angular range of 0 to 180°, preferably 0° to 90°,

- A pump which is fluidically connected to the inlet (16) and outlet (18).

10. System according to claim 9, characterized in that the holding device (50) is a frame with a plurality of frame elements (52), the frame having a groove (54) for receiving one or more flow cells which are connected to the holding elements (12) of the carrier plate (10) cooperates.

11 . System according to claim 9 or 10, characterized in that the holding device (50) is a frame with a plurality of frame elements (52), the frame having a groove (54) for receiving one or more flow cells which are connected to the holding elements (12) of the carrier plate (10) cooperates.

12. System according to any one of claims 9 to 11, characterized in that the holding device (50) with the one or more flow cells via rotary joints or a stepper motor within an angular range of 0 ° to 180 ° is pivotable.

13. System according to any one of claims 9 to 12, characterized in that a flow sensor is configured so that it determines the flow rate of the incubation medium in the sample chamber (20).

14. System according to any one of claims 9 to 13, characterized in that a temperature sensor is configured to determine the temperature in the sample chamber (20).

15. System according to any one of claims 9 to 12, characterized in that a

Microcontroller controls the flow rate, the temperature, the angle range or the inflow of incubation medium.

16. System according to claim 15, characterized in that the microcontroller controls a heating element or a fan for temperature control.

17. Use of a microfluidic flow cell according to any one of claims 1 to 8 or a system according to any one of claims 9 to 16 for the analysis or diagnosis of biofilms or cell cultures.