System and processes for detecting, classifying and dispensing particles

Abstract

System (10) for detecting, classifying, and dispensing particles, comprising: a microfluidic device (1), comprising a sample channel (100), a sorting and dispensing channel (130) intersecting the sample channel (100) and forming a sorting intersection (120). The sorting and dispensing channel (130) comprises a dispensing outlet (140) for the dispensing of classified particles. The system (10) further comprises a sorting pump (200), connected to the sorting channel (130) of the microfluidic device (1), arranged to provide a flow of liquid to the sorting and dispensing channel (130) at a pressure exceeding a burst pressure of the dispensing outlet (140). The system comprises a classification system (6) comprising a camera (600) for determining the visual properties of particles, the camera (600) has a field of view (601) comprising the sorting intersection (120) of the microfluidic device (1), and a sorting valve (134) which is controlled by the classification system (6).

Description

[0001] SYSTEM AND PROCESSES FOR DETECTING, CLASSIFYING AND DISPENSING PARTICLES

[0002] Field of the Invention

[0003] The present disclosure relates to systems and devices for detecting, classifying and dispensing particles, such as cells. In particular, it relates to systems comprising a microfluidic device enabling the flow and dispensing of cells and a classification system comprising a camera to enable visual identification and classification.

[0004] Background of the invention

[0005] In various biotechnology fields cells or other particles in a sample may need to be detected and sorted to enable downstream processes such as cell cultivation etc. Flow cytometry is a general term for the process of detecting characteristics of cells or other particles in a fluid sample.

[0006] Various flow cytometry devices exist on the market today. In general, a cell in a liquid sample flows through a detection region, and cells are thereafter sorted based on the detected characteristics of the cells. Typically, cells are stained with fluorescent markers or other detectable labels, such as antibodies, and cells detected as being of interest are separated by various means from the bulk of the sample. However, labelling of cells inherently modifies the properties of the cells and makes them unsuitable for downstream processes, such as culturing, drug screening etc.

[0007] Additionally, many flow cytometry devices capture sorted cells within a device, thereby necessitating the use of complex integrated systems to continue downstream processes or manual handling of sorted cells with, for example, pipettes. Devices and systems which conveniently and preferably, automatically integrate with the standard well plates used within cell culturing would be ideal. Such improved systems ideally fit within existing lab infrastructure, and minimise the potential for manual handling errors.

[0008] Summary of the invention

[0009] Accordingly, the present invention preferably seeks to mitigate, alleviate or eliminate one or more of the above-identified deficiencies in the art and disadvantages singly or in any combination and solves at least the above-mentioned problems by providing a system for detecting, classifying and dispensing particles. The system comprises a microfluidic device, comprising a sample channel along which a sample comprising the particles may flow, a sorting and dispensing channel which intersects the sample channel and thereby forms a sorting intersection. The sorting and dispensing channel comprising a dispensing portion downstream of the sorting intersection, wherein the dispensing portion comprises a dispensing outlet downstream of the sorting intersection, for the dispensing of detected, classified particles. The system further comprises a sorting pump connected to the sorting channel of the microfluidic device, arranged to provide a flow of liquid to the sorting and dispensing channel at a pressure exceeding a burst pressure of the dispensing outlet. The system comprises a classification system comprising a camera and a classification processor for determining the visual properties of particles. The camera has a field of view comprising the sorting intersection of the microfluidic device. The system comprises a sorting valve for controlling the flow of fluid in the sorting and dispensing channel, wherein the sorting valve is controlled by the classification system.

[0010] A process for detecting, classifying and dispensing particles is also provided.

[0011] Further advantageous embodiments are disclosed in the appended and dependent patent claims.

[0012] Brief description of the drawings

[0013] These and other aspects, features and advantages of which the invention is capable will be apparent and elucidated from the following description of embodiments of the present invention, reference being made to the accompanying drawings, in which

[0014] Fig. l is a schematic representation of a fluidic device for detecting, classifying and dispensing cells according to an aspect.

[0015] Fig. 2 is a view of a microfluidic device according to an aspect.

[0016] Fig. 3 is a schematic partial representation of a fluidic device showing a detailed view of the region A of figure 1, showing a sorting intersection according to an aspect.

[0017] Fig. 4a is a detailed view of a dispensing outlet according to an aspect.

[0018] Fig. 4b is a detailed view of a dispensing outlet and sorting intersection according to an aspect.

[0019] Fig. 5a is a detailed view of a dispensing outlet and sorting intersection. Particles are shown schematically by the solid black dots.

[0020] Fig. 5b is a detailed view of an alternative arrangement of a dispensing outlet and sorting intersection. Particles are shown schematically by the solid black dots. Fig. 6 is a schematic modular representation of components of a system for detecting, classifying and dispensing cells according to an aspect.

[0021] Fig. 7 is an image from a classification event whereby a spheroid has been detected within the field of view of the camera.

[0022] Detailed description

[0023] The following description describes a microfluidic device 1, and a system 10 for the detection, classification and dispensing of cells. Unless indicated otherwise, the term cells as used herein refers to single cells, clusters of multiple cells, spheroids, assembloids and / or organoids. The device 1 and associated systems 10 are also intended to be used to detect, classify, and dispense particles having a similar morphology to cells or spheroids, such as for example beads. Cells may be attached to beads, or beads may be detected, classified, and dispensed without attached cells. The following description refers primarily to particles which is intended to encompass various microparticles, such as cells, spheroids, organoids, or beads. The particles to be detected, classified, and dispensed via the system 10 are small, having a nominal diameter of less than 1 mm, such as less than 500 pm, such as less than about 250 pm. Exemplary particles for use with the system 10 and device 1 of the present disclosure are spheroids and / or miniature spheroids having a diameter of from about 100 pm to about 150 pm.

[0024] Figure 1 shows a schematic representation of a microfluidic device 1 and system 10 for dispensing particles. Figure 2 shows a view of the microfluidic device 1. The device 1 comprises a sample channel 100 for the flow of particles and sample media. The sample channel 100 comprises a first portion 100a referred to as the sample portion 100a, and a second portion 100b, referred to as the waste portion 100b. The sample portion 100a is upstream of a sorting intersection 120. The waste portion 100b is downstream of the sorting intersection 120. The length of the sample portion 100a is advantageously substantially equal to the length of the waste portion 100b. By matching the lengths of the two portions 100a, 100b of the sample channel 100 the fluidic resistance of the respective channel openings at the sorting intersection 120 is equivalent. Additionally, by matching the lengths of the two portions 100a, 100b reversable flow of sample, as will be described below, is possible.

[0025] The microfluidic device 1 further comprises a sorting and dispensing channel 130. The sorting and dispensing channel 130 is separate to the sample channel 100. The sorting and dispensing channel 130 comprises a first sorting channel portion 130a and a second dispensing channel portion 130b. The sorting channel portion 130a is upstream of the dispensing intersection 120. The dispensing channel portion 130b is downstream of the sorting intersection 120.

[0026] The sample channel 100 is provided with an inlet 101 in connection with a sample reservoir 105 comprising fluid sample. The fluid sample generally comprises the particles to be detected, classified and dispensed, dispersed within a sample media and in addition, any impurities which may be present. In particular, the sample portion 100a of the sample channel 100 is provided with the inlet 101 in connection with the sample reservoir 105. The sample reservoir 105 may be provided within the microfluidic device 1 i.e., on-chip, or separately and in connection with sample channel 100 of the microfluidic device 1.

[0027] The sample reservoir 105 is provided in connection with an input pump 210. The input pump 210 provides a positive pressure to the sample reservoir 105 to generate a fluid flow from the sample reservoir 105 to the sample channel 100. The input pump 210 and the sample reservoir 105 are provided upstream of the sorting intersection 120 in the fluidic configuration shown in figure 1.

[0028] The sorting intersection 120 is an intersection between the sample channel 100 and the sorting and dispensing channel 130. The microfluidic device 1 is arranged such that sample flows along the sample portion 100a to the sorting intersection 120. The sample channel 100 has an inlet 1001 to the sorting intersection 120, and an outlet 1002 from the sorting intersection 120. The inlet 1001 forms the end of the sample portion 100a of the sample channel 100. The outlet 1002 forms the start of the waste portion 100b of the sample channel 100. The sorting and dispensing channel 130 has an inlet 1301 to the sorting intersection 120, and an outlet 1302 from the sorting intersection 120. The inlet 1301 forms the end of the sorting portion 130a. The outlet 1302 forms the start of the dispensing portion 130b.

[0029] The dispensing portion 130b of the sorting and dispensing channel 130 comprises a dispensing outlet 140. The dispensing portion 130b connects the sample channel 100 such that particles may flow from the sample channel 100, and in particular, the sample portion 100a, of the sample channel 100 to the dispensing outlet 140 via the sorting intersection 120. The dispensing outlet 140 receives particles to be dispensed via the dispensing portion 130b of the sorting and dispensing channel 130. The dispensing outlet 140 is downstream of the sorting intersection 120. The dispensing outlet 140 is downstream of the outlet 1302 from the sorting intersection 120 into the dispensing portion 130b.

[0030] The sample channel 100 and the sorting and dispensing channel 130 may be arranged such that they are perpendicular / orthogonal each other at the sorting intersection 120 as shown in, for example, figures 3 and 5a. However, other arrangements of the sample channel 100 and dispensing channel 130 at the sorting intersection 120 are possible. For example, in the arrangement shown in figure 5b, the sample channel 100 extends aligned with the dispensing portion 130b of the dispensing channel 130. Such an arrangement may be preferable as due to the vertical alignment of the sample channel 100 in use, particles may preferentially sediment toward the dispensing portion 130b of the dispensing channel 130.

[0031] The sample channel 100 is arranged with respect to the sorting and dispensing channel 130 such that particles may be displaced from the sample channel 100 into the sorting and dispensing channel 130 at the sorting intersection 120. Specifically, particles are displaced from the sample portion 100a of the sample channel 100 into the dispensing portion 130b of the sorting and dispensing channel 130.

[0032] The dispensing outlet 140 is shown in detail in figure 3a and 3b. The dispensing outlet 140 is an opening connecting the dispensing portion 130b to atmosphere. The dispensing outlet 140 has an inlet 141 to the dispensing outlet 140 from the dispensing portion 130b of the sorting and dispensing channel 130 and an outlet 142 forming an opening to atmosphere. The fluid in the dispensing outlet 140 forms a meniscus 144 at the outlet 142. The outlet 142 forms a passive valve with a controllable bubble or burst pressure. The diameter of the outlet 142 in conjunction with the surface of the outlet 142 controls the bubble or burst pressure at the meniscus 144. The diameter of the outlet 142 may be from about 20 pm to about 300 pm, such as from about 50 pm to about 200 pm. The outlet 142 has a diameter greater than the particles of interest to be dispensed, and dimensions selected to provide a sufficiently high passive valve resistance.

[0033] A dispensing outlet 140 having a frustoconical interval form is shown in figure 4a. The frustoconical form of the dispensing outlet 140 shown in figure 4a is provided by a frustoconical internal sidewall 143 extending from a larger cross-section at an inlet 141 to the dispensing outlet 140 in connection with the dispensing portion 130b of the channel 130to a narrower cross-section at an outlet 142 to atmosphere from the dispensing outlet 140. The dispensing outlet 140 may have other internal geometries, such as cylindrical and need not be frustoconical. Additionally, the dispensing outlet 140 may be manufactured from, or provided with a coating of, hydrophobic material to increase the bubble / burst pressure.

[0034] As shown in figure 4b, the dispensing outlet 140 is ideally provided at the base of the microfluidic device 1, such that it may be arranged directly above a receptacle for receiving the particle to be dispensed. The dispensing outlet 140 as shown in figure 4b may be provided with the frustoconical form as shown in figure 4a.

[0035] Sample, comprising particles, sample media and any impurities present, is directed i.e., flows from the reservoir 105 to the sorting intersection 120 via the sample portion 100a of the sample channel 100.

[0036] The sorting portion 130a of the sorting and dispensing channel 130 is connected at an inlet 135 to a buffer reservoir 133. The sorting portion 130a of the channel 130 may decrease in cross-section from the inlet 135 toward the inlet 1301 to the sorting intersection 120. The decreasing cross-section controls the back pressure at the inlet 135.

[0037] The buffer reservoir 133 may be provided as a reservoir 133 on the microfluidic device 1, or may instead be provided as a separate reservoir 133 in connection with the microfluidic device 1. The buffer reservoir 133 may be maintained at a positive pressure with respect to atmospheric pressure via the provision of a sorting pump 200. The sorting pump 200 provides a positive pressure to the buffer fluid reservoir 133. The sorting pump 200, and the sorting valve 134 are arranged upstream of the sorting intersection 120. A sorting valve 134 is provided between the buffer reservoir 133 and the inlet 135 to the sorting and dispensing channel 130. The sorting valve 134 is ideally an active microfluidic valve, such as a solenoid valve. The sorting valve 134 enables precise control over the duration of time that the sorting and dispensing channel 130 is provided with a positive pressure and enables a stepchange in pressure and fluid flow within the sorting and dispensing channel 130 when compared to control of the sorting pump 200 pressure without the valve 134. Controlling the sorting pump 200 pressure alone would result in a slower change in pressure and fluid flow in the sorting and dispensing channel 130.

[0038] The sorting valve 134 may be opened for a duration of from about 10 ms to about 1000 ms. A shorter duration, such as from about 10 ms to about 50 ms, such as about 20 ms, is generally used with a higher-pressure dispensing pump 200 pressure leading to a jetting dispensing procedure. Longer durations, such as greater than about 100 ms, such as from about 100 ms to 1000 ms, are generally used with a relatively lower pressure dispensing pump pressure leading to droplet formation at the outlet 140. Both longer and shorter sorting valve 134 opening durations have been shown to successfully sort and dispense particles with the microfluidic device 1, and system 10.

[0039] Sample flowing in the sample channel 100 is analysed and particles to be dispensed are identified, as will be described in a later section of the description.

[0040] To displace particles comprised in the sample from the sample channel 100 to the dispensing outlet 140, a burst of fluid flow is provided to the sorting and dispensing channel 130. The fluid in the sorting portion 130a of the sorting and dispensing channel 130 is separate to the fluid in the sample channel 100. The burst of fluid flow displaces particles from the sample channel 100 into the dispensing portion 130b of the sorting and dispensing channel 130 at the sorting intersection 120. After the burst of fluid has entered and crossed the sorting intersection 120, the fluid from the sorting portion 130a and a portion of the sample fluid will have mixed. In the schematic arrangement shown in figure 1, the particles to be dispensed are shown displaced in a lateral plane from the sample channel 100 into the dispensing portion 130b of the sorting and dispensing channel 130 at the sorting intersection 120 i.e., sample and the particles flow in a single plane, and are displaced in the same plane. However, the representation in figure 1 is schematic and as shown in figures 2, and 4 other arrangements of the channels are possible and preferred. For example, the sorting and dispensing channel 130 is ideally arranged into the page of figure 1, such that it intersects the sample channel 100 from above and below, rather than laterally as shown in figure 1 i.e., the particles are displaced in an orthogonal plane to the plane of sample flow. Such an arrangement is shown in figure 2 and figure 4b where the sorting and dispensing channel 130 is arranged substantially vertically with respect to the earth. The dispensing channel is aligned with the Z axis of figure 2, and figure 4B, and the Z-axis is vertical with respect to the earth. Such an arrangement where the sorting and dispensing channel 130 intersects the sample channel 100 from above and below, rather than laterally, is particularly advantageous as the dispensing outlet 140 is ideally arranged at the base of the microfluidic device 1, and therefore enables a continuous sorting and dispensing channel 130 and dispensing outlet 140 arrangement without the need for any right-angle bends in the dispensing portion 130b of the sorting dispensing channel 130, or at the dispensing outlet 140 as is shown in the less preferable design of figure 4a.

[0041] As particles flow within the sample channel 100, when no sorting even is taking place, the sorting and dispensing channel 130 is typically free from a flow of fluid. The sorting valve 134 is closed, and the passive valve at the dispensing outlet 140 is sealed. That is, the meniscus 144 at the outlet 140 restricts flow of fluid. The pressure drop from the inlet 1302 to the dispensing outlet 140 is maintained at approximately 0 mbar.

[0042] When a sorting event is to take place, the particles are displaced from the sample channel 100 to the sorting and dispensing channel 130. In order to displace particles from the sample channel 100 to the dispensing portion 130b of the sorting and dispensing channel 130 at the sorting intersection 120, the sorting valve 134 is opened. The positive pressure provided to the buffer reservoir 133 is sufficient to burst the passive valve the dispensing outlet 140. Therefore, on opening the sorting valve 134, fluid flows along the sorting channel and dispensing channel 130, through the sample channel 100 at the sorting intersection 120 and thereby displaces particles from the sample channel 100 at the sorting intersection 120, to the dispensing portion 130b of the sorting and dispensing channel 130. The opening of the sorting valve 134 generates a step change in pressure at the inlet 1302 at the sorting intersection 120, the pressure introduces a flow along the sorting portion 130a, downstream of the valve 134, and the dispensing portion 130b downstream of the sorting intersection 120. The flow is sufficient to displace particles from the sample channel 100 at the sorting intersection 120 into the dispensing portion 130b of the sorting and dispensing channel 130. As shown in figure 1, the particles may be displaced laterally from the sample channel 100 to the dispensing channel 130. As described previously, the particles may advantageously be displaced in an orthogonal plane to the plane of flow through the sample channel 100. The sorting valve 134 is opened for a sufficient duration such that a volume of fluid comprising the particles which have been provided to the dispensing portion 130b flow along the dispensing portion 130b and out of the dispensing outlet 140. The particles continue to flow from dispensing portion 130b, of the sorting and dispensing channel 130 and are expelled from the outlet 142 to atmosphere of the dispensing outlet 140.

[0043] The sorting and dispensing channel 130 has a substantially lower fluidic resistance compared to the sample channel 100. This ensures that when the valve 134 is opened, fluid from the sorting portion 130a, of the sorting and dispensing channel 130 does not enter the sample channel 100, but continues from the sorting portion 130a of the sorting and dispensing channel 130 to the dispensing portion 130b. The fluidic resistance is determined by, for example, the length and / or cross-sectional area i.e., height and / or width of the sorting and dispensing channel 130 in comparison to the length of the sample channel 100. In particular, to reduce the fluidic resistance of the dispensing portion 130b in comparison to the sample channel 100, the length of the dispensing portion 130b of the sorting and dispensing channel 130 is advantageously significantly less than the length of the sample channel 100. As shown in figure 2, the length of the sample channel 100 may be increased by a spiral arrangement within the microfluidic device 1. The length may be increased by other techniques as are known within the field of microfluidics. The length of the sample channel 100 may be greater than about 5 times, such as greater than about 10 times, such as greater than about 20 times the length of the dispensing portion 130b of the sorting and dispensing channel 130. By increasing the length of the sample channel 100 with respect to the sorting and dispensing channel 130, and in particular with respect to the dispensing portion 130b, damp any fluctuations in pressure within the sample channel 100 on the influx of fluid during a sorting event. This improves the relative pressures within the system, and improves the classification process.

[0044] The fluidic resistance of the sorting and dispensing channel 130 being substantially less than the fluidic resistance of the sample channel 100 is especially important as the flow of relatively higher-pressure fluid during a sorting event causes turbulent, i.e., non-laminar, flow at the sorting intersection 120. By selecting respective channel dimensions which result in a relatively higher resistance of the sample channel 100 compared to the sorting and dispensing channel 130 the flow from the sorting and dispensing 130 during a sorting event does not enter the sample channel 100 at the sorting intersection 120.

[0045] After particles have been displaced into the dispensing portion 130b of the sorting and dispensing channel 130, and expelled from the dispensing outlet 140, the sorting valve 134 is closed. Closing the sorting valve 134 reduces the pressure provided at the inlet 1302 to the dispensing portion 130b of the dispensing and sorting channel 130. The passive valve at the dispensing outlet 140 thereafter closes as the pressure provided to the passive valve is not greater than the burst / bubble pressure of the passive valve. Therefore, on closing the sorting valve 134 after dispensing particles from the dispensing outlet 140, there is no fluid flow within the sorting portion 130a, and the dispensing portion 130b.

[0046] A collection reservoir 150 is provided downstream of the sorting intersection 120 in the fluidic arrangement shown in figures 1 and 2. The collection reservoir 150 is provided in connection with the waste portion 100b of the sample channel 100 downstream of the sorting intersection 120. That is, the sample channel 100 extends from the sample reservoir 105 to the collection reservoir 150 via the sorting intersection 120. The collection reservoir 150 may be provided within the microfluidic device 1 i.e., on-chip, or separately and in connection with sample channel 100 of the microfluidic device 1.

[0047] The waste portion 100b of the sample channel 100 is the continuation of the sample channel 100 downstream of the sorting intersection 120. Sample which has not been expelled from the dispensing outlet 140 therefore flows into the collection reservoir 150. The collection reservoir 150 is provided with an inlet 151 corresponding to the outlet 108 of the sample channel 100. The collection reservoir 150 is ideally provided in connection with a regulating pump 220. The regulating pump 220 provides an opposite, and therefore in general negative, pressure to the collection reservoir 150, and the waste portion 100b of the sample channel 100, such that fluid may flow from the inlet 101 of the sample channel 100 to the outlet 108 of the waste portion 100b of the sample channel 100.

[0048] The provision of the input pump 210 and the regulating pump 220 and their respective opposite flowrates or pressures ensures that sample flows within the sample channel 100 from the sample reservoir 105 to the collection reservoir 150 at a substantially constant rate, without inadvertent dropping at the outlet 140.

[0049] The regulating pump 220 is provided in communication with a sorting pressure sensor 221. The sorting pressure sensor 221 is arranged to measure the pressure within the sorting and dispensing channel 130. In particular, the sorting pressure sensor 221 is arranged to measure the pressure within the sorting portion 130a of the sorting and dispensing channel 130. Communication as used herein refers to the regulating pump 220 being in electrical communication with the sorting pressure sensor 221, and does not refer to a fluidic connection. The sorting pressure sensor 221 measures the pressure in the sorting and dispensing channel 130, generally the pressure within the sorting portion 130a, to ensure that the pressure at the sorting intersection 120 remains constant. A constant pressure is required to ensure that fluid pressure does not exceed the burst pressure of the dispensing outlet 140 and therefore, that fluid is not inadvertently displaced into the dispensing portion 130b of the channel 130 while the sorting valve 134 is closed i.e., dripping from the dispensing outlet 140. The sorting pressure sensor 221 may be provided in fluidic connection with the sorting portion 130a of the channel 130. The sorting pressure sensor 221 is arranged to measure the pressure in the sorting portion 130a which generally also corresponds to the pressure in dispensing portion 130b and ensures that the pressure does not need to be measured in the vicinity of particles to be dispensed.

[0050] The regulating pump 220 is controlled via the sorting pressure sensor 221. The pressure sensor 221 may be in electrical communication with the regulating pump 220. The regulating pump 220 may be controlled via a feedback loop, such as a Proportional, Integral, and / or Differential (P, PI, PD, PID) feedback control system controlling the pump output and measuring pressure via the sorting pressure sensor 221. Other known control system implementations are possible to combine with the regulating pump 220 and the sorting pressure sensor 221. A PID control system has been shown to effectively control the pressure at the sorting intersection 120.

[0051] As shown in figure 6, the control system may be implemented on a controller 300. The controller 300 is in electrical connection with the pumps 200, 210, 220, 230, the pressure sensor 221 and the valve 134. In figure 6 the controller is represented as comprising a first module 310 adapted to control the valve 134, the sorting pump 200, the input pump 210, and the focus pump 230. The controller 300 also comprises a second module 320 adapted to control the regulating pump 220 based on the pressure measured by the pressure sensor 221.

[0052] The input pump 210 and the regulating pump 220 may be controlled via a flowrate feedback loop, to maintain a predetermined a flowrate through the sample channel 100. Alternatively, or in addition, the input pump 210 and the regulating pump 220 may be controlled via a pressure feedback loop, to maintain a predetermined pressure in the sample channel 100. Likewise, and advantageously the focus pump 230 may be incorporated into the feedback control loop to control the flowrate and / or pressure in the sample channel 100. Ideally, the flowrate of the input pump 210 may be about 1 pL / min. As described above, if the flowrate of the input pump 210 is positive, the flowrate of the regulating pump 220 is generally negative. The corresponding flowrate of the regulating pump 220, if the input pump 210 flowrate is 1 pL / min, may be about -1 pL / min, or higher, such as from about -1 pL / min to about -5 pL / min, such as about -3 pL / min. The respective flowrates may be tuned to ensure that inadvertent dropping at the outlet 140 does not occur, that is that the pressure at the outlet 140 does not exceed the burst pressure.

[0053] The sample channel 100, and in particular the sample portion 100a, may be provided with inlets 113a, 113b connecting the sample channel 100 to at least one focusing channel 110a, 110b. The at least one focusing channel 110a, 110b provides a sheath flow of fluid to control the position and flowrate of particles within the sample channel 100. The at least one focusing channel 110a, 110b may be, as shown in figure 1, two separate channels 110a, 110b in connection with the sample channel 100. An inlet 113a of the at least one focusing channel 110a, 110b connects the focusing channel 110a, 100b to the sample channel 100. The inlets 113a, 113b, are generally provided downstream of the inlet 101 from the reservoir 105. If two focusing channels 110a, 110b are provided, the inlets 113a, 113b from the focusing channels 110a, 110b may be provided at opposing positions with respect to each other on the sample channel 100. If two focusing channels 110a, 110b are provided, the focusing channels 110a, 110b are ideally of equal length and channel dimensions, such that the flow rate and pressure of any fluid at the inlets 113a, 113b provided to the sample channel 100 is substantially equal.

[0054] The at least one focusing channel 110a, 110b is connected to a focus pump 230. The focus pump 230 provides a positive pressure to the at least one focusing channel 110a, 110b to flow liquid along the focusing channel(s) 110a, 110b into the sample channel 100. Ideally, the focus pump 230 is provided in connection with a focus reservoir 111. The focus reservoir 111 is a reservoir for receiving and holding a volume of liquid media. The focus reservoir 111 has at least one outlet 112 in connection with the focusing channel(s) 110a, 110b. The focus reservoir 111 need not be provided to the microfluidic device 1, but may be a separate reservoir 111. The outlet 112 from the focus reservoir 111 to the focusing channels 110a, 110b may be a Y-outlet having a single inlet from the focus reservoir 111 splitting into two outlets at the focusing channels 110a, 110b respectively. The flow of liquid media from the focus channel 110a, 110b additionally ensures that the particles do not clump together and thereby improves the detection, classification and dispensing performance of the system 10.

[0055] Particles to be dispensed are analysed by a visual classification process and system 6. The visual classification system 6 comprises a camera 600. The camera 600 is arranged such that a region comprising the sorting intersection 120 is within the camera’s field of view 601. As would be understood, the camera 600 is typically connected to a microscope. The field of view 601 includes at least the sorting intersection 120 and ideally a portion of the sample channel 100 upstream of the sorting intersection 120 i.e., the sample portion 100a. As the field of view comprises the sorting intersection 120, the field of view 601 therefore includes at least a portion of the sample channel 100, and at least a portion of the sorting and dispensing channel 130. The field of view ideally includes the dispensing portion 130b of the sorting and dispensing channel 130. The field of view may also advantageously include a portion of the sorting portion 130a of the sorting and dispensing channel 130 and a portion of the waste portion 100b of the sample channel 100. By provision of the field of view 601 at the sorting intersection 120, the system 10 as a whole, and in particular the visual classification system 6, can ensure that a particle which has been identified and classified travels into the correct channel downstream of the sorting intersection 120. That is, a particle which should enter the dispensing portion 130b of the sorting and dispensing channel 130 can be detected and monitored into the dispensing portion 130b. Likewise, a particle which should pass through the sorting intersection 120 into the waste portion 100b of the sample channel 100 can be monitored through the sorting intersection 120 into the waste portion 100b.

[0056] As shown in figures 2, 4b, 5a and 5b, the field of view 601 of the camera 600 ideally and advantageously comprises the dispensing outlet 140. In particular, the field of view 601 ideally comprises the meniscus 144 and the outlet to atmosphere 142.

[0057] By arranging the field of view 601 of the camera 600 to include the sorting intersection 120, and the dispensing outlet 140, particles to be detected and dispensed can be monitored by the visual classification system 6 from the sorting intersection 120 to the outlet to atmosphere 142 via the dispensing outlet 140. This improves control over a sorting and dispensing process and enables the system to detect successful or, in the event of an error, an incorrect dispensing process. For example, if a particle has been identified and sorted into the dispensing portion 130b of the sorting and dispensing channel 130 it can be tracked during an entire dispensing process to ensure correct dispensing.

[0058] The plane of the field of view 601 is advantageously a vertical plane with respect to the earth such that the visual classification system 6, camera 600, can monitor the dispensing of particles from the device 1, when a receptacle is provided beneath the device 1.

[0059] The camera 600 is connected to a classification processor 610. The classification processor 610 ideally comprises a machine learning system trained on the appearance of various particles and their characteristics. The classification processor 610 is trained to determine objects within the field of view 601 of the system 6. As described, the classification processor identifies and detects particles, the classification processor 610 ideally furthermore is trained to identify and detect single particles, such as single cells, single spheroids, single organoids etc., multiple particles, such as clumps of cells, clumps of spheroids, clumps of organoids etc., air bubbles and, where necessary, beads. The classification processor 610 may analyse both the static and dynamic characteristics of the particles as they pass within the field of view 601. Static characteristics refers to the size, shape and morphology in general of the identified particles. Dynamic characteristics refers to, for example, the flow rate through the field of view 601, that is the velocity of the particles through the field of view 601. The classification processor 610 is capable of identifying particles and determining particle-types based on the visual characteristics of the detected particles. The classification processor 610 may furthermore detect and identify a plurality of grouped particles, for example two or more individual spheroids, simultaneously. Figure 7 shows an image from a classification event. In figure 7, the classification processor 610 has detected a spheroid within the field of view 601 of the camera 600.

[0060] The classification processor 610 does not require, and the classification system 6 does not comprise, a means for detecting, for example fluorescent labels. This enables the detection, classification, and dispensing of label-free particles. The classification processor 610 detects and classifies particles without user input during a detection and dispensing process. As is typical with processors 610 trained via machine learning, a human operator may have trained the system prior to its usage with the microfluidic device 1 in a dispensing process.

[0061] As shown in figure 6, the classification system 6 is in communication with the sorting valve 134, and optionally, the sorting pump 200. The classification system is ideally in communication with the controller 300 such that the classification system 6 receives input from and can provide output to the controller 300. As shown in figure 6, the classification system 6 generally is in communication with the first module 310 of the controller 300. The communication between the classification system 6 and the pumps 200, 210, 230, via the first module 310, enables, for example, the flow rate of particles through the field of view 601 to be optimised for the classification processor 610. For example, higher viscosity fluids, or a sample comprising a relatively higher concentration of particles may require reduction in the flow rate through the sample channel 100 to ensure that the processor 610 has sufficient time to detect and classify particles. Additionally, or alternatively, the camera 600 may visually monitor the meniscus 144 at the dispensing outlet 140. The controller 300 may adjust the flowrate of, or pressure provided by, the input pump 210, the regulating pump 220, and / or the focus pump 230 in order to ensure that the meniscus 144 is maintained at an ideal predetermined height within the dispensing outlet 140. Once the classification processor 610 has detected and identified particles to be sorted into the dispensing portion 130b of the sorting and dispensing channel 130, the classification system 6 sends a signal to open the sorting valve 134 such that burst of pressure and fluid flow is provided to the sorting and dispensing channel 130. The signal may be sent via the first module 310 of the controller 300. The burst of pressure is provided when the particles are within the sorting intersection 120. As described above, the burst of pressure displaces the detected and identified particles into the dispensing portion 130b of the sorting and dispensing channel 130. The classification system 6 may advantageously continue to track / monitor, via the camera 600, the detected and identified particles within the dispensing portion 130b, and ideally to and out from the dispensing outlet 140. This enables the classification system 6 to ensure that the particles have entered and flow through the dispensing portion 130b toward, and out of the dispensing outlet 140.

[0062] Particles dispensed at the dispensing outlet 140, that is, particles of interest, are comprised within a droplet of sample media. The volume of the droplet is controlled by the opening duration of the sorting valve 134, and the pressure provided to the dispensing and sorting channel 130, via the sorting pump 200. A higher pressure, or longer open time of the valve 134 results in an increased droplet volume. In general, the volume of fluid which is provided to the dispensing and sorting channel 130 due to the opening of the valve 134 is expelled from the dispensing outlet 140. The dispensed volume may range from about 1 pL to about 50 pL, such as from about 10 pL to about 20 pL.

[0063] Particles which have been dispensed from the dispensing outlet 140 are ideally provided to a well plate. For example, a 384-well plate suitable for drug screening or subsequent culturing of particles. However, the microfluidic device 1 is capable of dispensing particles into any suitable receptacle. During a dispensing process, the dispensing outlet 140 may be aligned above a well in a well plate, the volume of liquid including the particle of interest is dispensed into a well in the well plate, the well plate may thereafter be displaced such that a new well is under the dispensing outlet 140. The well plate is ideally displaced via an automated stage, robot arm etc. capable of displacing the well plate in at least XY axes.

[0064] The well plate is filled sufficiently depending on the specific usage requirements, the well plate may be replaced for example by manually replacing the well plate by an operator or by an automated system to replace the well plate, such as a robotic arm or conveyor system. An automated system is therefore ideally connected to the classification system 6 such that parameters relating to the dispensed particles can be communicated to the automated system. If the classification system 6 detects and identifies particles which are not to be dispensed, based on their visual characteristics, the classification system 6 does not open the sorting valve 134 and the detected and identified particles which are not to be dispensed continue to flow within the sample channel 100 past the sorting intersection 120, into the waste portion 100b and subsequently into the collection reservoir 150. The particles provided to the collection reservoir 150 are the particles which were not provided to the dispensing portion 130b. A majority of the sample media and the liquids provided from the focus reservoir 111 to the sample channel 100 flow into the collection reservoir 150.

[0065] After a sufficient number of particles have been dispensed from the microfluidic device 1, the device 1 may be disposed of or washed and re-used depending on the specific usage requirements.

[0066] The flow of sample within the sample channel 100 is reversable. Sample which has been received in the collection reservoir 150 may subsequently be provided back to the sample channel 100, and therein, the sorting intersection 120. As the sample reservoir 105 forms one end of the sample channel 100, and the collection reservoir 150 forms the opposing end of the sample channel 100, the device 1 may be operated in reverse. That is, rather than the direction of flow as shown in figure 1, the sample comprising particles may flow in the opposite direction, from the collection reservoir 150 to the sample reservoir 105. Particles present in the sample media flow via the sorting intersection 120 and can be detected and sorted into the dispensing portion 130b of the sorting and dispensing channel 130 as described previously. In such an arrangement the regulating pump 220 acts as the input pump 210, and the input pump 210 acts as the regulating pump 220. Such an arrangement of the sample reservoir 105 and collection reservoir 150 enables multi-step dispensing processes, or processes where particles can be preferentially dispensed and if, for example, wells in a well plate remain empty the less preferential particles can be dispensed after the most preferential particles have been dispensed. The reversable arrangement described herein also enables reduced waste, as the sample media may be reused. The waste may be misidentified particles, or sample media which has entered the collection reservoir 150. As the fluid can flow from the collection reservoir 150 to the sorting intersection by reversing the pressure / flow of the pumps 210, 220 compared to the direction shown in figure 1, a detection and dispensing process may be run several times with the same sample media, provided with new particles for detection and dispensing, or even the same sample with misidentified particles. The dispensing process does not require human operator input and may be fully automated. A user may provide the sample to be analysed to the sample reservoir 105, and subsequently the microfluidic device 1 in conjunction with the visual classification system 6 may detect, classify and dispense particles without further operator input.

[0067] The input pump 210, regulating pump 220, and focus pump 230 are ideally micropumps providing a continuous, pulsation free, flow of liquid. Such micropumps having a pulsation free flow ensure that the flow rate of the liquid along the sample channel 100 may be maintained and controlled, such that the particles flow through the sample channel 100 at a substantially continuous rate. The output or inlet pressure provided by the input pump 210, regulating pump 220, and focus pump 230 may be in the range of 1 mbar to about 200 mbar, such as from about 5 mbar to about 100 mbar. Such relatively low pressures are ideal for providing a continuous flow of particles along the sample channel 100.

[0068] The sorting pump 200 is ideally a pump having a substantially higher output pressure than the input 210, regulating 220 and focus 230 pumps. In particular, the sorting pump 200 has a higher output pressure than the input pump 210. The sorting pump 200 may have an output pressure of from about 100 mbar to about 1000 mbar, such as about 200 mbar. The output pressure of the sorting pump 200 exceeds the burst / bubble pressure at the dispensing outlet 140.

[0069] The microfluidic device 1 may be manufactured from any suitable material such as glass or preferably a polymer. At least the sorting intersection 120 is ideally manufactured from an optically transparent material such that the camera 600 can image the sorting intersection 120. Suitable polymers may for example be PDMS, PC, PS, PVC, COC or PMMA. The sample channel 100, sorting and dispensing channel 130, and dispensing outlet 140 may be provided on or within a single piece of material. As is well known within the field, the device 1 may be sealed by a cover layer on at least one side such that the microfluidic channels are sealed. As described above, to enable dispensing of particles, the dispensing outlet 140 is open to atmosphere, and is therefore not sealed. The sample channel 100 and sorting and dispensing channel 130 may have a nominal width of less than about 1 mm, such as less than about 500 pm, such as about 400 pm.

[0070] The sorting pump 200, input pump 210, regulating pump 220, and focus pump 230 are each provided external to and in connection with the microfluidic device 1. The connection between each of the pumps and the respective fluidic element to which they are connected is an air and fluid-tight seal. The seal between the microfluidic device 1, and in particular the sample reservoir 105, the collection reservoir 150, the buffer reservoir 133, and the focus reservoir 111, and the respective pumps 200, 210, 220, 230 may be provided by a compliant element disposed between the device 1, or a respective reservoir 105, 150, 144, 111 and the outlet of the respective pump 200, 210, 220, 230.

[0071] The sample provided to the sample reservoir 105 comprises particles distributed in a liquid sample media. The sample media is typically a sample media suitable for cell culturing. The sample media ideally comprises a hydrogel matrix. A hydrogel matrix has been found to be ideal as the particles tend to remain evenly distributed within the hydrogel matrix during flow along the sample channel 100 and sorting at the sorting intersection 120 into the sorting and dispensing channel 130. The hydrogel matrix may be a shear-thinning hydrogel which has a lower viscosity when subjected to shear stress. The provision of a shear-thinning hydrogel enables the hydrogel to have a greater viscosity when provided to the low shearstress environment of the reservoir 105, such that the particles are maintained separate from each other. When the hydrogel flows through the higher shear-stress environment of the sample channel 100, the hydrogel has a reduced viscosity and therefore the particles can flow more readily along the sample channel 100 and within the dispensing portion 130b of the sorting and dispensing channel 130. Additionally, after dispensing to the low shear-stress environment of a well plate, the hydrogel has a greater viscosity, and the particles may be maintained in position within the wells. Such shear-thinning hydrogels may be referred to as shear-thinning and self-healing, also known as thixotropic. The hydrogel may be a gelling polysaccharide based shear-thinning and self-healing hydrogel. Other hydrogels, such as non- shear-thinning hydrogels, i.e., Newtonian hydrogels are also suitable as the liquid sample media.

[0072] The fluid provided to the sorting and dispensing channel 130, such as via the buffer reservoir 133, is ideally a sample media suitable for cell culturing. Generally, the fluid provided to the sorting and dispensing channel 130 has a lower viscosity than the sample media provided to the sample reservoir 105, and therein, provided to the sample channel 100. This reduces the pressure required to be provided by the sorting pump 200 for the relatively higher-pressure sorting flow.

[0073] To enable visual identification by the classification processor 610 in conjunction with the camera 600 the sample media, the fluid for the sorting and dispensing channel 130, and where present, the hydrogel matrix is substantially optically transparent. The hydrogel matrix is ideally biocompatible with cells, spheroids, or organoids such that the hydrogel can be used in both upstream preparation of cells etc., and downstream cultivation, growth etc. of cells etc. Although, the present invention has been described above with reference to specific embodiments, it is not intended to be limited to the specific form set forth herein. Rather, the invention is limited only by the accompanying claims.

[0074] In the claims, the term “comprises / comprising” does not exclude the presence of other elements or steps. Furthermore, although individually listed, a plurality of means, elements or method steps may be implemented by e.g. a single unit or processor. Additionally, although individual features may be included in different claims, these may possibly advantageously be combined, and the inclusion in different claims does not imply that a combination of features is not feasible and / or advantageous. In addition, singular references do not exclude a plurality. The terms “a”, “an”, “first”, “second” etc do not preclude a plurality. Reference signs in the claims are provided merely as a clarifying example and shall not be construed as limiting the scope of the claims in any way.

Claims

CLAIMS1. A system (10) for detecting, classifying, and dispensing particles, comprising:- a microfluidic device (1), the microfluidic device (1) comprising:- a sample channel (100) along which a sample comprising the particles may flow,- a sorting and dispensing channel (130) which intersects the sample channel (100) at a sorting intersection (120), wherein the sorting and dispensing channel (130) comprises a dispensing portion (130b) downstream of the sorting intersection (120), and wherein the dispensing portion (130b) comprises a dispensing outlet (140) downstream of the sorting intersection (120), the dispensing outlet (140) for the dispensing of detected, classified particles,- a sorting pump (200) connected to the sorting channel (130) of the microfluidic device (1), arranged to provide a flow of liquid to the sorting and dispensing channel (130) at a pressure exceeding a burst pressure of the dispensing outlet (140),- a classification system (6) comprising a camera (600) and a classification processor (610) for determining the visual properties of particles, wherein the camera (600) has a field of view (601) comprising the sorting intersection (120),- a sorting valve (134) for controlling the flow of fluid in the sorting and dispensing channel (130), the sorting valve (134) being controlled by the classification system (6).

2. The system (10) for detecting, classifying, and dispensing particles according to claim 1, wherein the sample channel (100) of the microfluidic device (1) has a substantially higher fluidic resistance than the sorting and dispensing channel (130).

3. The system (10) for detecting, classifying, and dispensing particles according to claim 1 or 2, wherein the particles are label-free.

4. The system (10) for detecting, classifying, and dispensing particles according to any of claims 1 to 3, wherein the classification processor (610) is configured to determine the shape, morphology and / or size of the particles.

5. The system (10) for detecting, classifying, and dispensing particles according to any of claims 1 to 4, wherein the field of view (601) of the camera (600) comprises the dispensing outlet (140).

6. The system (10) for detecting, classifying and dispensing particles according to any of claims 1 to 5, wherein the dispensing outlet (140) is arranged at the base of the microfluidic device (1) and open to atmosphere.

7. The system (10) for detecting, classifying, and dispensing particles according to any of claims 1 to 6, wherein the microfluidic device (1) is provided with a sample reservoir (105) in fluidic connection with the sample channel (100) upstream of the sorting intersection (120), and wherein the sample flows from the sample reservoir (105) to the sorting intersection (120).

8. The system (10) for detecting, classifying, and dispensing particles according to claim 7, wherein the microfluidic device (1) is provided with a collection reservoir (150) in fluidic connection with the sample channel (100), wherein the collection reservoir (150) provided downstream of the sorting intersection (120) such that sample not expelled from the dispensing outlet (140) is receivable in the collection reservoir (150).

9. The system (10) for detecting, classifying, and dispensing particles according to claim 7 and 8, wherein each of the sample reservoir (105) and the collection reservoir (150) are each provided in connection with a respective pump (210, 220), and wherein the flow of sample is reversable such that sample received in the collection reservoir (150) may subsequently be provided again to the sample channel (100) and sorting intersection (120).

10. The system (10) for detecting, classifying, and dispensing particles according to claim9, wherein the sample reservoir (105) is in fluidic connection with an input pump (210), and wherein the collection reservoir (150) is in fluidic connection with a regulating pump (220).

11. The system (10) for detecting, classifying, and dispensing particles according to claim10, wherein the pressure and / or flowrate of the regulating pump (220) is controllable based on the pressure in the sorting and dispensing channel (130), the pressure in the sorting and dispensing channel (130) being measurable via a sorting pressure sensor (221) for measuring the pressure in the sorting and dispensing channel (130).

12. The system (10) for detecting, classifying, and dispensing particles according to any of claims 1 to 11, wherein the microfluidic device (1) comprises at least one focussingchannel (110a, 110b) in fluidic connection with the sample channel (100), the at least one focussing channel (110a, 110b) for providing a flow of sheath liquid to the sample channel (100) and thereby controlling the position and flowrate of particles within the sample channel (100).

13. The system (10) for detecting, classifying, and dispensing particles according to any of claims 1 to 12, wherein the sample is a hydrogel sample media comprising the particles.

14. The system (10) for detecting, classifying, and dispensing particles according to claim 13, wherein the hydrogel is a shear-thinning and self-healing hydrogel.

15. A process for detecting, classifying, and dispensing particles, the process comprising:- introducing a sample comprising particles to be detected, classified, and dispensed to a sample channel (100) of a microfluidic device (1),- providing a camera (600) having a field of view (601) which comprises a sorting intersection (120) of the microfluidic device (1),- flowing the sample along the sample channel (100) to the sorting intersection (120) such that the particles are within a field of view (601) of a camera (600),- detecting and classifying the particles based on the morphology, shape, size of the particles via a visual classification processor (610) configured to determine visual characteristics of the particles, and if the particles are classified as being of interest,- providing a burst of liquid to a sorting and dispensing channel (130), wherein the sorting and dispensing channel (130) intersects the sample channel (100) at the sorting intersection (120), such that particles at the sorting intersection (120) enter a dispensing portion (130b) of the sorting and dispensing channel (130); and,- dispensing the particles of interest from a dispensing outlet (140) of the microfluidic device (1).

16. The process for detecting, classifying and dispensing particles according to claim 15, wherein the field of view (601) of the camera comprises the dispensing outlet (140) and wherein the process comprises:- monitoring the dispensed particles during dispensing from the dispensing outlet (140).

17. The process for detecting, classifying and dispensing particles according to claim 16, wherein the process comprises:- monitoring at least the height of a meniscus (144) at the dispensing outlet (140), and, - adjusting the flowrate and / or flow pressure of the sample within the sample channel (100).

18. The process for detecting, classifying and dispensing particles according to any of claims 15 to 17, wherein the sample channel (100) has a substantially higher fluidic resistance compared to the sorting and dispensing channel (130) such that the burst of fluid provided to the sorting and dispensing channel (130) does not enter the sample channel (100).

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