Methods and devices for clinical testing

Abstract

A method for determining the interaction between a drug and a cell type is provided, the method comprising: providing an array of first droplets, each droplet containing a cell type derived from a biological sample; providing an array of second droplets, each droplet containing one or more drugs at one or more predetermined concentrations; combining the array of first droplets and the array of second droplets to form an array of combined droplets; monitoring the properties of one or more cells in the combined droplets using an optical detection system configured to detect the interaction between the cell type and the drug.

Description

Technical Field

[0001] The present invention relates to methods and apparatus for determining the interaction between a drug and cells of a cell type, and in particular to the rapid and accurate profiling of antimicrobial susceptibility and the identity of the organism causing an infection. Background Art

[0002] Rapid and accurate profiling of the microorganisms causing an infection is a major challenge in the healthcare field. Blood culture analysis remains the gold standard for diagnosing infections such as sepsis. However, this method is generally too slow and cumbersome to have any significant impact on the initial management of patients. Patients are typically treated immediately with broad-spectrum antibiotics while waiting for blood culture results. There is a need for a diagnostic test that can rapidly capture clinically relevant organisms and characterize antimicrobial resistance properties such as the minimum inhibitory concentration (MIC) of a particular drug.

[0003] Microfluidic platforms have been proposed to address this need, but to date no method has been proven to be practical and reliable in a clinical setting. In particular, the ability to perform simultaneous multiplex assays on multiple different drugs and cells has proven to be erratic.

[0004] Determining the MIC using microfluidics has been shown to employ continuous flow microfluidics, for example, Choi et al., "Rapid antibiotic susceptibility testing by tracking single cell growth in a microfluidic agarose channel system", Lab Chip, 2013, 13, 280 - 287. However, continuous flow microfluidics does not allow for the extensive and refined test panel possible with a microdroplet manipulation platform.

[0005] Lyu et al., "Phenotyping antibiotic resistance with single-cell resolution for the detection of heteroresistance", Sensors and Actuators B: Chemical 270 2018 369 - 404, describes a microfluidic method for quantitatively phenotyping heterogeneous resistance by encapsulating single bacterial cells from a heterogeneous population into microfluidic droplets. However, the number of droplets that can be manipulated simultaneously, as well as the number and concentration of antibiotics that can be evaluated simultaneously, are limited. The throughput is limited in part due to the absence of a mechanism for discarding empty droplets, which must be carried out together with those droplets containing cells in this method.

[0006] Similar limitations also exist in Sabhachandani et al., “Integrated microfluidic platform for rapid antimicrobial susceptibility testing and bacterial growth analysis using bead-based biosensor via fluorescence imaging”, Microchimica Acta 184 2017, 12, 4619-4628, which describes a droplet-based microfluidic method for phenotypic antimicrobial susceptibility testing (AST). Only low-viability droplet counting can be achieved, and unwanted droplets cannot be removed from the surface of the microfluidic chip.

[0007] There is clearly a need for a method of characterizing antimicrobial resistance properties such as MIC that is fast and accurate enough to be practical in a clinical setting. Based on the prior art, no method has been shown to be able to reliably do this. The methods and devices of the present invention are set forth in this context. SUMMARY OF THE INVENTION

[0008] According to a first aspect of the present invention, there is provided a method for determining the interaction between a drug and a cell type, the method comprising:

[0009] providing an array of first microdroplets, each microdroplet containing a cell type derived from a biological sample;

[0010] providing an array of second microdroplets, each microdroplet containing one or more predetermined concentrations of one or more drugs,

[0011] combining the array of first microdroplets and the array of second microdroplets to form an array of combined microdroplets;

[0012] monitoring the properties of one or more cells in the combined microdroplets using an optical detection system to detect the interaction between the cell type and the drug.

[0013] In some embodiments, the array of second microdroplets comprises microdroplets having multiple different concentrations for each drug.

[0014] In some embodiments, each microdroplet may contain one or more cells.

[0015] In some embodiments, the one or more cells may be of a single type or strain and / or a mixture.

[0016] In some embodiments, the step of providing the array of first microdroplets comprises:

[0017] Emulsify a biological sample with an immiscible carrier fluid to form aqueous first microdroplets, where at least some contain cells of one or more cell types;

[0018] Load the first microdroplets onto a microfluidic chip configured to manipulate the microdroplets using real or virtual electrowetting electrodes;

[0019] Determine that the microdroplets containing cells contain at least one live cell for proliferation; and

[0020] Generate an array of the first microdroplets from the at least one live cell.

[0021] In some embodiments, providing the array of the first microdroplets includes:

[0022] Move a first portion of the first microdroplets to a cell proliferation location configured to provide aerobic conditions;

[0023] Move a second portion of the first microdroplets to a second cell proliferation location, the second cell proliferation location being separate from the first cell proliferation location and configured to provide anaerobic conditions; and

[0024] Monitor and record the cell behavior of the first and second portions of the first microdroplets to determine whether the first microdroplets contain live aerobic cells or live anaerobic cells.

[0025] In some embodiments, select the environmental conditions at the first and second cell proliferation locations to promote the proliferation of cells of the cell type, the environmental conditions including temperature and environmental fluid composition.

[0026] In some embodiments, the steps of providing an array of second aqueous microdroplets include one or more of the following:

[0027] Emulsify one or more drugs with an immiscible carrier fluid to form aqueous second microdroplets, each microdroplet containing at least one drug type;

[0028] Load the second microdroplets onto a microfluidic chip configured to manipulate the microdroplets using real or virtual electrowetting electrodes;

[0029] Perform a series of dilution operations on the second microdroplets to create an array.

[0030] In some embodiments, the method further includes assaying a subsample of one or more cells from one or more microdroplets.

[0031] In some embodiments, the assay is one or more of the following: Gram staining, destructive lysis assay, oxidase test, or wheat germ agglutinin (WGA) assay.

[0032] In some embodiments, the assay is performed before combining the first and second arrays.

[0033] In some embodiments, the method further comprises performing at least one cell identification assay on cells from a biological sample using a classification algorithm.

[0034] In some embodiments, the cell identification step comprises analyzing one or more characteristics of cells from a biological sample, the one or more characteristics including at least one of the following: cell motility, cell shape, and cell proliferation behavior.

[0035] In some embodiments, the method further comprises determining the minimum inhibitory concentration (MIC) of at least one drug on cells of a cell type based on the characteristics of one or more cells detected over time in the merged droplets.

[0036] In some embodiments, the drug is an antibiotic or an antifungal drug for treating sepsis.

[0037] In some embodiments, the method further comprises determining an antibiotic or antifungal treatment regimen for treating sepsis based at least in part on the determined MIC.

[0038] In some embodiments, the first and / or second droplet further comprises a growth medium. The medium can be a cell growth medium and is selected from one or more of the following, but is not limited to, RPMI 1640, EMEM, DMEM, Ham's F12, Ham's F10, F12-K, HAT medium, or a modified version thereof.

[0039] In some embodiments, the method can further comprise the step of splitting the merged droplet containing one or more cells to form a clonal colony.

[0040] In some embodiments, one or more cells can be stained for viability or detection studies and / or express a fluorescent protein. For example, nucleic acid stains such as propidium iodide, TO-PRO TM -3 iodide, Zombie Green TM, or membrane stains can be used. In some embodiments, nucleic acid or membrane stains can be used to count the number of cells. In some embodiments, a dead stain can be used for viability measurement.

[0041] According to another aspect of the present invention, there is provided a microfluidic chip device configured to perform the method of any of the foregoing embodiments, the device comprising:

[0042] A sorting component configured to separate droplets containing cells from empty droplets;

[0043] A droplet manipulation component configured to manipulate droplets using real or virtual electrowetting electrodes;

[0044] An optical detection system configured to monitor droplets contained in a microfluidic chip through one or more detection windows.

[0045] In some embodiments, the device further includes a cell culture component configured to hold a first droplet and provide conditions favorable for cell proliferation.

[0046] In some embodiments, the device further includes a sample preparation component configured to generate a droplet emulsion from a biological sample in an immiscible carrier fluid.

[0047] In some embodiments, a detection system can be provided to detect one or more droplets. The one or more droplets can utilize light or optical spectroscopy, such as fluorescence spectroscopy. In some embodiments, the detector can be configured to detect the fluorescence of one or more droplets. In some embodiments, the detector can be a fluorescence detector.

[0048] In some embodiments, the droplet manipulation component includes one or more OEWOD structures, the OEWOD structures comprising:

[0049] A first composite wall comprising:

[0050] A first substrate;

[0051] A first transparent conductor layer located on the substrate, the thickness of the first transparent conductor layer ranging from 70 to 250 nm; and

[0052] A first dielectric layer located on the photosensitive layer, the thickness of the first dielectric layer ranging from 30 to 280 nm;

[0053] A second composite wall comprising:

[0054] A second substrate;

[0055] A second conductor layer located on the substrate, the thickness of the second conductor layer ranging from 70 to 250 nm;

[0056] A photosensitive layer located on the second conductor layer and activated by electromagnetic radiation in a wavelength range of 400 - 850 nm, the thickness of the photosensitive layer ranging from 300 - 1500 nm, and

[0057] Optionally, a second dielectric layer located on the second conductor layer, the thickness of the second dielectric layer ranging from 30 to 280 nm

[0058] wherein the exposed surfaces of the first and second dielectric layers are configured to be spaced 1 - 180 μm apart to define a microfluidic space suitable for containing droplets;

[0059] An A / C power supply for providing a voltage across first and second composite walls connecting a first and a second conductor layer;

[0060] At least one electromagnetic radiation source having an energy higher than the bandgap of the photosensitive layer, adapted to impinge on the photosensitive layer to induce corresponding virtual electrowetting positions on the surface of the first dielectric layer; and

[0061] A device for manipulating the point of impingement of the electromagnetic radiation on the photosensitive layer so as to change the arrangement of the virtual electrowetting positions, thereby generating at least one electrowetting path along which the microdroplets move.

[0062] According to another aspect of the present invention, there is provided an array of microdroplets, the array comprising:

[0063] A plurality of drug microdroplets, each drug microdroplet containing one or more drugs from a group of different drug types within a predetermined concentration range; and

[0064] Control microdroplets, the number of which is less than or equal to the number of microdroplets in the plurality of drug microdroplets, the control microdroplets not containing drugs. Description of the Drawings

[0065] Figure 1 Shows a first part of an exemplary experimental workflow, in which sample cells are emulsified into a plurality of cell-containing microdroplets, which are then loaded onto a microfluidic chip;

[0066] Figure 2 Shows a second part of the experimental workflow, in which cell behavior is monitored to identify viable cells suitable for generating an array of first microdroplets, and optional assays are performed on one or more cells from the sample;

[0067] Figure 3 Shows a third part of the experimental workflow, in which the cultured cells are split into an array of first microdroplets;

[0068] Figure 4 Shows a fourth part of the experimental workflow, in which an array of second microdroplets containing different concentrations of drugs is prepared;

[0069] Figure 5 Shows a fifth part of the experimental workflow, in which the first and second arrays are combined and the cell behavior in the combined microdroplets is monitored to characterize the interaction;

[0070] Figure 6 Shows a side view of an exemplary configuration of a microfluidic chip for performing the method of the present invention;

[0071] Figure 7 Shows Figure 6Top view of an embodiment configuration of a microfluidic chip and illustrates an embodiment workflow that can be performed thereon;

[0072] Figure 8 An embodiment array generated by the method according to the present invention is shown, the array comprising a range of droplet sizes, each droplet having a range of bacteria.

[0073] Figure 9 Experimental results of bacterial growth in microdroplets obtained by the method according to the present invention are shown.

[0074] Figure 10 An embodiment of panel merging performed using the method and device according to the present invention is shown.

[0075] Figure 11 A set of embodiment experimental results obtained using the method and device according to the present invention is shown, which allows for MIC determination between an Escherichia coli strain and gentamicin antibiotic. DETAILED DESCRIPTION OF THE INVENTION

[0077] To further illustrate various aspects of the present invention, the specific embodiments of the present invention will now be described in detail with reference to the accompanying drawings.

[0078] The present invention provides methods and devices for determining the interaction between cells and drugs and thereby obtaining information that can be used to develop treatment regimens using said drugs.

[0079] REFERENCE Figure 1 , illustrates a first part of an embodiment workflow of the method according to the present invention, the workflow comprising a plurality of steps. Although the steps of the embodiment workflow are provided in a specific order, those skilled in the art will understand that the method of the present invention can be performed in any number of different orders.

[0080] In a first step 1, one or more sample cells are suspended in a nutrient solution, such as Luria - Bertani (LB) medium, tryptic soy broth (TSB), Mueller - Hinton medium, or Super Optimal Broth (SOB).

[0081] In a second step 3, the cells are emulsified and dispensed into a fluorocarbon oil to form a plurality of microdroplets. Typically, the fluorocarbon oil is one of HFE - 7500, HFE - 7700, FC - 40, or FC - 70. Such oils are selected to contain a suitable fluorinated surfactant, such as RAN - 008, Picosurf 1, Picosurf 2, or dSurf. The oil can optionally be further treated with a nutrient solution to provide a more favorable environment for the emulsified cells. Those skilled in the art will understand that the specific nutrient solution used will depend on the particular cell type being studied.

[0082] Initially, the emulsion contains both cell-containing droplets and empty droplets.

[0083] In a third step 5, the droplets are loaded onto a device such as a microfluidic chip. For example, the droplets can be loaded onto a chip including an optically-mediated electrowetting-on-device (oEWOD) stack structure.

[0084] In one embodiment, an emulsifier is integrated into a device such as a microfluidic chip. For example, the emulsifier can be integrated into a chip including an optically-mediated electrowetting-on-device (oEWOD) stack structure.

[0085] In one embodiment, the emulsification is vortex emulsification. In another embodiment, the emulsification is stepwise emulsification.

[0086] The microfluidic chip classifies the first droplets into cell-containing droplets and empty droplets 7, and the empty droplets are discarded 9. Discarding the empty droplets can include completely removing them from the chip or storing them in a holding area of the microfluidic chip. The classification can be performed by optical inspection of each droplet. Each cell-containing droplet can contain one or more cells. In some embodiments, sorting can be facilitated by automated software and the droplets can be manipulated along the surface of the microfluidic chip by oEWOD-induced forces.

[0087] The cell-containing droplets are moved to a cell proliferation location 11 on the chip surface. The cell proliferation location can include a first cell proliferation location and a second cell proliferation location. The first cell proliferation location includes a target area of the chip configured to provide environmental conditions favorable for aerobic cell proliferation, and the second cell proliferation location includes a target area of the chip configured to provide environmental conditions favorable for anaerobic cell proliferation.

[0088] Once in place, one or more cells remain in place 13 at the target area. Cell proliferation is allowed during a culturing step 15. In some embodiments, the culturing step requires the cells to remain in place at one or more target areas for a specific length of time, which will depend on the generation time of the particular pathogen being studied. Under ideal experimental conditions, the culturing step may require the cells to remain in place for between 15 minutes and 1 hour. However, it is recognized that various factors may extend this incubation time in practice, but the culturing phase is not expected to exceed 12 hours. Those skilled in the art will understand that the specific length of time the cells remain at one or more target areas will vary depending on the exact type of cells being studied and the number of generation cycles required to obtain a sufficient number of sample cells.

[0089] At any point in the workflow, the cells contained in the droplets can be manipulated in any number of ways according to the needs of a particular sampling assay. Such manipulation can include altering the electrowetting conditions of the droplet such that the droplet dewets or partially dewets from the surface. As used herein, the term "dewetting" refers to a change in the contact angle between the droplet and the chip surface such that the droplet is pulled away from the surface.

[0090] The oEWOD forces can also be used to agitate and "stir" the droplets to disperse the cells contained therein, or to stretch and elongate the droplets to form smaller daughter droplets (if single cells need to be assayed from a cultured colony). This process can be aided by keeping the mother droplet on the surface of the target area wet or partially wet. The cell occupancy of the daughter droplets can then be examined, and if the desired cell distribution is not achieved, the droplets can be re-merged and split again.

[0091] Reference Figure 2 , after allowing the cells to proliferate for a sufficient length of time, the droplets are optically interrogated to determine which cells are viable for creating the array 17. In some embodiments, viable cells are those determined to have a suitable proliferation rate, while cells determined to have undergone a small number of divisions are classified as non-viable. In other embodiments, cells determined to have undergone a small number of divisions can be determined to be fastidious - i.e., cells that grow slowly, or cells that grow only when special nutrients are present in the growth medium or specific conditions are met. In embodiments where fastidious cells are determined to be present, these cells can be further incubated under suitable conditions.

[0092] The droplets containing viable cells are separated 19 from the other droplets, and the remaining droplets containing non-viable cells are discarded 21, or further incubated as described above. If it is determined at this point that no cells are viable, the process is stopped and steps 1-17 are repeated with a new set of one or more sample cells, or it is determined that the original sample does not contain viable cells. In some embodiments where it is determined that the original sample does not contain viable cells, the sample examination is stopped and the result is reported as negative.

[0093] Optionally, if viable cells are determined to be present, the droplets containing these viable cells can be split into two parts 23, where the first part is assayed 25 to characterize the cells contained therein, such as a Gram stain test or image-based cell identification, and the second part is further cultured to generate more cells for additional assays on the chip. Image-based cell identification can be performed on the first or second part of the droplets, or can be performed on all droplets containing viable cells without the need to split the droplets as in 23. The assay 25 can provide information for determining a set of drugs to test against the cells.

[0094] In embodiments where the determination 25 is an image-based cell identification, the image-based identification can be performed by a human or it can be performed automatically using a software algorithm. Those skilled in the art will understand that, using a database of known cells, a software algorithm can be trained to classify cells into known classes based on features from which the algorithm is derived from the database.

[0095] Reference Figure 3 , the microdroplets 27 containing live cells are further cultured to produce a sufficient number of cells to test a set of different types of drugs at different concentrations. Once it is determined that a sufficient number of cells have been produced, the microdroplets are multiplexed 29 to obtain a plurality of first microdroplets, each first microdroplet containing at least one live cell, which are then formed into an array 31. Optionally, at this stage, one or more microdroplets can be selected for further evaluation in the form of an assay, which can be supplementary to or in place of 25. The microdroplets can be further split to construct a panel of sub-microdroplets, each sub-microdroplet containing at least one cell. Then one or more microdroplets from the assay array can be combined with the sub-microdroplets, the array containing microdroplets with the appropriate assay reagents at the appropriate concentration for the assay in question, while continuing to culture the remaining microdroplets of 31.

[0096] The manipulations required to form the assay array can be performed on-chip or off-chip. In embodiments where the assay array is prepared off-chip, the assay array is then brought onto the same chip as the first array. In embodiments where the assay array of microdroplets is prepared off-chip, the assay array can optionally be stored in a cartridge, where the cartridge is shared among multiple chips and can be dispensed with the required assay array as needed. In some embodiments, the assay array can include a set of pre-dropletized assay reagents stored on-chip, each assay reagent being accompanied by one or more control droplets without the assay reagent.

[0097] In some embodiments, the assay array of microdroplets is "pre-loaded" onto the chip, and the chip is stored under conditions suitable for maintaining the integrity and activity of the assay reagents. For example, such conditions can be a controlled temperature. In some embodiments, the temperature can be controlled to be about 4 °C, selectively in the assay reagent storage area.

[0098] In some embodiments, the first microdroplets are further split 33 such that the microdroplets in the array 35 each initially contain only a single live cell.

[0099] In some embodiments, whenever the minimum cell number has been reached, the microdroplets containing live cells can be repeatedly split and combined with empty microdroplets containing culture medium to prevent the cells from entering the stationary phase of the cell growth curve. Those skilled in the art will understand that the minimum cell number at which the microdroplets containing live cells can be split and combined with empty droplets containing culture medium can vary depending on the particular cell type being studied.

[0100] Reference Figure 4 Separate from the array of the first microdroplets, the array 43 of the second microdroplets is formed by combining 41 microdroplets 37 containing a known concentration / volume of a drug with microdroplets 39 containing a known concentration / volume of a diluent. Alternatively, the array 43 of the second microdroplets can be formed by a series of alternating combining and splitting operations. Thus, an array including microdroplets with a known concentration of a drug is created, including microdroplets containing 0% concentration of the drug that serve as controls. As described above, if an assay is performed on the first microdroplets, the drug contained in the array of the second microdroplets can be based on one or more results obtained from the assay.

[0101] The operations required to form the array 43 of the second microdroplets can be performed on-chip or off-chip. In an embodiment, where the second array of microdroplets is prepared off-chip and then brought onto the same chip as the first array. In an embodiment, where the second array of microdroplets is prepared off-chip, the second array can optionally be stored in a cartridge, where the cartridge is shared among multiple chips and the required second array can be dispensed on demand. In some embodiments, the second array can include a set of pre-dropletized drugs stored on-chip, each drug present at two or more dilution levels, accompanied by one or more control droplets without the drug.

[0102] In some embodiments, the second array of microdroplets is "pre-loaded" onto the chip and the chip is stored under conditions suitable for maintaining the integrity and pharmacological activity of the drug. For example, such conditions can be a controlled temperature. In some examples, the temperature can be controlled at about 4°C, selectively in the drug storage area.

[0103] In some embodiments, the second array contains between one and ten different drugs. In other embodiments, the second array contains between one and fifteen different drugs. In other embodiments, the second array contains between one and twenty different drugs.

[0104] In some embodiments, the second array can contain, for example, microdroplets of different dilutions containing one or more of oxacillin, vancomycin, imipenem, gentamicin, ciprofloxacin, cefoxitin, metronidazole, ampicillin, daptomycin, linezolid, nystatin, or voriconazole.

[0105] Those skilled in the art will further understand that the second array can contain one or more control microdroplets without the drug. These control microdroplets can further include a growth medium required for cell culture.

[0106] Reference Figure 5, an array 45 of first microdroplets is merged 47 with an array 49 of second microdroplets. The merged array 51 is then cultured, and one or more cells contained within the merged microdroplets are monitored optically or otherwise to determine the antimicrobial resistance characteristics of the cells. For example, the MIC of the cells associated with each drug can be determined, and it can be determined whether the drug is bacteriostatic, bactericidal, fungistatic, or fungicidal against the cells of that cell type.

[0107] Reference Figure 6 , illustrates an exemplary configuration of a microfluidic chip including an oEWOD stack suitable for implementing the method according to the present invention.

[0108] The exemplary device is suitable for manipulating aqueous microdroplets 53 that have been emulsified in oil, such as hydrocarbon oil or fluorocarbon oil, having a viscosity of 5 centistokes or less at 250 °C and having a diameter, for example, between 20 μm and 50 μm in their unconstrained state. In other embodiments, the device can be suitable for droplets having an unconstrained diameter between 4 μm and 100 μm. In some embodiments, the oil can be fluorocarbon oil HFE7500 having 2% RAN, or it can also be hydrogenated RTM6 containing up to 5% ABIL.

[0109] The oEWOD stack of the device can include top 55a and bottom 55b glass plates, each 500 μm thick and coated with a transparent layer 57 of conductive indium tin oxide (ITO) having a thickness of 130 nm. Each conductive indium tin oxide (ITO) layer 57 is connected to an A / C power supply 59, and the ITO layer on the bottom glass plate 55b is grounded. The bottom glass plate 55b is coated with an 800 nm thick amorphous silicon layer 61. The top glass plate 55a and the amorphous silicon layer 61 are each coated with a 160 nm thick layer of high purity alumina or Hafnia 63, which are in turn coated with a single layer of poly(3-(trimethoxysilyl)propyl methacrylate) 65 to render the surface of the high purity alumina or Hafnia layer 63 hydrophobic. In some embodiments, the bottom plate 55b can be made of an opaque material such as silicon.

[0110] The top glass plate 55a and the amorphous silicon layer 61 are spaced 8 μm apart using spacers (not shown), such that the microdroplets undergo a certain degree of compression when introduced into the device cavity. In some embodiments, the distance between 55a and 61 is between 5 - 50 μm, between 5 - 40 μm, between 7 - 30 μm, between 5 - 20 μm, or between 1 - 10 μm. Thus, those skilled in the art will understand that the size of the spacers used will depend on the desired size of the distance between 55a and 61. The image of the reflective pixelated screen illuminated by the LED light source 67 is typically set above the top glass plate 55a, and visible light (wavelength 660 or 830 nm) at a level of 0.01 W / cm2 is emitted from each diode 69 and causes an impact on the amorphous silicon layer 61 through propagation in the direction of the multiple downward arrows of the top glass plate 55a and the intermediate layer.

[0111] At each impact point, a photoexcitation region 71 that generates charge is created in the amorphous silicon layer 61, which causes a changed liquid - solid contact angle on the high - purity alumina or Hafnia layer 63 at the corresponding electrowetting position 73. These changed properties provide the capillary force required to advance the microdroplet 53 from one electrowetting position 73 to another. The LED light source 67 is controlled by a microprocessor 75, which determines which diodes 69 in the array are illuminated at any given time through a pre - programmed algorithm.

[0112] Advantageously, this configuration provides a flexible platform for simultaneously manipulating a large number of microdroplets with high accuracy. In particular, since there is no need to manipulate the microdroplets through a predefined path (which is a limitation of traditional microfluidic platforms that use static electrode arrays or flow paths to affect the direction of microdroplet travel), the platform can be advantageous. Eliminating these limitations enables complex and precise operations that are necessary for on - chip sorting of microdroplets and for the formation and merging of large arrays for implementing the method according to the present invention.

[0113] Further specific details of the microfluidic chip suitable for implementing the method of the present invention can be found in our published patent WO 2018 / 234445, which is incorporated herein by reference.

[0114] The device of the present invention can also implement environmental control features to create target regions on the chip surface that are particularly suitable for cell proliferation, such as: controlled temperature, regions of different flow, controlling the carrier fluid to continuously supply nutrients to the cultured cells, and controlling the local gas concentration in the carrier fluid surrounding the cultured cells. In addition, the carrier fluid can contain surfactants or fluorosurfactants. Surfactants can be particularly advantageous since surfactants can be used to stabilize emulsion droplets. The carrier fluid can be an oil. The oil carrier fluid can include fluorocarbon oils, such as HFE7500.

[0115] For example, a cell culture can be located in a low flow region and surrounded by a faster flow region that contains nutrients and chemicals and supplies the nutrients and chemicals to the culture to promote growth. These mechanisms may constitute Figure 1 part of the aerobic and anaerobic conditions described in step 11 of

[0116] Figure 7 An exemplary workflow performed on the surface of an oEWOD microfluidic chip device in accordance with aspects of the present invention is illustrated.

[0117] The illustrated view is of the surface of an oEWOD microfluidic chip configured to simultaneously manipulate multiple microdrops containing individual emulsified cell samples and reagents between different locations on the surface via real or virtual electrowetting electrodes.

[0118] At the start of the workflow, fluid inlet 77 receives an emulsion 79 of a mixture of empty and cell-containing first microdrops in a fluorocarbon oil carrier fluid.

[0119] These first microdrops are then transferred, e.g., via the oEWOD structure of the chip, to a sorting region 81 where they are sorted into empty microdrops 83 and cell-containing microdrops 85. Thereafter, each cell-containing microdrop 85 is transferred to a merging region 87.

[0120] One or more cells are held for a predetermined period of time on a target region under conditions that promote cell growth and division to form cell colonies within the first microdrops. As previously described, these first microdrops then undergo further manipulation to form a first array.

[0121] At the end of this stage, a second inlet 89 receives second microdrops. The second microdrops can be an emulsion of fluorocarbon oil and one or more drugs, each microdrop containing one or more drugs at known concentrations in an aqueous solution.

[0122] The second microdrops are then merged with the cell-containing first microdrops 85 in the merging region 87 to form merged microdrops 91 which are then held for a predetermined time. For example, once merged, the merged microdrops can be incubated at a temperature of 37 °C for 5 to 30 minutes. During incubation, the droplets are monitored by an optical detection system.

[0123] Thus, the disclosed device advantageously allows manipulation of microdrops over a wide range of sizes and is digitally controlled, providing dynamically reprogrammable operating steps. Compared to traditional methods, the microfluidic substrate of the device has no patterned electrodes, removing several complex low-yield manufacturing steps and simplifying electrical interconnections. This also eliminates device failures caused by dielectric breakdown between adjacent electrodes.

[0124] Compared with traditional methods, the resulting device structure allows for a more refined and integrated workflow, such as independently controlling the carrier phase and droplets, and allows for a greater density of droplets to be controlled within the area of the microfluidic chip surface.

[0125] Experimental details

[0126] A number of experimental results obtained using the devices and methods of the present invention will now be described.

[0127] 1. Bacterial growth in droplets:

[0128] For this experiment, the bacterium Escherichia coli DH5-α (with a multi-copy vector encoding the green fluorescent protein GFPmut3 and the ampicillin resistance gene (bla) conferring resistance to 100 μg / ml ampicillin) was cultured in a flask of Luria-Bertani (LB) liquid medium supplemented with 100 μg / ml ampicillin at 37 °C.

[0129] To estimate the bacterial concentration, the optical density (OD) of the culture was measured at 600 nm (OD600 = 1.6 to 1.9), and a concentration of 20x10 6 bacteria per 10 μl was reached. This corresponds to approximately 1 cell per 10 μm droplet.

[0130] Droplets were generated using vortex emulsification to produce 50 μl of a 40 μl oil carrier phase / 10 μl bacterial suspension.

[0131] The 0.6 μl emulsion consisting of the immiscible fluorocarbon oil Fluorinert TM FC-40 as the carrier phase (containing 5% RAN fluorosurfactant to facilitate droplet formation) plus the bacterial suspension was added to an oEWOD device with 1 μm spacers and incubated.

[0132] Figure 8 A shows an array containing a series of droplet sizes with a wide range of starting concentrations (number of bacteria per droplet) at 0 minutes.

[0133] Figure 8 B shows the same array after incubation at 40 °C for 50 minutes, with visible growth (bacterial replication).

[0134] Figure 8 C shows the same array after incubation at 40 °C for 100 minutes, with further growth of the bacteria clearly visible when compared to Figure 8 A.

[0135] Figure 8 D shows the same array after incubation at 40 °C for 150 minutes, with further growth of the bacteria clearly visible.

[0136] 2. Tracking Growth in Droplets:

[0137] Monitor bacterial growth in droplets by optical inspection and use three different data analysis methods to analyze the optical data:

[0138] i. Average Intensity - This method involves only picking droplets with one or more bacterial cells and measuring the average fluorescence intensity of the entire droplet.

[0139] ii. Droplet Brightness Fraction - This method involves only picking droplets with one or more bacterial cells and selecting an intensity threshold that is above the background but below that of most bacteria. Then measure the area with intensity greater than the threshold as a fraction of the total droplet area.

[0140] iii. Spot Count per Droplet - This method involves only picking droplets with one or more bacterial cells and selecting the above threshold. A spot is defined as a connected region with intensity above the threshold.

[0141] Figure 9 Bacterial growth tracked using each of the three methods described above in the experiment described previously is shown. The two colors represent different ranges of droplet sizes; each point plotted is the average of one frame. The spot size is the number of droplets included, i.e., a small spot indicates a small number of droplets of this size in this frame. The upward trend becomes statistically significant (p < 0.05) after about 60 minutes (at this data collection rate; only moderately dependent on the analysis method), eventually reaching p < 2e - 5.

[0142] 3. Panel Merging and MIC Determination:

[0143] Figure 10 An example of panel merging performed on the device according to the present invention is shown. Droplets are paired together such that the merged droplets all have approximately uniform sizes. Every third droplet is marked as a control and remains unpaired. In this embodiment, the smaller droplets contain the antibiotic gentamicin at a concentration of 500 ng / mL in tryptic soy broth (TSB), while each larger droplet contains 1 - 3 bacteria in TSB.

[0144] Figure 11 How the concentration of the antibiotic gentamicin is reduced to the level at which the Escherichia coli sample strain shows resistance is shown. A robust growth behavior pattern is observed within three hours of reducing the concentration, allowing the determination of the MIC. In this specific case, the MIC is determined to be approximately 300 pg / mL.

[0145] As used herein, the term "array" when referring to an array of droplets is intended to specifically refer to droplets containing the substance to be assayed and does not include any additional droplets that are used as controls but are not actually tested.

[0146] As used herein, the term drug refers to a pharmaceutical preparation containing at least one pharmaceutically active compound, or a candidate pharmaceutical preparation or a candidate drug for a preparation.

[0147] As used herein, the term sample / biological sample refers to any human, animal, environmental (natural, artificial or modified) or even food sample containing at least one cell type. The sample / biological sample can be selected from: feces, peripheral blood, serum, plasma, ascites, urine, cerebrospinal fluid (CSF), sputum, saliva, bone marrow, synovial fluid, aqueous humor, amniotic fluid, earwax, breast milk, bronchoalveolar lavage fluid, semen, prostatic fluid, Cowper's fluid or pre-ejaculatory fluid, female ejaculation, sweat, fecal matter, hair, tears, cyst fluid, pleural and peritoneal fluid, pericardial fluid, lymph fluid, chyme, chyle, bile, interstitial fluid, menstruation, pus, sebum, vomit, vaginal secretion, mammary secretion, mucosal secretion, stool water, pancreatic juice, lavage fluid from the sinus cavity, broncho-pulmonary aspirate, blastocyst cavity fluid and cord blood.

[0148] Environmental samples can be selected from: water samples, air samples, soil samples or surface swabs of surfaces.

[0149] As used herein, the term bacteriostatic agent / bacteriostatic reagent refers to a drug that inhibits the reproduction of bacteria but does not necessarily kill them. Similarly, the term fungistatic agent / fungistatic reagent refers to a drug that inhibits the reproduction of fungi but does not necessarily kill them.

[0150] As used herein, the term bactericidal agent / bactericidal reagent refers to a drug that kills bacteria. Similarly, the term fungicidal agent / fungicidal reagent refers to a drug that kills fungi.

[0151] The term aerobic conditions as used herein refers to oxidative conditions in which aerobic organisms can survive and grow.

[0152] As used herein, the term anaerobic conditions refers to conditions with reduced oxygen concentration under which anaerobic organisms can survive and grow.

[0153] As used herein, the term environmental conditions includes pH, temperature, pressure, permeability, salt concentration, type of culture medium and concentration of any additional nutrient supplements that can be added.

[0154] The term classification algorithm as used herein refers to an algorithm that can automatically perform image analysis. The operation of such algorithms may be as simple as intensity-based threshold determination, or they may be machine learning operations such as logistic regression, random forest, decision tree, or support vector machine (SVM), or they may be as complex as convolutional neural network (CNN) recognition algorithms. Such algorithms are known in the art.

[0155] In view of the present disclosure, various other aspects and embodiments of the present invention will be apparent to those skilled in the art.

[0156] As used herein, "and / or" shall be regarded as a specific disclosure of each of two specific features or components with or without the other. For example, "A and / or B" shall be regarded as a specific disclosure of (i) A, (ii) B, and (iii) A and B, as if each were listed separately herein.

[0157] Unless the context otherwise requires, the description and definition of the above features are not limited to any particular aspect or embodiment of the present invention and apply equally to all aspects and embodiments described.

[0158] Those skilled in the art will further understand that although the present invention has been described by way of example with reference to several embodiments, the present invention is not limited to the disclosed embodiments and alternative embodiments can be constructed without departing from the scope of the present invention as defined in the appended claims.

Claims

1. A microfluidic chip device configured to perform a method for determining the interaction between a drug and a cell type, the method comprising: Providing an array of first microdroplets, comprising the steps of: Emulsifying a biological sample with an immiscible carrier fluid to form aqueous first microdroplets, wherein at least some of the first droplets contain cells of one or more of said cell types; Loading the first microdroplets onto a microfluidic chip configured to manipulate the microdroplets using real or virtual electro-wetting electrodes; Classifying each microdroplet in the first array of microdroplets as a cell-containing microdroplet and an empty microdroplet, wherein the empty microdroplets are discarded; Determining that the cell-containing microdroplets contain at least one live cell for proliferation; Generating an array of first microdroplets from the at least one live cell; Providing an array of second microdroplets, each microdroplet containing one or more drugs at a predetermined concentration; Merging the array of cell-containing first microdroplets and the array of second microdroplets to form an array of merged microdroplets; and Monitoring the properties of one or more cells in the merged microdroplets using an optical detection system to detect the interaction between the cell type and the drug; The device includes: A sorting component configured to separate cell-containing microdroplets from empty microdroplets; A microdroplet manipulation component configured to manipulate microdroplets using real or virtual electro-wetting electrodes; An optical detection system configured to monitor the microdroplets contained in the microfluidic chip through one or more detection windows; The microdroplet operation component includes one or more OEWOD structures, and the one or more OEWOD structures include: A first composite wall, which includes: A first substrate; A first transparent conductor layer located on the substrate, the thickness of the first transparent conductor layer ranging from 70 to 250 nm; and A first dielectric layer located on the conductor layer, the thickness of the first dielectric layer ranging from 30 to 280 nm; A second composite wall, which includes: A second substrate; A second conductor layer located on the substrate, the thickness of the second conductor layer ranging from 70 to 250 nm; and A photosensitive layer located on the second conductor layer and activated by electromagnetic radiation in the wavelength range of 400 - 850 nm, the thickness of the photosensitive layer ranging from 300 to 1500 nm; An A / C power source for providing a voltage across the first and second composite walls connecting the first and second conductor layers; At least one electromagnetic radiation source having energy higher than the bandgap of the photosensitive layer, adapted to impinge on the photosensitive layer to induce corresponding virtual electro-wetting positions on the surface of the first dielectric layer; and A device for manipulating the impinging point of the electromagnetic radiation on the photosensitive layer so as to change the arrangement of the virtual electro-wetting positions, thereby generating at least one electro-wetting path such that the microdroplets move along the path.

2. The device according to claim 1, further comprising a cell culture component configured to hold the first microdroplets and provide conditions favorable for cell proliferation.

3. The device according to claim 1 or 2, further comprising a sample preparation assembly configured to generate a droplet emulsion from the biological sample in an immiscible carrier fluid.

4. The device according to claim 1 or 2, wherein The second composite wall further comprises a second dielectric layer on the photosensitive layer, the thickness of the second dielectric layer ranging from 30 to 280 nm, wherein the exposed surfaces of the first dielectric layer and the second dielectric layer or the photosensitive layer are configured to be spaced 1-180 μm apart to define a microfluidic space suitable for containing droplets.

Images