Improvements in or relating to methods or devices for detecting interactions between biological entities and molecules

Abstract

A method for detecting an interaction between a biological entity and a molecule, the method comprising: providing an array of a first microdroplet in a microfluidic chip, each microdroplet containing at least one bead, each bead having a photocleavable molecule attached thereto; providing an array of a second microdroplet in the microfluidic chip, each microdroplet containing a biological entity; retaining the entire first and second arrays of microdroplets; illuminating at least a subset of the first microdroplets containing at least one bead with an illumination source configured to photocleave the molecule; thereafter merging at least a subset of the first microdroplets with at least a subset of the second microdroplets to form an array of merged microdroplets; and detecting a change in an optical signal from the merged microdroplets with an optical system to indicate an interaction between the biological entity and the molecule. An apparatus for detecting an interaction between a biological entity and a molecule is also provided.

Description

[Technical field]

[0001] The present invention relates to a method for detecting an interaction between a biological entity and a molecule. More specifically, the present invention relates to a method for detecting a change in an optical signal from a microdroplet using an optical system to indicate an interaction between the biological entity and a molecule. The present invention also provides an apparatus for detecting an interaction between the biological entity and a molecule. [Background technology]

[0002] background Conventional high throughput screening (HTS) is a method for scientific experimentation particularly used in drug discovery and relevant to the fields of biology and chemistry. HTS typically uses automated equipment to rapidly test thousands to millions of samples for biological activity at the model organism, cell, pathway, or molecular level. In its most common form, HTS is used to screen 10 or 20 of known structures. 3 ~10 8 Screen small molecule compounds in parallel. Other substances such as chemical mixtures, natural product, oligonucleotides, peptides, and antibodies may also be screened.

[0003] A subset of HTS methods is the DNA (deoxyribonucleic acid) encoded library (DEL) screening method. DELs are collections of small molecules covalently linked to DNA, carrying unique information about the identity and structure of each library member. DNA-encoded chemical libraries (DELs) have been widely adopted by major pharmaceutical companies and are used in a large number of drug discovery programs. The application of DEL technology is advantageous in the early stages of drug discovery due to the reduction in cost, time, and storage space for target compound identification.

[0004] Further miniaturization of high performance screening techniques aims to further reduce space, reagents, consumables, and target materials, moving away from plate-based sample analysis. Screens based on DNA-encoded chemical libraries are moving towards bead-bound libraries that can be integrated into microfluidic devices. Bead-bound libraries contain a large number of microcarrier beads, which act as binding substrates for drug compounds. Along with a single drug compound, each bead carries one or more copies of a synthetic DNA tag, the DNA sequence of which encodes the identity of the drug compound associated with that bead. In some cases, a set of multiple different DNA tags can be present on each bead, with the presence of each tag indicating the completion of a particular step in the synthesis process.

[0005] In order to assay these compounds on cells or other biological entities, they need to be released from the beads. For this purpose, the compounds can be held on the beads using a photocleavable linker molecule. When this linker molecule is irradiated with light of the correct wavelength and fluence for the appropriate duration, the linker breaks, releasing the compound from the bead into the surrounding solution.

[0006] The DEL screening method can be implemented in microfluidic devices to allow for functional screens of DNA-encoded compound beads. Some devices are capable of dispensing library beads into picoliter-scale assay reagent droplets, photochemical cleavage of compounds from beads, laser-induced fluorescence-based assay detection, and fluorescence-activated droplet sorting to isolate hits.

[0007] Advantageously, when compound beads are contained within a small reaction volume such as a microdroplet, localized concentration of compound within the microdroplet can be much higher than with a single bead contained within a larger volume such as that provided by a microwell plate. Microbeads are limited in the binding capacity they can have for compounds, and therefore by encapsulating microbeads within a smaller volume, this capacity limitation does not necessarily translate into limited compound concentration in subsequent analytical testing. This is particularly relevant for drug screens, since the volume of the container constrains whether a drug dose exceeds the EC50 of the drug.

[0008] Using conventional microfluidic droplet formation devices, cells, beads, and other reagents can be packaged together and encapsulated in droplets as they are formed. Once a droplet is formed in a microfluidic channel, it is technically challenging to add any additional material to the droplet. For this reason, most microfluidic devices are constrained to encapsulate the entire target material in the droplet at the time of initial droplet formation. Thus, the statistics of the encapsulation distribution determine the contents of the droplet, and only a small fraction of the droplet can contain the entire target material.

[0009] A challenge with using microdroplets to assay bead-bound DNA-encoded libraries is that in conventional microdroplet fluidics, ultraviolet (UV) illumination of the droplets is used to cleave small molecules from the bead library as it passes through the microfluidic channel. This UV illumination of the microdroplets necessarily illuminates the beads as well as any biological material within the microdroplets. This can cause DNA damage and mutations in the biological material, which can be detrimental if downstream genetic analysis is required.

[0010] Therefore, there is a need for an efficient, high performance screening technique in droplet microfluidic devices, which can perform sample analysis on biological materials without damaging the biological materials within the microfluidic device during the illumination step. Summary of the Invention [Problem to be solved by the invention]

[0011] It is against this background that the present invention was born. [Means for solving the problem]

[0012] According to one aspect of the invention, there is provided a method for detecting an interaction between a biological entity and a molecule, the method comprising the steps of: providing a first array of microdroplets in a microfluidic chip, each microdroplet containing at least one bead, each bead having a photocleavable molecule bound thereto; providing a second array of microdroplets in the microfluidic chip, each microdroplet containing at least one biological entity; holding the first and second arrays of microdroplets; illuminating at least a subset of the first microdroplets containing at least one bead with an illumination source configured to photocleave the molecule; thereafter merging at least a subset of the first array of microdroplets with at least a subset of the second array of microdroplets to form an array of merged microdroplets; and detecting, using an optical system, a change in an optical signal from the merged microdroplets to indicate an interaction between the biological entity and the molecule.

[0013] The method of the present invention allows for selective and localized illumination of at least a subset of the first microdroplets with an illumination source configured for photocleavage of molecules from beads, while avoiding illumination of the array of second microdroplets with illumination configured for photocleavage.

[0014] The merging steps or operations described herein are particularly applicable to arrays of microdroplets when utilizing electrowetting-on-dielectric (EWOD) and / or optical electrowetting-on-dielectric (oEWOD) techniques. The merging step may include microdroplets of mismatched size. For example, it may be necessary to encapsulate biological entities such as cells within larger droplets to ease the geometric constraints of the droplet generation process and / or to provide a larger source of nutrients to the biological entities. Merging with small microdroplets containing beads can be performed to keep the size of the merged droplets as small as possible and allow for a higher dose of the released compound. The merging step may include a tripartite merging, whereby merging microdroplets in one or more of the steps may be mismatched in size. In an additional example, a first microdroplet contains a drug loaded onto beads, a second microdroplet contains a reporter entity, which can be either a cell or a bead, and a third microdroplet contains a cell of interest. These three microdroplets can be merged in whatever order best suits the workflow. The first merger produces a larger microdroplet, which is then merged with a smaller third microdroplet. In another example, a microdroplet loaded with a stimulant is first merged with a microdroplet containing cells. The merged microdroplet is then merged with a microdroplet containing beads.

[0015] Microdroplet arrays in flow cells or chambers, or microdroplets contained within conventional channel-based microfluidics, can be used to image and classify microdroplets, but it is often difficult and inefficient to perform a step of merging two or more different arrays of microdroplets within a flow cell, chamber, or conventional channel-based microfluidics.

[0016] Additionally or alternatively, flow-through microfluidic devices can be used to cleave molecules from beads in microdroplets. However, flow-through devices provide only short periods of time for illuminating the microdroplets to photocleave the molecules from the beads. Thus, when using flow-through microfluidic devices, a high-power illumination source is often required to cleave the molecules in the microdroplets. This often also has a detrimental effect on other surrounding microdroplets containing cells that are exposed to the high-power illumination. For example, the high power of the illumination source can damage or kill any cells contained within the microdroplets. In particular, this damage can include photoinduced mutagenesis. In contrast, the steps of the present invention allow the illumination source to be operated for longer periods of time and at lower illumination power to cleave molecules from beads in the array of the first microdroplets without damaging the cells in the array of the second microdroplets.

[0017] According to the invention, each microdroplet in the array of second microdroplets contains at least one biological entity, and thus, by selectively illuminating only the first microdroplets, damage to the biological entities in the second set of microdroplets by an illumination source configured for photodissection can be avoided.

[0018] The method of the present invention allows for the preparation of an array of first and second microdroplets in a microfluidic chip before the first microdroplet is illuminated with an illumination source configured for photocleavage. The step of preparing the microdroplets in a microfluidic chip can be a time-consuming process. Thus, by preparing the microdroplets in the microfluidic chip before photocleavage, the imaging step can follow immediately after the photocleavage step, greatly minimizing the time between photocleavage and merging of the first and second microdroplets. This is beneficial because it minimizes the time that the cleaved molecules spend in the first microdroplet after cleavage from the bead, minimizing the possibility of the cleaved molecules leaking out of the microdroplet, and thus maximizing the time that the biological entity is in proximity to the photocleaved molecules.

[0019] In some embodiments, a subset of the first microdroplets can be illuminated with an illumination source configured for photo-dissection, hi some embodiments, the entire array of the first microdroplets can be illuminated with an illumination source configured for photo-dissection.

[0020] In some embodiments, merging of the first array of microdroplets with the second array of microdroplets can be performed simultaneously or nearly simultaneously for all pairs of microdroplets across the entire microfluidic chip, hi some embodiments, this merging can be performed on a field-by-field basis.

[0021] In some embodiments, the merging step may also include mixing or agitating at least a subset of the first array of microdroplets with at least a subset of the second array of microdroplets to form the array of merged microdroplets.

[0022] In addition, the methods of the invention disclosed herein can include multiple merging steps. For example, the first or second array of microdroplets can be merged with multiple different arrays of microdroplets before merging with the second or first array of microdroplets, and these arrays can contain specific nutrients or minerals. In another example, the first and second microdroplets can be merged together to form an array of merged microdroplets. At this stage, either the merged or pre-merged microdroplets can be additionally merged with one or more additional arrays of microdroplets, and the additional arrays of microdroplets can contain other substances such as nutrients, additives, or minerals. Such additives can include density modifiers, humectants, desiccants, surfactants, or crowding agents. The additional microdroplets can be further used to add reporter entities such as reporter beads, detection reagents, antibodies, stimulatory agents, cofactors, cytokines, substrates, reporter cells, costimulatory cells, bacteria, or viruses. The additional droplets may be merged at any step during the analytical assay, including between the merging of the first and second microdroplets, before the merging of the first and second microdroplets, and after the merging of the first and second microdroplets. Additionally, fourth, fifth and further sets of additional microdroplets may be merged at any stage during the analytical assay.

[0023] In some embodiments, retaining the entire first and second arrays of microdroplets can be accomplished using an optically mediated force, hi some embodiments, merging at least a subset of the first array of microdroplets with at least a subset of the second array of microdroplets can be accomplished using an optically mediated force.

[0024] In some embodiments, an optically mediated force can be generated upon illumination of at least a portion of the microfluidic chip with generation of an electric field across the microfluidic chip. In some embodiments, by utilizing different illumination wavelengths, a single light source can be used for both photodissection and optically mediated microdroplet control. In some embodiments, two light sources can be used, one optimized to provide an optically mediated force and the other configured for photodissection. In some embodiments, undesired optically mediated forces from a light-dissection illumination source can be avoided by modulating the illumination and generation of the electric field, so that optically mediated forces and light-dissection illumination are not generated simultaneously. In some embodiments, this modulation can be achieved by a controller. By modulating the optically mediated force and the light-dissection illumination, the arrays of the first and second microdroplets can be controlled while preventing interference from an illumination source configured for photodissection.

[0025] In some embodiments, each bead has a surface on which photocleavable molecules are disposed. In some embodiments, the method includes illuminating at least a subset of the first microdroplets containing at least one bead with an illumination source configured to photocleave the molecules from the surface of the bead.

[0026] In some embodiments, the bead material can be polystyrene, TentaGel®, hydrogel, resin, or other. In some embodiments, the beads can be magnetic. In some embodiments, the beads can be non-magnetic. The bead material can be selected depending on the desired density of cleavable molecules on the surface of the beads. In some embodiments, when forming beads from more porous materials such as TentaGel®, the cleavable molecules can be suspended within the beads as well as on the surface of the beads.

[0027] In some embodiments, the cleavable molecule may be a drug molecule, a peptide, a protein, an antibody, or any suitable small molecule.

[0028] In some embodiments, the method may include detecting a change in an optical signal from the merged microdroplet. In the context of the invention disclosed herein, it should be understood that the optical signal may emanate from any portion of the microdroplet. For example, in some embodiments, the optical signal may emanate from the entirety of the merged microdroplet. In some embodiments, the superordinate optical signal may be emitted by the contents of the microdroplet. In some embodiments, the method may include detecting a change in an optical signal from the biological entity using an optical system to indicate an interaction between the biological entity and the molecule.

[0029] In some embodiments, the method may include detecting a change in the optical signal from a reporter, which may be attached to an additional macrobead. In some embodiments, the reporter may be attached to the same bead that carries the photocleavable molecule. In some embodiments, the reporter may include multiple microbeads. In some embodiments, the reporter may include additional biological entities of other types. The optical system may detect a change in the optical signal, which is indicative of an interaction between the biological entity and the molecule. The reporter may be, but is not limited to, a protein, a peptide, a nucleic acid such as DNA, or a fluorescently tagged molecule. The reporter may also be a secreted molecule from a cell or a tagged dye that can change color upon a change in pH when the biological entity interacts with the molecule. The reporter may be combined with the microdroplet during a subsequent merging operation.

[0030] In some preferred embodiments, the biological entity can be a cell, or part of a cell, or a virus, or an enzyme. Additionally or alternatively, the biological entity can be, but is not limited to, an antibody or antibody fragment thereof, an antigen, a receptor, a ligand, a substrate, a nucleic acid such as DNA, RNA (ribonucleic acid), a cell or part of an artificial cell, an extracellular vesicle, a liposome, a polymer, a tissue sample, a bacteriophage, a cytokine, and / or a protein.

[0031] In some embodiments, the biological entity can be a single cell. In some embodiments, the biological entity can be a multicellular organism such as a fungus or a single-cell organism such as a bacterium. In some embodiments, each of the second microdroplets provided to the microfluidic chip can include the same biological entity. In some embodiments, the second microdroplets can include a set of biological entities that are genetically modified to differ from one another. In some embodiments, each of the second microdroplets provided to the microfluidic chip can include a different biological entity. The array of second microdroplets can include a number of different cell lines to investigate interactions of molecules with different cell lines in a single microdroplet provided to the microfluidic chip.

[0032] In some embodiments, the method may further comprise sorting the first and / or second microdroplets on the microfluidic chip, but before providing an array of the first and / or second microdroplets in the microfluidic chip. For example, sorting may be used to ensure that a desired number of biological entities or beads are contained in the microdroplets that then form the array, and to prevent empty microdroplets from being fed to the array, which avoids wasting space on the chip. In some embodiments, the microdroplets may be sorted before being fed into the microfluidic chip to ensure that each microdroplet contains the desired contents. In some embodiments, the biological entities and / or reporter entities may be sorted before being encapsulated in the droplets. For example, FACS (fluorescence-activated cell sorting) may be used to select biological entities with specific surface markers before being encapsulated in the microdroplets.

[0033] In some embodiments, the rows of the array of second microdroplets may be interleaved with the rows of the array of first microdroplets to help minimize the distance that must be traveled before merging.

[0034] In some embodiments, each of the second microdroplets can contain a single biological entity. In some embodiments, each of the second microdroplets can contain multiple biological entities. It may be desirable to have multiple biological entities, such as multiple cells, within a single microdroplet to overcome issues of heterogeneity between single cells at the level of drug response or expression.

[0035] In some embodiments, each microdroplet in the array of first microdroplets can contain a bead, with the same cleavable molecule or molecules bound to the bead. It may be advantageous to generate multiple microdroplets containing the same beads and cleavable molecules to perform multiple iterations of the same analytical assay. In some embodiments, each microdroplet in the array of first microdroplets can contain a bead, with different cleavable molecules or molecules bound to the bead or disposed on the surface of the bead. It may be advantageous to have an array of microdroplets, each containing a different cleavable molecule, to enable detection of interactions between biological entities and different molecules within a single microdroplet delivery to a microfluidic chip.

[0036] In some embodiments, each of the first microdroplets can contain a single bead. In some embodiments, each of the first microdroplets can contain multiple beads. The beads contained within a single microdroplet can have the same type of cleavable molecule disposed on the surface of the bead. Alternatively, multiple beads contained within a single microdroplet can have different cleavable molecules disposed on the surface of the bead. In some embodiments, multiple beads within the same droplet can be used to investigate competitive reactions or interference effects between different molecules and biological entities.

[0037] In some embodiments, a positive control and / or a negative control can be performed on the microfluidic chip. A positive control can include providing a microdroplet containing a known molecule at a known concentration. A positive control can include providing a microdroplet containing beads to which photocleavable fluorescent or colorimetric dye molecules are attached to assess the success of the photocleavage event. A negative control can include a microdroplet containing beads, where no cleavable molecules are disposed on the surface of the beads. A negative control can include beads with inactive cleavable molecules, which are known to have no effect on the biological entity being assayed.

[0038] In some embodiments, the method may further include a step of classifying the merged microdroplets using the detected optical signal. In some embodiments, the detected optical signal may indicate whether the desired interaction has occurred. In some embodiments, sorting the microdroplets before merging may ensure that each merged microdroplet on the microfluidic chip contains both beads and biological cells. Without sorting the microdroplets before merging, the merged microdroplets may be sorted in a similar manner for content, but Poisson statistics increase the time required to load the desired content into an array of droplets with all elements. Without the sorting step, the proportion of droplets containing a single bead and a single biological cell is limited by the Poisson distributed payout of entities among the droplets. By sorting the droplets to load only those containing a single entity, the throughput of assaying for droplets containing the correct substance may be increased. Similarly, it is advantageous to be able to classify droplets containing two, three or more entities. Such a sorting operation can be controlled by an optical inspection step, which identifies the contents of the droplets by imaging followed by computer analysis to count the number of beads or cells contained in the droplets. Such a sorting process is useful for sorting both beads and cells, as well as other items, within the droplets. Alternatively, the sorting process can be used to sort the size of the droplets. In some embodiments, the optical inspection step can include, but is not limited to, bright field imaging, dark field imaging, or fluorescent imaging.

[0039] In some embodiments, each bead can include a molecular tag on its surface. In some embodiments, the beads contained in the first set of microdroplets can be provided with a library of cleavable molecules attached to their surface. In some embodiments, the beads contained in the first set of microdroplets can further include a molecular tag, which can help identify the molecule involved in the interaction detected during the detection step. In some embodiments, the tag can be a nucleic acid tag, or a protein tag, or a small molecule tag, or a synthetic tag. In some embodiments, the molecular tag can be a DNA tag or an RNA tag. In some embodiments, the molecular tag can be a small interfering RNA (siRNA) tag or a messenger RNA (mRNA) tag. In some embodiments, the molecular tag can be a drug molecule.

[0040] In some embodiments, the array of the first microdroplets and the array of the second microdroplets can be held in an interdigitated array. The interdigitated array allows the first microdroplets and the second microdroplets to be in close proximity to each other. The proximity of the first microdroplets and the second microdroplets minimizes the time between cleavage of the molecules from the beads and merging with the microdroplets containing the biological entities. This is advantageous because the cleaved molecules may leak from the first microdroplets into the surrounding phase after they are released from the surface of the beads. In addition, minimizing the time between photocleavage of the molecules from the beads and the merging step increases the efficiency of the overall method. Furthermore, the proximity of the first microdroplets and the second microdroplets can minimize the movement between the microdroplets containing the cells and the microdroplets containing the beads, and this reduced movement can therefore reduce the risk of the cleaved molecules leaking out of the first microdroplets.

[0041] Alternatively, the first and second arrays of microdroplets can be maintained as two separate arrays.

[0042] In some embodiments, an array of additional microdroplets can be provided on the microfluidic chip. In some embodiments, each of the additional microdroplets can comprise at least one reporter entity. In some embodiments, the reporter entity can be a reporter cell.

[0043] In some embodiments, the illumination light source may be applied to the first microdroplets for a period of 1 to 300 seconds. In some embodiments, the illumination light source may be applied to the first microdroplets for more than 1 second, more than 10 seconds, more than 20 seconds, more than 30 seconds, more than 40 seconds, more than 50 seconds, more than 60 seconds, more than 70 seconds, more than 80 seconds, more than 90 seconds, more than 100 seconds, more than 110 seconds, more than 120 seconds, more than 130 seconds, more than 140 seconds, more than 150 seconds, more than 160 seconds, more than 170 seconds, more than 180 seconds, more than 190 seconds, more than 200 seconds, more than 220 seconds, more than 230 seconds, more than 240 seconds, more than 250 seconds, more than 260 seconds, more than 270 seconds, more than 280 seconds, or more than 290 seconds. In some embodiments, the illumination source may be applied to the first microdroplet for less than 300 seconds, less than 290 seconds, less than 280 seconds, less than 270 seconds, less than 260 seconds, less than 250 seconds, less than 240 seconds, less than 230 seconds, less than 220 seconds, less than 210 seconds, less than 200 seconds, less than 190 seconds, less than 180 seconds, less than 170 seconds, less than 160 seconds, less than 150 seconds, less than 140 seconds, less than 130 seconds, less than 120 seconds, less than 110 seconds, less than 100 seconds, less than 90 seconds, less than 80 seconds, less than 70 seconds, less than 60 seconds, less than 50 seconds, less than 40 seconds, less than 30 seconds, less than 20 seconds, or less than 10 seconds. In some preferred embodiments, the illumination source may be applied to the subset of first microdroplets for more than 5 minutes, more than 6 minutes, more than 7 minutes, more than 8 minutes, more than 9 minutes, more than 10 minutes, more than 15 minutes, more than 20 minutes, more than 25 minutes, more than 30 minutes, more than 40 minutes, more than 50 minutes, more than 60 minutes, more than 100 minutes, more than 140 minutes, more than 180 minutes, or more than 240 minutes.

[0044] In some embodiments, the preferred illumination duration depends on the intensity of the illumination source. For example, if the microdroplet is illuminated with lower intensity illumination, the illumination source must be directed at the first microdroplet for a longer period of time to provide sufficient energy to photocleave the molecules from the bead surface. In some embodiments, the illumination source is directed at the first microdroplet for a preferred period of time between 10 seconds and 60 seconds.

[0045] In some embodiments, a longer period of illumination can result in an increased number of cleavable molecules being photocleaved from the surface of the bead. By controlling the number of molecules cleaved from the surface of the bead, the concentration of cleaved molecules that contact the biological entity in the merging step can be controlled. This can facilitate the detection of interactions between the biological entity and the molecule at different doses.

[0046] Unless otherwise specified, "dose" or "dose amount" disclosed in and within the context of the present invention should be understood to include exposing a biological entity, such as a cell, to a molecule, such as a drug molecule, at a particular concentration. In bulk assays, dose-response curves can be obtained by treating cells with different concentrations of the same compound. This can facilitate the calculation of the drug concentration that achieves the maximum response, Emax, and the half-maximal response, EC50.

[0047] To achieve a tunable dose, the number of cleavable molecules released from the beads can be varied by varying the duration and / or intensity of the photodissection illumination applied to the beads contained within the first microdroplet.

[0048] A dose-response curve can be obtained by exposing the biological entity in the second microdroplet to increasing concentrations of molecules, such as drug molecules. In some embodiments, this can be achieved by sequential merging steps, in which a second microdroplet is sequentially merged with a plurality of first microdroplets at sequential doses of the cleaved molecule. Additionally, a dose-response curve can be obtained by merging a droplet containing a sample of a drug molecule with a droplet containing a diluent to dilute the drug molecule and provide a panel of different doses.

[0049] In some embodiments, the method may further include varying the time each of the first microdroplets is illuminated, or in other embodiments, the method may further include varying the time a subset of the first microdroplets are illuminated. In some embodiments, by varying the time each microdroplet is illuminated while maintaining the same illumination intensity, an array of first microdroplets containing varying numbers of cleaved molecules can be generated. Thus, each of the first microdroplets can contain different concentrations of the cleaved molecules, and each first microdroplet can be merged with an identical second microdroplet. This allows biological entities to be exposed to different concentrations of cleaved molecules during a single analytical assay without the need for sequential photocleavage steps.

[0050] In some embodiments, the illumination source may be directed at one or more of the first microdroplets with a total power of 0.7 to 400 mW. In some embodiments, the illumination source directed at one or more of the first microdroplets may have a power of more than 0.7 mW, more than 1 mW, more than 10 mW, more than 50 mW, more than 100 mW, more than 150 mW, more than 200 mW, more than 250 mW, more than 300 mW, or more than 350 mW. In some embodiments, the illumination source directed at one or more of the first microdroplets may have a power of less than 400 mW, less than 350 mW, less than 300 mW, less than 250 mW, less than 200 mW, less than 150 mW, less than 100 mW, less than 50 mW, less than 10 mW, or less than 1 mW. In some embodiments, it may be preferred to direct the illumination source at one or more of the first microdroplets with a power of 0.7 to 400 mW. In some embodiments, the illumination source directed to one or more of the first microdroplets can have a power output of greater than 0.7 mW, greater than 1 mW, greater than 5 mW, greater than 10 mW, greater than 15 mW, greater than 20 mW, greater than 25 mW, greater than 30 mW, or greater than 35 mW. In some embodiments, the illumination source directed to one or more of the first microdroplets can have a power output of less than 40 mW, less than 35 mW, less than 30 mW, less than 25 mW, less than 20 mW, less than 15 mW, less than 10 mW, less than 5 mW, or less than 1 mW. In some embodiments, the illumination source directed to at least a subset of the first microdroplets can have a total power output of at least 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 2.0, 3.0, 4.0, 5.0, 10.0, or 100.0 watts.

[0051] In some embodiments, the intensity of the photocleavage illumination can be selected according to the energy required to cleave the bond between the cleavable molecule and the bead. In some embodiments, it may be beneficial to use an illumination source with a higher intensity to minimize the time required to cleave the molecule or molecules from the surface of the bead. This can maximize the efficiency of the method and minimize the total time of the analytical assay. In some embodiments, it may be beneficial to use an illumination source with a lower intensity for a longer period of time to avoid damaging components of the microdroplet medium that may be sensitive to photocleavage illumination.

[0052] In some embodiments, the illumination source may illuminate one or more of the first microdroplets at a wavelength between 360 and 380 nm.

[0053] In some embodiments, the illumination source that illuminates one or more of the first microdroplets can have a wavelength greater than 200 nm, greater than 220 nm, greater than 240 nm, greater than 260 nm, greater than 280 nm, greater than 300 nm, greater than 320 nm, greater than 340 nm, greater than 360 nm, greater than 380 nm, greater than 400 nm, or greater than 420 nm. In some embodiments, the illumination source that illuminates one or more of the first microdroplets can have a wavelength less than 420 nm, less than 400 nm, less than 380 nm, less than 360 nm, less than 340 nm, less than 320 nm, less than 300 nm, less than 280 nm, less than 260 nm, less than 240 nm, less than 220 nm, or less than 200 nm. In some embodiments, the preferred wavelength can be 365 nm.

[0054] In some embodiments, the illumination source that illuminates one or more of the first microdroplets can have a wavelength greater than 360 nm, greater than 362 nm, greater than 364 nm, greater than 366 nm, greater than 368 nm, greater than 370 nm, greater than 372 nm, greater than 374 nm, greater than 376 nm, or greater than 378 nm. In some embodiments, the illumination source that illuminates one or more of the first microdroplets can have a wavelength less than 380 nm, less than 378 nm, less than 376 nm, less than 374 nm, less than 372 nm, less than 370 nm, less than 368 nm, less than 366 nm, less than 364 nm, or less than 362 nm. In some embodiments, the preferred wavelength can be 365 nm.

[0055] The illumination light source can illuminate the microdroplets with other wavelengths, for example, the illumination light source can illuminate the first microdroplets with a wavelength corresponding to blue light. In some embodiments, the illumination light source can illuminate one or more first microdroplets with different wavelengths to cleave the photocleavable molecules from the beads. The illumination light source can illuminate the microdroplets with a UV (ultraviolet) wavelength spectrum to cleave the photocleavable molecules from the beads.

[0056] In some embodiments, the method may further include varying the intensity of illumination on each of the first microdroplets. In some embodiments, the method may further include varying the illumination on at least a subset of the first microdroplets in a grayscale pattern. In some embodiments, the intensity of an illumination source configured for photocleavage may be varied such that each of the first microdroplets releases a varying number of molecules from the surface of the bead. This may facilitate varying the concentration of molecules to be cleaved and exposing biological entities to molecules during a single analytical assay without the need for sequential photocleavage steps.

[0057] In some embodiments, varying the duration or intensity of light dissection illumination on at least a subset of the first microdroplets can be used to generate a concentration gradient of the cleavable molecule throughout at least a subset of the first microdroplets, which can be used to efficiently investigate the effect of varying doses on a biological entity without the need to introduce beads with varying numbers of cleavable molecules into the device, which can be time consuming and impractical.

[0058] In some embodiments, the intensity of the illumination for photo-dissection can be varied to provide a grayscale pattern across at least a subset of the first microdroplets. The grayscale pattern is formed when the light-dissection illumination is applied to at least a subset of the first microdroplets for the same length of time, but with different illumination intensities across the subset of microdroplets. For example, the illumination across the subset of the first microdroplets can vary from full intensity to 50% intensity to 25% intensity. The gradient of illumination across the microdroplets corresponds to a concentration gradient of the number of dissected molecules within the microdroplets after photodissection has occurred.

[0059] In some embodiments, a concentration gradient can be obtained by illuminating the microdroplets in at least a subset of the first microdroplets with the same illumination intensity, but for different lengths of time across the subset of microdroplets.

[0060] In some embodiments, the number of microdroplets that can be simultaneously illuminated depends on the field of view of an optical system configured to provide the light-section illumination, which can include an objective lens.

[0061] To tailor the energy delivered to the microdroplets for photodisruption, the power applied to the microdroplets and / or the duration of illumination of the microdroplets can be varied, allowing photodisruption to be performed efficiently but without the use of high power illumination that would damage the contents of the microdroplets.

[0062] In some embodiments, it may be advantageous to parallelize the method by maximizing the number of microdroplets that are illuminated simultaneously, thereby saving time and maximizing the efficiency of the method.

[0063] In some embodiments, the method may further include varying the intensity or duration of illumination used to hold the microdroplets in the array, where the illumination may be applied in a grayscale pattern to at least a subset of the first and / or second microdroplets. In some embodiments, the microdroplets may be held and manipulated with optically mediated forces, allowing the intensity of the controlled illumination light source applied to each microdroplet, or to a subset of the first and / or second microdroplets, to be varied. In some embodiments, it may be advantageous to hold and / or control the microdroplets with weaker forces in certain regions of the microfluidic chip. For example, the efficiency with which the microdroplets can be formed into an array under weaker optically mediated control when the microdroplets are delivered into the microfluidic chip may be promoted. In subsequent stages of the analytical assay, it may be advantageous to hold and / or control the microdroplets with stronger forces. In some embodiments, the controlled light source may be used with a lower illumination intensity, for example 80% of the illumination intensity, in the region of the chip where the microdroplets are initially delivered. In other areas of the chip that require stronger retention, the illumination intensity of the controlled light source can be 100%.

[0064] In some embodiments, the method may further include splitting at least a subset of the first and / or second microdroplets. In some embodiments, after initial illumination of the first microdroplets with an illumination source configured for photocleavage, the first microdroplets may be split prior to the merging step. In some embodiments, the first microdroplets are split such that the cleaved molecules are contained within one portion of the microdroplet and the beaded cleaved molecules are contained within another portion of the microdroplet.

[0065] In some embodiments, a medium can be added into the microfluidic chip. In some embodiments, after splitting, a medium can be added into the microfluidic chip to increase the volume of the split microdroplets by merging the split microdroplets with the medium. After the splitting step, the microdroplets containing the cleaved molecules can be merged with the microdroplets containing the biological entities. In some embodiments, after the splitting step, the microdroplets containing the beads can be used in a subsequent photo-cleaving step. When splitting the microdroplets into multiple parts using optical electrowetting, information encoding the identity of the original droplets containing the beads can be maintained, and the identity of the original beads can then be linked to the results of an analytical assay performed with the split droplets. The original beads can then be selected for additional analytical assays or recovery based on the results of the analytical assay.

[0066] In some embodiments, the same bead can be exposed to photocleavage illumination for successive periods of time to allow additional cleavable molecules to be released from the bead surface. The number of molecules released from the bead by successive illuminations with the same photocleavage illumination decreases exponentially with each illumination. Thus, to release the same amount of molecules from the bead in a subsequent step, subsequent illumination times and / or illumination intensities may need to be greater. It may be advantageous to photocleave molecules from the same bead in successive steps to increase the efficiency of space usage on the microfluidic chip and minimize the number of beads required to achieve a desired dose of biological entities.

[0067] In some embodiments, detection of a change in the optical signal from the merged microdroplet exceeding a predetermined threshold level can be further configured to measure the concentration or number of molecules released from the bead.

[0068] In some embodiments, the optical signal from the merged microdroplets may be a fluorescent signal that indicates a positive response when it exceeds a predetermined threshold of fluorescent signal, which may be compared to a model system to determine the concentration or number of molecules released from the beads.

[0069] In some preferred embodiments, the optical signal may be a fluorescent signal, or a fluorescence resonance energy transfer (FRET) signal, or a homogeneous time resolved fluorescence (HTRF) signal, or a luminescence signal, or a chemiluminescence signal.

[0070] In some embodiments, the optical signal can be detected by a fluorescent assay on the beads or cells, a luminescent assay on the beads or cells, or a bright or dark field image of the cell morphology or number. A positive hit can be a dark or bright state, or can indicate cell morphology or proliferation. The result of the assay can be a binary result, or a quantifiable event in response to the assay. The result can be a measurement of the cell's response to the released small molecule, and can indicate the effectiveness of the drug. Alternatively, the labeled antibody and / or catch reagent can be released to participate in an ELISA (enzyme-linked immunosorbent assay) style sandwich assay, for example for cytokine detection. In some embodiments, the HTRF signal can be advantageous because it can have a reduced background (noise) compared to other types of signals. In some embodiments, the HTRF signal can have an improved signal-to-noise ratio.

[0071] Intracellular imaging can also be performed, showing signals within parts of a cell or cell contents, examples of which include the nucleus, the Golgi apparatus, and / or mitochondria.

[0072] In some embodiments, the array of first microdroplets may be provided on the microfluidic chip before the array of second droplets.

[0073] In some embodiments, the method can further include sequentially introducing the second array of microdroplets and the first array of microdroplets into the microfluidic chip, hi some embodiments, droplets containing cells can be loaded before droplets containing beads, or vice versa.

[0074] In some embodiments, the method may further comprise the step of simultaneously introducing the first and second arrays of microdroplets into the microfluidic chip.

[0075] The arrays of first and second microdroplets can be loaded simultaneously through different inlets. Simultaneous loading of the first and second microdroplets into the microfluidic chip can minimize loading time and increase device performance.

[0076] In some embodiments, the microdroplets can contain cell culture medium. In some embodiments, the cell culture medium can include EMEM (Eagle's minimal essential medium), DMEM (Dulbecco's modified Eagle medium), RPMI (Roswell Park Memorial Institute) medium, F-12 medium. In some embodiments, the microdroplets can contain modified cell culture medium. For example, the cell culture medium can contain additional antibodies, buffers including pH buffers, and / or conditioned media. In some embodiments, the microdroplets can include density modifiers such as, but not limited to, Optiprep.

[0077] Additionally or alternatively, the microdroplets can contain additives, such as, but not limited to, supplements, density modifiers, agonist molecules, antagonists, moisturizers, drying agents, cofactors, stimulators, cytokines, inhibitors, or crowding agents.

[0078] In some embodiments, the method may further include providing a dispersion medium into the microfluidic chip. The dispersion medium may be an oil. In some embodiments, a surfactant exchange may be performed to reduce the surfactant level by exchanging the oil. In some embodiments, the device may be pre-primed with a low surfactant oil compared to the oil used for emulsifying the loaded microdroplets. A lower concentration of surfactant in the dispersion medium may help reduce leakage of photocleaved molecules into the dispersion medium surrounding the microdroplets. In some embodiments, the oil may be conditioned with cell culture medium to improve cell survival. In some embodiments, dissolved oxygen and / or carbon dioxide may be mixed into the oil to supplement the gas provided to the cell-containing microdroplets to extend cell survival in the device.

[0079] In some embodiments, the first microdroplet array and / or the second microdroplet array can include microdroplets of different sizes, hi some embodiments, the first microdroplet array and / or the second microdroplet array can include droplets with diameters between 5 and 200 μm.

[0080] The diameter of the first and / or second microdroplets can be greater than 5 μm, greater than 10 μm, greater than 20 μm, greater than 30 μm, greater than 40 μm, greater than 50 μm, greater than 60 μm, greater than 70 μm, greater than 80 μm, greater than 90 μm, greater than 100 μm, greater than 110 μm, greater than 120 μm, greater than 130 μm, greater than 140 μm, greater than 150 μm, greater than 160 μm, greater than 170 μm, greater than 180 μm, or greater than 190 μm. In some embodiments, the first and / or second microdroplets may have a diameter of less than 200 μm, less than 190 μm, less than 180 μm, less than 170 μm, less than 160 μm, less than 150 μm, less than 140 μm, less than 130 μm, less than 120 μm, less than 110 μm, less than 100 μm, less than 90 μm, less than 80 μm, less than 70 μm, less than 60 μm, less than 50 μm, less than 40 μm, less than 30 μm, less than 20 μm, or less than 10 μm.

[0081] The size of the microdroplets can be used to vary the concentration of cleaved molecules exposed to the biological entities during the merging step. Smaller molecules can be used to expose the biological entities to a higher concentration of cleaved molecules. The minimum size of the microdroplets depends on the minimum volume of cell culture medium required to maintain the viability of the biological entities within the microdroplets throughout the duration of the analytical assay. The maximum size of the microdroplets can depend on the useful concentration of cleaved molecules.

[0082] In some embodiments, a plurality of different sized microdroplets can be used to form the first and / or second arrays, hi some embodiments, the volume of the microdroplets can be increased after merging, and thus the size can be increased during the analytical assay.

[0083] In some embodiments, by utilizing smaller microdroplets, the microdroplets can be delivered more efficiently into the microfluidic chip. Smaller droplets require less space on the microfluidic chip. When using smaller microdroplets, it may be necessary to increase the number of biological entities used to form the microdroplets. This may be necessary to avoid having a large number of empty microdroplets in the microfluidic chip, which requires time to sort and remove from the chip, reducing the efficiency of the analytical assay.

[0084] In some embodiments, the first array of microdroplets and / or the second array of microdroplets may include microdroplets of approximately the same size.

[0085] In some embodiments, the method may further include dispensing the microdroplets into a container such as a well plate. In some embodiments, dispensing into the well plate allows beads and / or cells corresponding to positive hits to be collected. The well plate may contain a buffer, or an aqueous solution, or a DNA stabilization solution. Such a solution may also contain appropriate primers for sequencing. The droplets in the emulsion may be ruptured in the well plate, leaving the beads in the aqueous solution. Alternatively, the beads may be dispensed into oil and allowed to dry nearly before adding the aqueous solution.

[0086] Beads can be dispensed into individual wells in a well plate to maintain phenotypic information linked to the biological entity, or multiple beads can be pooled together for dispensing, in which case multiple beads are dispensed into a single well in a well plate.

[0087] In some embodiments, one or more droplets may be selected for collection based on the optical measurement.

[0088] In some preferred embodiments, optical measurements are made on droplets split off from the original droplet, and the original droplet is selected for collection accordingly.

[0089] According to an additional aspect of the invention, there is provided an apparatus for detecting interactions between a biological entity and a molecule, the apparatus comprising: a microfluidic chip; an illumination source; a controller; an optical system; and a dispensing system, the microfluidic chip comprising a microfluidic space and an inlet for introducing a first array of microdroplets and a second array of microdroplets into the microfluidic space, each of the first microdroplets containing a bead, each bead having a photocleavable molecule bound to the bead, each of the second microdroplets containing at least one biological entity, and the first array of microdroplets and the second array of microdroplets being coupled to each other. and are merged to form an array of merged microdroplets in the microfluidic space, an illumination light source illuminates at least a subset of the first microdroplets containing at least one bead in the microfluidic space, the illumination light source is further configured to photocleave the molecule, the controller is configured to control the time or intensity or wavelength of the illumination light source applied to the first microdroplets, the optical system is configured to detect a change in the optical signal from the merged microdroplets to indicate an interaction between the biological entity and the molecule, and the dispensing system is configured to expel the droplets from the microfluidic space into a container.

[0090] In some embodiments, the microfluidic device may be an optical electrowetting-on-dielectric (oEWOD) device, or an electrowetting-on-dielectric (EWOD) device, or a dielectrophoresis (DEP) device.

[0091] In some embodiments, the illumination source can be an ultraviolet illumination source. In some embodiments, the illumination source configured to photocleave the molecules from the surface of the bead can be a blue light source or a white light source. In some embodiments, the illumination source configured to photocleave the molecules from the surface of the bead can be any illumination source capable of achieving sufficient energy to photocleave the molecules from the surface of the bead.

[0092] In some embodiments, the apparatus may further comprise a homogeneous time resolved fluorescence (HTRF) reader.

[0093] In some embodiments, the environment surrounding the microdroplets can be controlled. For example, the temperature of the microdroplets can be maintained at 37° C. by passing an electric current through the microfluidic chip. In some embodiments, the microdroplet emulsion can be cooled off-chip. In some embodiments, the biological entities contained within the array of second microdroplets can be cooled before being delivered to the microfluidic chip. In some embodiments, there can be a recovery period for recovering the biological entities before detecting the optical change. In some embodiments, the microfluidic chip can include a Peltier system that facilitates cooling of the microdroplets while they are within the microfluidic chip. In some embodiments, the microdroplets can be cooled to 20° C. to 25° C. Cooling the microdroplets slows the consumption of nutrients by the biological entities contained within the microdroplets. Cooling the microdroplets slows the secretion rate and production of waste products produced by the biological entities contained within the microdroplets. Slowing the consumption of nutrients and / or the production of waste products extends the survival of the biological entities within the microfluidic chip.

[0094] According to another aspect of the invention, there is provided a cartridge comprising: a reservoir containing a liquid sample; an emulsification apparatus in fluid communication with the reservoir; an inlet channel downstream of the emulsification apparatus; a device according to any aspect of the invention; and a pump system, wherein the emulsification apparatus is configured to produce a medium comprising an emulsion of aqueous microdroplets in an immiscible dispersion medium, the inlet channel being configured to receive the medium comprising an emulsion of aqueous microdroplets in the immiscible dispersion medium from the emulsification apparatus, said device comprising an inlet port therefor, said device being in fluid communication with the inlet channel, and wherein the pump system directs a flow of the liquid sample to the emulsification apparatus and / or directs a flow of the medium comprising an emulsion of aqueous microdroplets in the immiscible dispersion medium through said device.

[0095] Suitably, the aqueous fluid in the cartridge may be a biological fluid such as a cell culture medium, and may be cells, beads, particles, drugs, biomolecules, or other biological entities. These entities may be viruses, DNA or RNA molecules, stimulants, cytokines, nutrients, and oil-soluble gases. For this reason, the design of the cartridge flow channels and structures may be optimized to preserve the dispersion and integrity of the biological fluid, particularly by the coordinated selection of uniform thrust diameters and minimal fluid shear forces.

[0096] In some embodiments, the cartridge may further comprise one or more valves at the input port of the device which control the flow of the medium, comprising an emulsion of aqueous microdroplets in an immiscible dispersion medium, through the device.

[0097] In some embodiments, the emulsifier may be a step emulsifier. In some embodiments, a plurality of emulsifiers may be provided, each emulsifier having an inlet passage.

[0098] In some embodiments, the pumping system may include, but is not limited to, a pump, a head reservoir, a pressure accumulator, and / or a pressure source. Additionally, it will be apparent to those skilled in the art that other pumping systems may be known that may be used to induce the flow of the liquid sample to the emulsification device and / or induce the flow of the medium through the device.

[0099] Numerous techniques are currently known in the art for forming aqueous emulsions of microdroplets surrounded by immiscible carrier fluids. These techniques include cross-flow emulsion generators, T-junction generators, and step emulsifiers. Cross-flow emulsion generators, T-junction emulsion generators, and other related devices are typically used to create variable size microdroplets. The size distribution of the microdroplets depends on the flow conditions created at the junction where the oil and aqueous materials intersect. In addition, the size of the microdroplets depends on the fluid properties such as the interfacial tension and viscosity of the flowing fluids. Therefore, the flow rate of the fluids entering these types of emulsion generators needs to be precisely controlled and adjusted to provide droplets of uniform and repeatable size distribution in the oEWOD device.

[0100] It is advantageous for the step emulsifier to produce an emulsion with a droplet size distribution that is minimally dependent on the flow rate at the emulsion junction. The droplet size is determined primarily by the physical dimensions of the nozzle as well as the material properties of the flowing fluid. While both step emulsifiers and other emulsifiers are sensitive to the properties of the flowing fluid, the degree of dependence on interfacial tension and viscosity is greatly reduced in the step emulsifier. Thus, there is no need to precisely control and adjust flow parameters to modify the droplet size distribution exiting the emulsifier. The step emulsifier can be operated with a simple fixed flow rate or fixed pressure system. The step emulsifier is particularly suited for operation with an oEWOD device because it avoids the need for optical access to the inspection and emulsification apparatus in a location that may otherwise overlap with the optical assembly used for operation of the oEWOD device. The step emulsifier avoids the complexity and cost of implementing multiple inspection and droplet size monitoring apparatus to monitor and control multiple operating emulsification apparatus within one cartridge assembly. Thus, multiple independent step emulsifiers can be connected to different inlets on the oEWOD device to provide a fluidically isolated emulsion production input path between the aqueous input and the oEWOD device. The use of fluidically isolated input paths allows the oEWOD device to receive a set of independent emulsion inputs from different aqueous input materials without the possibility of cross-contamination between the different aqueous input materials.

[0101] In some embodiments, a cartridge assembly can include up to 8 emulsification devices. In some embodiments, a cartridge assembly can include at least 1, 2, 3, 4, 5, 6, or 7 emulsification devices. In some embodiments, a cartridge assembly can include 8 to 12 emulsification devices. In some embodiments, a cartridge assembly can include 12 to 20, 20 to 30, 30 to 50, or 50 to 100 emulsification devices.

[0102] The emulsifiers may be user-interchangeable, allowing the user to select the type of emulsifier appropriate for the purpose intended. For example, the user may configure a cartridge with emulsifiers that provide microdroplets of a particular range of sizes. The user may select a set of emulsifiers, each providing microdroplets with a different size range or sub-section of the size range. In some embodiments, the emulsifiers may be configured to generate microdroplets with a volume in the range of 14 pL to 180 pL, or in the range of 180 pL to 500 pL, or in the range of 500 pL to 1.2 nL. The emulsifiers may also be configured to generate microdroplets with a volume less than 14 pL, particularly in the range of 10 fL to 50 fL, or in the range of 50 fL to 14 pL. In some embodiments, the emulsifiers may be configured to generate microdroplets with a volume greater than 1.2 nL, including at least the range of 1.2 nL to 4 nL. If the emulsifier is a step emulsifier, the volume of the microdroplets can be varied by modifying the geometry of the emulsifier nozzle, in particular by varying the nozzle height along the minor axis of a rectangular nozzle.

[0103] Furthermore, the operation of a set of step emulsifier nozzles in a single emulsifier can be parallelized, thereby connecting multiple emulsifier nozzles to a single aqueous input. These connected nozzles can operate independently with varying speeds determined by the complex interactions between the interconnected junctions. The emulsifiers can all generate microdroplets of approximately uniform size determined by the physical size of the nozzles. This allows a large number of generators to operate in parallel at low flow rates, eliminating the deleterious effects of shear that can damage cells and other biological entities. This also allows the emulsifier to continue to generate emulsions despite partial blockage or blocking of some nozzles, which is a result of biological material, including particles, passing through narrow nozzle openings.

[0104] According to one aspect of the invention, there is provided a species screened by the device, apparatus, cartridge or method disclosed herein.

[0105] According to one aspect of the present invention, a species selected by the device, apparatus, cartridge, or method disclosed herein is provided.

[0106] According to one aspect of the present invention, there is provided a species sequestered by a device, apparatus, cartridge, or method disclosed herein.

[0107] According to one aspect of the invention there is provided a species produced by the device, apparatus, cartridge or method disclosed herein.

[0108] These species can be chemical, biochemical or biological in nature.

[0109] For example, the invention can provide agonists / antagonists identified by the screening, selection and / or isolation methods disclosed herein. The invention can provide agonists / antagonists for use in therapy to entities identified by the screening, selection and / or isolation methods disclosed herein. The entities can be chemical, biochemical or biological in nature.

[0110] According to one aspect of the present invention, there is provided a method of using the device, apparatus, cartridge, method or species disclosed herein.

[0111] According to one aspect of the present invention there is provided a method of use of the device, apparatus, cartridge, method or species disclosed herein in therapy.

[0112] The present invention may provide for the use of the devices, apparatus, cartridges, methods, or species disclosed herein in the production of an article of manufacture, which may be chemical, biochemical, or biological in nature.

[0113] The method of use may be peptide synthesis. The method of use may be synthetic biology. The method of use may be cell line design or development. The method of use may be cell therapy. The method of use may be drug discovery. The method of use may be antibody discovery.

[0114] According to one aspect of the invention there is provided a method of use of a device, apparatus, cartridge, method or species disclosed herein in an assay.

[0115] The analysis can be a physical analysis, a chemical analysis, or a biological analysis.

[0116] The use may be intracellular imaging.The use may be high contrast imaging.

[0117] This use may be diagnostic.

[0118] The use may be a biological assay. The biological assay may be a screen. The biological assay may be an ELISA.

[0119] The use may be cellular secretion.

[0120] This use can be QC (quality control) safety profiling.

[0121] The invention will now be more particularly described, by way of example only, with reference to the accompanying drawings in which: [Brief description of the drawings]

[0122] [Figure 1] FIG. 1 shows a schematic diagram of photocleavage of molecules bound to the surface of beads within a microdroplet. [Diagram 2] FIG. 13 is a schematic illustrating the merging of microdroplets containing photocleaved molecules with microdroplets containing biological entities to produce merged microdroplets. [Diagram 3] 3A and 3B provide an array of light sprites and light sectioning illumination patterns that can hold droplets within the array. [Figure 4] FIG. 4A shows various droplets containing cells and single beads sorted into rows, and FIG. 4B shows the droplet merging operation. [Diagram 5] 5A and 5B show an array of droplets sorted by alternating rows of droplets containing cells with rows of droplets containing single beads. [Figure 6] FIG. 1 provides an array of light sprites with varying intensity from column to column. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0123] The present invention discloses herein a method and apparatus for detecting interactions between a biological entity 24 and a molecule 16. An array of first microdroplets 12 and second microdroplets 22 is prepared in a microfluidic chip, and the entire array of first and second microdroplets is held in the microfluidic chip. The microdroplets are sorted prior to being prepared in the microfluidic chip, such that only microdroplets having a desired content are prepared in the microfluidic chip.

[0124] Referring to FIG. 1, a first microfluidic 12 is shown containing beads 14, the beads 14 having a surface on which a plurality of cleavable molecules 16 are disposed. The material of the beads 14 can be polystyrene, TetraGel®, hydrogel, resin, or other. When the beads 14 are of a polystyrene material, the cleavable molecules 16 can be disposed on the surface of the beads 14. When the beads are of a more porous material, such as TetraGel®, the cleavable molecules 16 can be in suspension within the beads 14. The beads 14 can be magnetic or non-magnetic. The material of the beads 14 can be selected depending on the density of the cleavable molecules 16 on the surface of the beads 14. The first microdroplet 12 can contain a plurality of beads 14. The cleavable molecules 16 can be drug molecules, proteins or peptides, antibodies, or any suitable small molecules. In addition, the surface of the beads 14 can comprise one or more DNA tags, which can aid in the identification of the cleavable molecules 16 involved in the interaction at a later stage of the analytical assay. The microdroplets contain a fluid 20. The fluid 20 can include one or more of the following, but is not limited to: surfactants, cell culture media such as EMEM or DMEM or RPMI or F-12, antibodies, buffers, conditioned media, and / or density modifiers.

[0125] As shown in Figure 1, an illumination source is applied to a first microdroplet 12 within the microfluidic chip, which photocleaves the cleavable molecule 16 from the surface of the bead 14. The illumination source can be an ultraviolet illumination source, a blue light source, a white light source, or any other illumination source capable of achieving sufficient energy to break the bond between the linker that attaches the cleavable molecule 16 to the surface of the bead 14. The DNA tag 18 attached to the bead 14 is not cleaved by the illumination and is retained on the bead 14. The bead 14 with the DNA tag 18 attached can be dispensed outside the microfluidic chip in a subsequent step and read out to identify which cleavable molecule 16 was originally attached to the bead 14.

[0126] As shown in FIG. 1, the entire molecule 16 can be cleaved from the surface of the bead 14. Alternatively, the time and / or intensity of illumination can be adjusted so that only a portion of the molecule 16 is cleaved from the surface of the bead 14. Subsequent photocleavage steps can be performed to increase the concentration of the cleaved molecule 16 in the first microdroplet 12. The first microdroplet 12 can be split so that the cleaved molecule 16 is contained in one part of the microdroplet and the bead 14 is contained in another part of the microdroplet. The volume of the split microdroplet can be increased by adding medium to the microfluidic chip and merging the split microdroplet with the medium. After the splitting step, the microdroplet containing the cleaved molecule 16 can be merged with the second microdroplet 22. If the remaining molecules 16 are attached to the surface of the bead 14, after the splitting step, the microdroplet containing the bead 14 can be used in a subsequent photocleavage step.

[0127] 2, a first microdroplet 12 containing the cleaved molecule 16 can be merged with a second microdroplet containing at least one biological entity 24 to form a merged microdroplet 26. An array of first microdroplets 12 can be merged simultaneously or near simultaneously with an array of second microdroplets 22 across the entire microfluidic chip or on a field-of-view basis. Merging the first and second microdroplets 12 and 22 can include mixing and / or agitating to form the merged microdroplet 26. The composition of the fluid 20 in the first and second microdroplets can be the same or different compositions.

[0128] The biological entities 24 can be cells, viruses, protein samples, antibody samples, functionalized microbeads, or enzymes. The second microdroplets 22 are not exposed to the photocleavage illumination, thus protecting the biological entities 24 from DNA damage and mutations, which could be detrimental if downstream genetic analysis is required. During the merging step, the biological entities 24 come into contact with the cleaved molecules 16 in the microdroplets 12. The concentration of cleaved molecules 16 that the biological entities 24 come into contact with depends on the amount of molecules 16 released from the beads 14 during the photocleavage step, as shown in FIG. 1.

[0129] It is advantageous to minimize the time between the photo-dissection step shown in FIG. 1 and the merging step shown in FIG. 2. Once the cleaved molecules 16 are released into the fluid 20 in the first microdroplet 12, there is a possibility that the cleaved molecules 16 may leak out of the microdroplet 12 into the surrounding phase. To minimize the time between the photo-dissection step and the merging step, both the first microdroplet 12 and the second microdroplet 22 may be provided to the microfluidic chip before the photo-dissection step. The photo-dissection step may be performed in a selective and localized manner, as shown in FIG. 1, whereby the first microdroplet 12 may be illuminated by the photo-dissection illumination while the second microdroplet 22 is not illuminated by the photo-dissection illumination. In some embodiments, the arrays of the first microdroplets 12 and the second microdroplets 22 may be arranged in an interdigitated array to minimize the time between the photo-dissection step and the merging step.

[0130] The light-dissection step shown in FIG. 1 can be performed on an array of first microdroplets 12 with varying intensity and / or duration of light-dissection illumination. An array of first microdroplets 12 can be generated with different concentrations of cleaved molecules 16 in the first microdroplets 12 across the array of first microdroplets 12. During the merging step, an array of identical microdroplets 22 can be merged with the array of first microdroplets 12 to expose the biological entities 24 in the second microdroplets 22 to different doses of cleaved molecules 16. This allows the effect of dose to be investigated without having to prepare beads 14 with varying numbers of cleaved molecules 16 attached to their surfaces, which can be investigated during a single preparation of the microdroplets in the microfluidic chip.

[0131] Alternatively, or in addition, the merged microdroplet 26 can be subjected to sequential merging steps with multiple first microdroplets 12, thereby exposing the biological entity 24 to sequential doses of the cleaved molecule 16.

[0132] A change in the optical signal can be detected from the merged microdroplet 26 using an optical system to indicate an interaction between the biological entity 24 and the cleaved molecule 16. The detected optical signal can emanate from the entire merged microdroplet 26 or can emanate from contents within the merged microdroplet 26. The merged microdroplet 26 can be sorted using the detected optical signal.

[0133] The optical signal can be, but is not limited to, a fluorescent signal, or a FRET signal, or a HTRF signal. The optical signal can be detected by a fluorescent assay on the beads 14 or the biological entities 24, a luminescence assay on the beads 14 or the biological entities 24, or by the form or number of bright-field or dark-field images of the biological entities 24. The luminescence assay on the beads 14 or the biological entities 24 can be used with a reporter system. For example, the microdroplets 12, 22 can contain one or more binding partners, which can be brought together when the microdroplets are merged. These binding partners can be, for example, capture moieties such as antibodies and / or biological entities 24. One or more binding partners can be brought together to provide a molecular entity or molecular system with functional activity. For example, parts of an enzyme can be brought together and bound together to form an entire enzyme with functional activity. The enzyme can then generate a luminescence signal in the presence of a substrate to generate a catalytic reaction. The luminescent signal produced by the enzymatic reaction can then be detected by a luminescence assay.

[0134] The luminescence analytical test can be one or more of the following techniques, but is not limited to: chemiluminescence; enhanced chemiluminescence (ECL); bioluminescence; bioluminescence resonance energy transfer (BRET); flash luminescence; and glow luminescence.

[0135] Additionally or alternatively, the optical signal from the merged microdroplets 26 may be a fluorescent signal that indicates a positive response when it exceeds a predetermined threshold of fluorescence. The fluorescent signal may be compared to a model system to determine the concentration of cleaved molecules 16 or the number of cleaved molecules 16 released from the beads 14.

[0136] 3A and 3B, a light sprite 30 is shown that can hold droplets in an array. As shown in FIGS. 3A and 3B, the light sprite 30 is shown as a square, but one skilled in the art will appreciate that the light sprite 30 can take any form, shape, or configuration. FIG. 3A shows the holding position of the microdroplets, where the microdroplets are paired in rows and columns containing cells 24 and beads loaded with photocleavable compounds. Referring to FIG. 3B, a light sectioning illumination spot can then be selectively directed at the spaces designated for the microdroplets containing beads and not the spaces designated for the microdroplets containing cells, and the light sectioning illumination spot can be a UV light.

[0137] 4A, a plurality of microdroplets 12, 22 are provided sorted into rows containing one or more cells 24 and a single bead 14. In another example, not shown in the accompanying drawings, the cell-containing microdroplets 22 can be specifically selected during loading to ensure that all of these microdroplets contain a single cell 24.

[0138] In the example shown in Figure 4A, microdroplets 22 containing cells and microdroplets 12 containing beads 14 are held in separate arrays. An illumination source, such as UV light, can then be directed at the microdroplets 12 containing beads 14 to trigger the release of the photocleavable compound from the beads 14. As shown in Figure 4B, the microdroplets 22 containing cells 24 and the microdroplets 12 containing beads 14 are moved together and merged to introduce the photocleaved compound released from the beads 14 to the cells 24.

[0139] Referring to Figure 5A, there is shown an array of microdroplets 22, 12 sorted into a row of droplets containing cells 24 and a row of droplets containing single beads 14. Figure 5B shows a portion of the array shown in Figure 5A. In Figure 5B, the microdroplets 22, 12 have been selected and paired together, with each microdroplet 22 and each microdroplet 12, prior to the merging operation.

[0140] Referring to Figure 6, an array of light-section illumination spots 30 is shown that vary in intensity from row to row. When light-section illumination is applied to a microdroplet 12 containing beads 14, the gradient in intensity of the light-section spots 30 produces a gradient in the concentration of drug released from the beads 14 by photocleavage. The intensity of the light-section spots 30 can be varied in any or repeated manner as desired. Two example arrays with different intensity steps between rows are shown in the same microfluidic chip.

[0141] By way of example only, if the microdroplet is illuminated with lower intensity illumination, the illumination light source may need to be directed at the first microdroplet for a longer period of time to provide sufficient energy to photocleave the molecule from the bead surface.

[0142] In another example, longer illumination can result in an increase in the number of photocleavable molecules that are photocleaved from the surface of the bead. By controlling the number of molecules that are cleaved from the surface of the bead, the concentration of cleaved molecules that contact the biological entity in the merging step can be controlled. This can facilitate detection of interactions between biological entities and molecules at different molecular doses.

[0143] Varying the duration or intensity of the light cleavage illumination applied to at least a subset of the first microdroplets can be used to generate a concentration gradient of cleaved molecules throughout at least a subset of the first microdroplets. Applying a higher intensity to at least a subset of the first microdroplets results in a higher concentration of cleaved molecules. The concentration gradient can be used to vary the number of cleavable molecules carried by the beads to efficiently investigate the effect of variable doses on cells without the need to introduce beads into the device. In some cases, varying the intensity of the illumination applied to at least a subset of the first microdroplets and / or varying the duration of illumination applied to at least a subset of the first microdroplets can be advantageous to control the light cleavage of molecules on the beads.

[0144] Various additional aspects and embodiments of the present invention will be apparent to those skilled in the art in view of this disclosure.

[0145] As used herein, "and / or" should be interpreted as a specific disclosure of each of the two specified features or components with the other or without the other. For example, "A and / or B" should be interpreted as a specific disclosure of each of (i) A, (ii) B, and (iii) A and B, just as if each were individually set forth herein.

[0146] Unless otherwise indicated by context, the feature descriptions and definitions set forth above are not limited to any particular aspect or embodiment of the present invention, but apply equally to all aspects and embodiments described.

[0147] Furthermore, while the present invention has been described by way of example with reference to certain embodiments, it will be understood by those skilled in the art that the invention is not limited to the disclosed embodiments and that alternative embodiments may be constructed without departing from the scope of the present invention as defined in the appended claims.

Claims

1. A method for detecting an interaction between a biological entity and a molecule, comprising: preparing an array of first microdroplets in a microfluidic chip, each of the first microdroplets containing at least one bead, each of the beads having the photolabile molecule bound thereto; preparing an array of second microdroplets in the microfluidic chip, each of the second microdroplets containing at least one biological entity; holding the entire array of the first microdroplets and the array of the second microdroplets; illuminating at least a subset of the first microdroplets containing the at least one bead with an illumination light source configured to photolyze the molecule; subsequently, merging at least a subset of the first microdroplets with at least a subset of the second microdroplets to form an array of merged microdroplets; detecting a change in an optical signal from the merged microdroplets using an optical system to detect an interaction between the biological entity and the molecule and a method comprising the steps of.

2. The method according to claim 1, wherein the biological entity is a cell or a part of a cell or a virus or an enzyme.

3. The method according to claim 1, further comprising classifying the first microdroplets and / or the second microdroplets before preparing the array of the first microdroplets and / or the array of the second microdroplets in the microfluidic chip.

4. The method according to claim 1, further comprising classifying the merged microdroplets using the detected optical signal.

5. The method according to claim 1, wherein each of the beads further comprises a molecular tag on the surface of the bead, and the molecular tag is a nucleic acid tag, or a protein tag, or a small molecule tag, or a synthetic tag.

6. The method according to claim 1, wherein the array of the first microdroplets and the array of the second microdroplets are held in an alternating entry pattern.

7. The method according to claim 1, further comprising preparing an array of additional microdroplets in the microfluidic chip, each of the additional microdroplets containing at least one reporter entity.

8. The method according to claim 1, wherein the light of the illumination light source is applied to the first microdroplets for a period of 1 to 300 seconds.

9. The method according to claim 1, further comprising the step of varying the time for illuminating at least a subset of the first microdroplets.

10. The method according to claim 1, wherein the light of the illumination light source is applied to one or more of the first microdroplets at an intensity of 0.7 to 400 mW.

11. The method according to claim 1, wherein the light of the illumination light source is applied to one or more of the first microdroplets at a wavelength of 360 to 380 nm.

12. The method according to claim 1, further comprising the step of varying the intensity of the illumination applied to at least a subset of the first microdroplets.

13. The method according to claim 1, further comprising the step of dividing at least a subset of the first microdroplets or at least a subset of the second microdroplets.

14. The method according to claim 1, wherein detecting that the change in the optical signal from the merged microdroplets exceeds a predetermined threshold is further configured to measure the concentration or number of the molecules released from the beads.

15. The method according to claim 1, wherein the optical signal is a fluorescence signal or a fluorescence resonance energy transfer (FRET) signal or a homogeneous time-resolved fluorescence (HTRF) signal or a luminescence signal.

16. The method according to claim 1, wherein the first microdroplets are prepared in the microfluidic chip before the array of the second microdroplets.

17. The method according to claim 1, wherein the microdroplets contain a cell culture medium.

18. The method according to claim 1, further comprising the step of supplying a carrier fluid into the microfluidic chip.

19. The method according to claim 1, further comprising the step of dispensing the microdroplets into a well plate, and the microdroplets are selected for recovery based on optical measurement.

20. The method according to claim 19, wherein the optical measurement is performed on the microdroplets divided from the original microdroplets, and the original microdroplets are selected for recovery according to the optical measurement.

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