WO2019193126A1 - Structure microfluidique pour effectuer des dosages biologiques et puce pourvue d'une telle structure - Google Patents

Structure microfluidique pour effectuer des dosages biologiques et puce pourvue d'une telle structure Download PDF

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Publication number
WO2019193126A1
WO2019193126A1 PCT/EP2019/058570 EP2019058570W WO2019193126A1 WO 2019193126 A1 WO2019193126 A1 WO 2019193126A1 EP 2019058570 W EP2019058570 W EP 2019058570W WO 2019193126 A1 WO2019193126 A1 WO 2019193126A1
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Prior art keywords
channel
assay
microfluidic structure
channels
biofilm
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PCT/EP2019/058570
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English (en)
Inventor
Eduard Torrents Serra
Josep SAMITIER MARTÍ
María José LÓPEZ MARTÍNEZ
Núria BLANCO CABRA
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Fundació Institut De Bioenginyeria De Catalunya (Ibec)
Universitat De Barcelona (Ub)
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Publication of WO2019193126A1 publication Critical patent/WO2019193126A1/fr

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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/08—Geometry, shape and general structure
    • B01L2300/0809—Geometry, shape and general structure rectangular shaped
    • B01L2300/0816—Cards, e.g. flat sample carriers usually with flow in two horizontal directions
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/08—Geometry, shape and general structure
    • B01L2300/0861—Configuration of multiple channels and/or chambers in a single devices
    • B01L2300/0864—Configuration of multiple channels and/or chambers in a single devices comprising only one inlet and multiple receiving wells, e.g. for separation, splitting
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/08—Geometry, shape and general structure
    • B01L2300/0861—Configuration of multiple channels and/or chambers in a single devices
    • B01L2300/0877—Flow chambers
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
    • B01L3/502761—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip specially adapted for handling suspended solids or molecules independently from the bulk fluid flow, e.g. for trapping or sorting beads, for physically stretching molecules

Definitions

  • the present invention relates to a microfluidic structure for carrying out biological assays. More specifically, the present invention seeks to guarantee reproducibility in biological growth assays and more specifically in the growth of biofilms and their treatment. Another objective is to reduce the assembly time, the test times and the sample volumes involved in the tests. A further objective is to guarantee the stability and uniformity of biofilms during the assays.
  • Biofilms Bacteria form biofilms by adhering to surfaces and developing complex communities.
  • a biofilm is a multicellular aggregate of cells encased in an extracellular polymeric matrix. Biofilms may form on living or non-living surfaces and can be prevalent in natural, industrial and hospital settings.
  • biofilms In the field of medicine, infections associated with the biofilm growth usually are challenging to eradicate. It is mostly due to the fact that mature biofilms display tolerance towards antibiotics and the immune response.
  • the biofilm matrix provides microorganisms with a protective shield that contributes significantly to several clinical challenges, including symptomatic inflammation, antibiotic resistance, recurrence, and the spread of infectious emboli.
  • cells can survive under harsh environments, for example, at high temperatures or in the presence of antibiotics.
  • the biofilm matrix improves a microbe’s opportunities for proliferation inside the body. Further, biofilms often form on the inert surfaces of implanted devices such as catheters, prosthetic cardiac valves and intrauterine devices, causing prevalent infections.
  • biofilms have become problematic in several industries due to the ability to form on plants and during industrial processes. Bacteria can survive long periods of time in water, animal manure, and soil, causing biofilm formation on plants or in the processing equipment. The build-up of biofilms can affect the heat flow across a surface and increase surface corrosion and frictional resistance of fluids. These can lead to a loss of energy in a system and overall loss of products. Along with economic problems, biofilm formation on food poses a health risk to consumers due to the ability to make the food more resistant to disinfectants.
  • the microbial cells growing in a biofilm are physiologically distinct from planktonic cells of the same organism, which, by contrast, are single-cells that may float or swim in a liquid medium.
  • planktonic cells of the same organism which, by contrast, are single-cells that may float or swim in a liquid medium.
  • a key characteristic sought in these devices is the reproducibility between channels, i.e. that the same or practically the same conditions must be achieved in all the channels.
  • Another objective pursued is to minimize the effect of manipulation by laboratory technicians. Indeed, part of the experimental procedures is done manually, specially injections of samples, and it is desired that these devices allow to minimize the differences in the flows that can be produced by the great variability that may arise in such manipulations.
  • Another objective sought is the simplification and the cost reduction of the experimental assembly and shorten the volumes of fluids involved, both by the occupation of space and by the storage of the waste generated. Additionally, it is important the reduction of material used in drug screening.
  • the Technical University of Denmark has designed a plate comprising three independent inlets, each connected to a single assay channel, each one connected to an outlet port, thus providing three independent parallel circuits on a single plate (Tolker-Nielsen T, Sternberg C,“Growing and analyzing biofilms in flow chambers” Curr Protoc Microbiol. 201 1 Chapter 1 , 1 B.2.1 -1 B.2.17).
  • the inlet and outlet ports, the assay channel and the connections between the ports and the assay channels are arranged longitudinally.
  • the particular topology of this device entails difficulties to guarantee the reproducibility of the tests between channels, since it is necessary to guarantee equal conditions at the entrances and exits.
  • the DTU mitigate their possible biofilm disturbances by increasing the length of their test chambers.
  • US2007202589A1 discloses a microfluidic structure for carrying out biological assays, which comprises a plurality of assay channels arranged in parallel, a first inlet port for inserting a first fluid, a second inlet port for inserting a second fluid and a common area with several culture cells places.
  • the inlet ports are both connected to a common channel, which outlet is connected to a distribution connector, perpendicular to the common channel.
  • the resultant topology provides an addition of the flows in the common channel but implies an uneven distribution of the flows in the assay channels. This uneven distribution is due to both the upstream and downstream conditions of the flows with respect to the assay channels.
  • One severe drawback of this design is that it is very difficult to guarantee homogeneity and thus reproducibility simultaneously in all the assay channels.
  • US5635358A and US2010170796A1 disclose an analytical device for carrying out biological assays, which comprises a plurality of assay channels arranged in parallel and a port for inserting a fluid, and a distributing channel system which connects the inlet port with the assay channels.
  • the distributing channel system is based in splitting three times the main channel such that eight channels are obtained that simulates a biological system, specifically the circulatory system.
  • these devices are not adapted to carry out biofilms assays, since the resulting shear stress, even for small flows, is too high.
  • US6440645B1 discloses a device having a surface microstructure of channels. All the embodiments disclose therein comprise two or more inlet ports connected to a common connection point, which is linked to a channel which ends in an outlet port. The device is aimed at the treatment of one sample obtained by mixing the fluids coming from the inlet ports.
  • the present invention proposes a microfluidic structure for carrying out biological assays, which comprises at least a first assay channel and a second assay channel, the assay channels being coplanar and arranged in parallel, a first inlet port for inserting a first fluid, a second inlet port for inserting a second fluid, a common outlet, a distributing channel system which connects the inlet ports with the first and second assay channels, and wherein the distributing channel system comprises a common connection point, a first supply channel connecting the first inlet port with the common connection point, a second supply channel connecting the second inlet port with the common connection point, a first distribution channel connecting the common connection point with the first assay channel, a second distribution channel separate from the first distribution channel connecting the common connection point with the second assay channel, and a first outlet channel connecting the first assay channel with the common outlet and a second outlet channel separate from the first outlet channel connecting the second assay channel with the common outlet.
  • This particular topology allows to overcome the drawbacks of the prior art devices cited in the background section.
  • this topology allows to obtain balanced flow conditions in the assay channels in assays involving two fluids, namely a biological sample and a growth medium, which, as already said, is a key factor to create similar or almost similar conditions in all the assay channels.
  • this topology can be implemented in a common base and in a very compact manner.
  • the claimed topology allows to obtain identical or near identical fluid conditions simultaneously in all the assay channels and on the other hand it allows to minimize the effect of the application of particular flow sequences such as those disclosed in the background section, that is, sequences where a fluid is injected manually in a short duration of time.
  • This topology makes it possible to reduce the impact on the injection of the way in which each laboratory technician performs the injection or, said in other words, it allows the conditions in the test chambers to be independent of the particular conditions in which the fluid injections are carried out without the disturbance of a formed biofilm. These conditions guarantee the uniformity in all our test channels.
  • the invention is well suited to carry out a sequence of fluid flows such are those disclosed with reference to Fig. 4, as described in more detail below.
  • the microfluidic structure has three or more assay channels.
  • the assay channels are planar and have a rectangular cross- section, such that a length L, a width W and a height H are defined per channel.
  • the connection point is a chamber having a cross-section area comprised between 1 ⁇ 2-S AC and 3/2-S AC , S AC being the cross-section area of each assay channel. More preferably the chamber has a cross-section area equal to S AC . In the present specification chamber is also called‘pre-chamber’.
  • the chamber is cylindrical and planar, such that is coplanar with the assay channels.
  • a hydraulic path is defined for each assay channel, each of the hydraulic path going from the common connection point to the common outlet having the same hydraulic losses for a determined flow rate.
  • the height H is greater than 150 pm.
  • the width W is comprised between 1 .9 mm and 2.1 mm.
  • the length L is comprised between 8 and 12 mm.
  • the invention also relates to a chip comprising a microfluidic structure according to the invention, which comprises a base where the inlets, the channels and the outlets are defined, and a cover.
  • the base is made of a material selected from PDMS (polydimethylsiloxane), PMMA (poly(methyl methacrylate)), polycarbonate, polystyrene, polypropylene, COC (cyclic olefin copolymer, such as TOPAS ® ), COP (cyclic olefin polymer, such as ZEONOR) and the cover is made of glass, silicone, titanium, iron, steel, copper, aluminium, or hydroxyapatite, among other suitable materials. More particularly, the base is made of a material selected from PDMS, PMMA, COC or COP (TOPAS ® ) and the cover is made of glass, COP (TOPAS ® ) or COC.
  • the inlets and outlets have their axis perpendicular to the base.
  • the chip comprises two or more microfluidic structures, such that each microfluidic structure is independent thus preventing contamination between structures.
  • a further aspect of the invention is related to a method of forming a biofilm starting from a biological sample, using the microfluidic structure of the invention, the method comprising the following steps:
  • Fig. 1 shows a plant view of the microfluidic structure according to a first embodiment.
  • Fig. 2 shows a plant view of the microfluidic structure according to a second embodiment.
  • Fig. 3 shows a plant view of the microfluidic structure according to an embodiment where the inlet and outlet ports have their axis perpendicular to the general plane of the assay channels.
  • Fig. 4 is a time diagram of the flows sequence of a biofilm assay for which the microfluidic structure is well suited.
  • Fig. 5 is a confocal laser scanning microscopy (CLSM) image of the sum and orthogonal views of a Pseudomonas aeruginosa PA01 formed biofilm stained with the LIVE/DEAD BactLight Bacterial Viability kit (Thermo Fisher Scientific) in a channel having a minimum height of 50 microns.
  • CLSM confocal laser scanning microscopy
  • Fig. 6 is a confocal laser scanning microscopy (CLSM) image of the sum and orthogonal views of a Pseudomonas aeruginosa PA01 formed biofilm stained with the LIVE/DEAD BactLight Bacterial Viability kit (Thermo Fisher Scientific) in a channel having a minimum height of 100 microns.
  • CLSM confocal laser scanning microscopy
  • Fig. 7 is a confocal laser scanning microscopy (CLSM) image of the sum and orthogonal views of a Pseudomonas aeruginosa PA01 formed biofilm stained with the LIVE/DEAD BactLight Bacterial Viability kit (Thermo Fisher Scientific) in a channel having a minimum height of 150 microns.
  • CLSM confocal laser scanning microscopy
  • Fig. 8 is a confocal laser scanning microscopy (CLSM) image of the sum and orthogonal views of a Pseudomonas aeruginosa PA01 formed biofilm stained with the LIVE/DEAD BactLight Bacterial Viability kit (Thermo Fisher Scientific) in a channel having a minimum height of 150 microns and in a microfluidic structure having a pre-chamber (V).
  • a microfluidic structure with a chamber size of 10 mm long x 2 mm wide was used, with an estimated flow rate of 3.8 mI/min in each chamber and a velocity of growth medium of 13.6 mm/s.
  • the biomass of this biofilm is 29.55 pm 3 /pm 2 , and the average thickness is 37.82 pm.
  • the measurements were made by Comstat2 software.
  • Fig. 9A shows an exploded view of the microfluidic structure.
  • Fig. 9B is an enlarged photograph of an embodiment of the microfluidic structure of the invention, in which the structure of the invention is present in quadruplicate.
  • Fig. 10A is a confocal laser scanning microscopy image of the sum and orthogonal views of a Pseudomonas aeruginosa PA01 formed biofilm.
  • a microfluidic structure with a chamber size of 10 mm long x 2 mm wide x 150 microns height with pre-chamber was used, with an estimated flow rate of 3.8 mI/min in each chamber and a velocity of growth medium of 13.6 mm/s.
  • Figs. 10B and 10C are graphs of the biomass and average thickness of the biofilms shown in Fig. 10A.
  • Fig. 1 1 A is a confocal laser scanning microscopy image of the sum and orthogonal views of a Staphylococcus aureus CECT86 formed biofilm.
  • a microfluidic structure with a chamber size of 10 mm long x 2 mm wide x 150 microns height with pre-chamber was used, with an estimated flow rate of 3.8 mI/min in each chamber and a velocity of growth medium of 13.6 mm/s.
  • Figs. 1 1 B and 1 1 C are graphs of the biomass and average thickness of the biofilms shown in Fig. 1 1 A.
  • Fig. 12 shows biofilm biomass of Pseudomonas aeruginosa cystic fibrosis isolate (PAET1 ) (Control) after treatment with 20 pg/ml ciprofloxacin (cpx 20).
  • the inventive microfluidic structure 1 for carrying out biological assays comprises a first assay channel C1 , a second assay channel C2, and a third assay channel C3 that are coplanar, identical and arranged in parallel.
  • the structure comprises a first inlet port 11 for inserting a first fluid, which is the growth medium, and a second inlet port I2 for inserting a second fluid, for example the biological sample or drug molecules test.
  • the microfluidic structure comprises a distributing channel system D which connects the inlet ports 11 , I2 with the three assay channels C1 , C2 and C3.
  • the distributing channel system D comprises a common connection point V, a first supply channel IP1 connecting the first inlet port 11 with the common connection point V and a second supply channel IP2 connecting the second inlet port I2 with the common connection point V.
  • a first distribution channel PC1 connects the common connection point V with the first assay channel C1
  • a second distribution channel PC2 separate from the first distribution channel PC1 connects the common connection point V with the second assay channel C2
  • a third distribution channel PC3 separate from the other distribution channels PC1 , PC2 connects the common connection point V with the third assay channel C3.
  • a first C01 , second C02 and third C03 separate outlet channels connect the assay channels to the common outlet 01 .
  • the assay channels C1 , C2, C3 are planar and have a rectangular cross-section, such that a length L, a width W and a height H are defined per channel C1 , C2, C3, L, W and H being most preferably the same for the three assay channels.
  • connection point V is a chamber having a cross- section area S A c which is the cross-section area of each assay channel C1 , C2, C3.
  • the chamber V is cylindrical and planar, such that is coplanar with the assay channels C1 , C2, C3.
  • the chamber V is connected to the inlets with two L-shaped supply channels.
  • the inlets 11 , I2 and the outlet 01 have a circular shape having the axis perpendicular to the general plane of the microfluidic structure.
  • volume can be reduced at least to 50 ml for a biofilm assay.
  • the hydraulic path defined for each assay channel C1 , C2, C3, going from the common connection point V to the common outlet 01 have the same hydraulic losses for a determined flow rate. This is achieved by balancing the linear losses and the losses caused by the direction changes and section changes along the different paths.
  • the height H of the channels is greater than 150 pm.
  • a structure like the one claimed in which this minimum height is established leads to conditions of shear stress that guarantee the survival of this particular biofilm, or biofilms having similar characteristics. This has been demonstrated by the inventors as can be seen in Figs. 5 to 7.
  • Figs. 5 and 6 the formation of biofilms for channel heights of 50 and 100 pm, respectively, can be seen in green. However, the green color is not uniform and dead bacteria can be seen in red, which rules out the trials. However, when a height of 150 pm is established, and as shown in Fig. 7, red spots are no longer seen, that is, the biofilm survives.
  • Fig. 10A is an image of confocal laser scanning microscopy of the sum and orthogonal views of a Pseudomonas aeruginosa PA01 formed biofilm or different channels stained with the LIVE/DEAD BactLight Bacterial Viability kit (Thermo Fisher Scientific).
  • Figs 10B and 10C are graphs of the biomass and average thickness of the biofilm, calculated with Comstat2 software. The three images and bars are from different channels, showing the uniformity of the biofilm obtained with the microfluidic structure of the present invention.
  • Fig. 1 1 A is an image of confocal laser scanning microscopy of the sum and orthogonal views of a Staphylococcus aureus CECT86 formed biofilm stained with the LIVE/DEAD BactLight Bacterial Viability kit (Thermo Fisher Scientific).
  • Figs 1 1 B and 1 1 C are graphs of the biomass and average thickness of the biofilm, calculated with Comstat2 software. The three images and bars are from different channels, showing again the uniformity of the biofilm obtained with the inventive microfluidic structure.
  • Preferred dimensions of the microfluidic structure are a width W about 2 mm and a length about 10 mm. These dimensions are sufficient to allow the formation of sufficient biofilm for observation with a confocal or fluorescent microscope.
  • the resulting structure is very small, but has proven to be very efficient for biofilm growth, stability and analysis.
  • the inlets and outlets have their axis perpendicular to the base, such that an easy connection is ensured. This arrangement also prevents contamination upstream.
  • the chip supports four microfluidic structures such as disclosed above, such that each microfluidic structure are independent thus preventing contamination between structures.
  • This particular embodiment allows to carry out multiplexing analysis, while using only one microfluidic structure.
  • Fig. 9A shows an exploded view of the microfluidic structure.
  • the structure can be obtained from two pieces only, a first substrate part L1 , such as a polymer in which the different channels are practiced, on top of which there is a glass cover L2.
  • Fig. 9B is an enlarged photograph of an embodiment of the microfluidic structure of the invention, in which the structure 1 of the invention is present in quadruplicate. This structure allows for multiple tests with a single disposable microfluidic structure, in which the same conditions are also available in all devices, which allows valid comparisons between treatments.
  • the manufacture of the microfluidic structure consists of a multistep procedure designed to obtain a deep chamber using photolithographic and soft-lithographic techniques. All procedures are carried out in a clean room facility. Master is designed using CAD software and manufactured over a glass slide. The manufacturing process starts with three consecutive solvent baths of acetone, isopropanol and ethanol. Following the cleaning protocol, next step is pattern Ordyl SY 550. This negative photoresist is laminated on the top of a glass slide using a hot/cold laminator to get a smooth attached film surface (50 pm-high). A second layer of Ordyl SY 550 is laminated on the top of the previous one in order to get 100 pm high. Then, the slide is exposed through an acetate photomask to UV light (5 s, 24 mW cm 2 , 345 nm) in the mask aligner and subsequently placed on a hot plate at 65 °C for 3 minutes.
  • UV light 5 s, 24 mW cm 2 , 345
  • the 3D master mould manufacturing is finished by developing the Ordyl film using the developer for 3 minutes.
  • a PDMS prepolymer mixture (a curing agent-to-PDMS ratio of 1 :10, Sylgard ® 184, Dow Corning) is placed in a desiccator with vacuum applied in order to remove bubbles. Then, the mixture is poured on top of the Ordyl master to fabricate a PDMS mould and heated at 65 °C overnight.
  • the casted PDMS is peeled off carefully and inlet and outlet holes are made using a Harris Uni-Core 1 mm puncher.
  • PDMS structures are permanent bonded to a cover-glass slide using 02 plasma to activate surface and immediately pressed together. PDMS deep chamber chip is ready to use.
  • the obtained microfluidic structure has a chamber size of 10 mm long x 2 mm wide x 150 pm.
  • the illustrated and described structures have turned out to be specially adapted to realize a protocol for the growth of biofilms as the one depicted by means of a flow vs time graph shown in Fig. 4.
  • the procedure consists in first establishing a constant flow of nutrient medium, for example TSB (Tryptic Soy Broth) or LB (Luria Bertani medium), and by means of a pump, whose flow rate can be precisely controlled. This is done using the second inlet 12, for example.
  • This flow rate Q is shown as a constant function that is interrupted to give rise to the inoculation of bacteria or biofilm treatment with antibacterial drugs. This inoculation has been represented as a peak, and represents a manual injection by a laboratory operator.
  • the invention allows to control this inoculation stage without a biofilm disturbance. Indeed, by providing the particular topology of the invention, where the connection point is pre-chamber V, it is achieved that the volume of bacteria inoculated reaches in a balanced manner the three test channels, which will allow a valid comparison to be made between them.
  • the system is left at rest, about two hours, without flow, to allow the bacteria that start the biofilm to adhere to the glass cover of the assay channels C1 , C2, C3.
  • This step involves a high volume of nutrient, so that the reduction in dimensions that has been possible thanks to the particular configuration of the microfluidic structure according to the invention.
  • a pre-analysis treatment is carried out.
  • the biofilm structure is crucial.
  • the nutrient flow is interrupted and a treatment is injected.
  • This injection is usually done manually and is a particularly delicate stage for which the microfluidic structure of the invention has proved to be specially adapted. If a microfluidic structure as shown in Fig. 1 1 is used, then one or more independent treatments can be carried out.
  • an aspect of the invention is related to a method of forming a biofilm using the microfluidic structure of the invention comprising the steps as explained in section Description of the invention.
  • the resulting formed biofilm in the microfluidic structure can be further analysed in connection with other biological techniques such as microscopy or direct measurement of the biofilm rate by e.g. an impedance system.
  • aspects of the biofilm that can be analysed are morphology, grow, biomass, roughness, thickness, or bacterial interactions.
  • the method of forming a biofilm further comprises applying a treatment to the formed biofilm, so that the method comprises the additional steps following step (iv):
  • Pseudomonas aeruginosa PAET1 biofilm were cultivated for 3 days at room temperature in flow cell chambers with channel dimension of 10 mm long x 2 mm wide x 150 pm high; then the flow was stopped and treatment with 20 pg/ml of free ciprofloxacin (CPX) was added for 1 additional day without flow.
  • Biofilms were stained with a mixture of 6 mM SYTO 9 and 30 mM propidium iodide at room temperature in the dark for 30 min, according to the specifications of the LIVE/DEAD BacLight Bacterial Viability kit (Molecular Probes).
  • the present invention arises from the need to be able to carry out tests with biofilms, guaranteeing the reproducibility of the tests, while facilitating the handling, reducing the volumes involved, the experimentation times and therefore the costs.
  • the microfluidic structure of the invention can be used in basic research for studying bacteria in an in vivo- like environment, mimicking natural biological conditions in a three- dimensional culture system. Remarkably, it can also be used in the pharma industry for high- throughput screening of anti-biofilm molecules, and in clinical settings in a microbiological service to evaluate the best antimicrobial treatment for given person (personalised medicine). It can be used as a platform to evaluate biofilm growth of different bacterial species, including clinical isolates from patients infected with bacteria forming biofilms.
  • the antibiofilm activity of various compounds can be evaluated simultaneously in a high-throughput way with the structure of the invention.
  • Growth complex mixed biofilms can be tested together with eukaryote cells to mimic the realistic conditions during infections.
  • Different bacterial species can be co-cultured and screened in a single biofilm chip, since infectious diseases are not typically due to a single bacterial growth.
  • the microfluidic structure allows the biofilm growth without cross contamination among the different channels.
  • the microfluidic structure can house hundreds of continuous-flow biofilms allowing independent treatment of them with different drugs, together with their optical characterization and analysis by coupled technologies such as confocal laser scanning microscopy (CLSM).
  • CLSM confocal laser scanning microscopy
  • microfluidic structure can be combined with existing quantification tools, such as confocal and fluorescence microscopy, to enable real-time monitoring of biofilm developments.
  • the microfluidic structure according to the invention can be easily used in an automated analysis unit.
  • biological fluid analysis devices consisting of two subunits, namely a first subunit comprising the pumps and the control electronics of the pumping parameters and a second subunit designed to house the disposable unit in which the biological samples are grown.
  • the subunit housing the disposable unit generally comprises an insertion slot which leads to a housing conveniently provided with the fluid supply ports.
  • the chip object of the present invention it is only necessary to adapt the dimensions of said slot and the housing, so that the chip of the present invention can be easily used with machines available on the market.
  • microstructure and chip described can be very advantageous in any type of microfluidic assay in which it is essential to guarantee reproducibility for other cell analysis (yeast, animal cells, etc.).
  • cell live assays such as cell interaction, cell rolling, cell adhesion, cell migration, cell aggregation, cell invasion, cell exposure and differentiation, analysis of chemoattractants, wound healing assays, bacteria interaction with cells, planktonic growth of bacteria, bacteria co-culture, single cell analysis among others.

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Abstract

Une structure microfluidique (1) pour effectuer des dosages biologiques, qui comprend un premier canal de dosage (C1) et un second canal de dosage (C2) coplanaire, identiques et disposés en parallèle, un premier orifice d'entrée (I1) pour introduire un premier fluide, un second orifice d'entrée (I2) pour introduire un second fluide, une sortie commune (O1), et un système de canaux de distribution (D) comprenant un point de connexion commun ou une pré-chambre (V), un premier canal d'alimentation (IP1) reliant le premier orifice d'entrée (I1) au point de connexion commun (V), un second canal d'alimentation (IP2) reliant le second orifice d'entrée (I2) avec le point de connexion commun (V), un premier canal de distribution (PC1) reliant le point de connexion commun (V) au premier canal de dosage (C1), un second canal de distribution (PC2) séparé du premier canal de distribution (PC1) reliant le point de connexion commun (V) au second canal de dosage (C2), et un premier canal de sortie (CO1) reliant le premier canal de dosage (C1) à la sortie commune (O1) et un second canal de sortie (CO2) séparé du premier canal de sortie (CO1) reliant le second canal de dosage (C2) à la sortie commune (O1). L'invention concerne également une puce (2) ayant deux de ces structures microfluidiques (1) ou plus et un procédé dans lequel une telle structure microfluidique est utilisée.
PCT/EP2019/058570 2018-04-06 2019-04-04 Structure microfluidique pour effectuer des dosages biologiques et puce pourvue d'une telle structure WO2019193126A1 (fr)

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WO2023222631A1 (fr) * 2022-05-17 2023-11-23 Centre National De La Recherche Scientifique Puce microfluidique pour croissance cellulaire

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