Patent Description:
A new application of cell sorting technology is the production of cell therapies. Many newer cell therapies require the sorting of large numbers of cells. For example, many new autologous T-cell therapies require sorting of relatively rare subsets of T-lymphocyte cells from peripheral blood mononuclear cells (PBMCs). Typically, large numbers of cells must be sorted in a reasonably short amount of time (e.g. <NUM><NUM> cells in around an hour), and the desired cells (which may typically compose <NUM>-<NUM>% of the total PBMCs) must be recovered with high purity, yield and viability. There is currently no sorter technology capable of fulfilling these requirements, in the form of either a research instrument or a cell therapy manufacturing instrument.

Other disadvantages of current cell sorting instruments are that they are not suitable for GMP (good manufacturing practice) production of therapeutic products, since they are not considered 'safe by design' for the operator or patient. This is because the fluid-wetted components are difficult to separate from the instrument into an enclosed single-use consumable, and they produce aerosols which may harm the operator.

The solutions described herein provide a particle sorting technology that is suitable for sorting a large number of cells, at high viability, yield and purity, in a short amount of time, in an enclosed microfluidic chip that may be integrated into a single-use consumable.

Many microfluidic particle sorting technologies have been invented in the last couple of decades, although few have reached commercial application. A common theme is that of a 'single-junction sorter', where an inlet channel bifurcates into two output channels: a 'sort' channel and a 'waste' channel. Particles entering the inlet channel are focused into the centre of the input channel, typically by a hydrodynamic focusing region, and the outputs are hydrodynamically biased towards the waste channel, such that the centre streamline flows into the waste. An actuator, which is positioned at or upstream of the bifurcation point, selectively exerts a force on a desired particle (or on the fluid around the desired particle) in order to move it away from the centre streamline and into the sort channel. Microfluidic particle sorters with various actuators have been demonstrated, for example actuators based on standing surface acoustic waves, transient surface acoustic waves, piezo-actuated displacement, micromechanical valves, optical tweezers, electrophoresis, dieletrophoresis and thermal vapour bubbles created by laser absorption or by electrical heating.

The focusing of the particles into the centre of the inlet channel is an important part of many particle sorters for two reasons: firstly it allows a greater precision of optical measurement of the particles by a focused laser beam; secondly it allows a smaller deflection of the particle by the actuator to push the particle from waste stream to sort stream. Alternatives to hydrodynamic focusing are acoustic focusing, described by <CIT> and inertial focusing, described by <CIT>.

Microfluidic particle sorters employing thermal vapour bubble actuators have been demonstrated. The bubbles are created by electrical heating. The thermal vapour bubble actuator is placed within a side channel. The effect of the side channel is to focus and amplify the fluid displacement caused by the bubble, so that a particle can be sorted by the transient displacement directly caused by the bubble itself.

However, the side channel is disadvantageous in that it complicates the microfluidic chip, requiring space on the chip and its own inlet or fill port.

In the case of fragile particles, such as biological cells, there is an upper limit on the sort rate that can be achieved with any single-junction sorter. This upper limit is of the order of the maximum shear stress that the particle can withstand without damage. For mammalian cells, this rate is around <NUM>,<NUM>-<NUM>. Therefore, no single-junction sorter of this format can achieve the specified sort rate of <NUM><NUM> cells in an hour, or <NUM>,<NUM> per second.

The attempt to parallelize particle sorters within a microfluidic chip (in order to increase the sort rate) has encountered formidable technological challenges that stem from the need to parallelize the optical instrumentation to measure the array of parallel sorters on the chip. For example, for laser-illuminated fluorescence measurement in a parallel sorter, the laser foci have to be split or parallelized, and simultaneously aligned with the array of microfluidic sorters. Then the collection optics has to be parallelized, either by scanning across the array or providing an array detector for each emission wavelength channel. To collect light from a sorter at high sensitivity requires a high-numerical-aperture objective lens. However, an array of parallel sorters on chip occupies a wider field of view than a single channel. Therefore to achieve an equivalent light collection efficiency requires a proportionally larger and more expensive objective lens, as well as larger and more expensive filters and other elements of the optical system. A one-dimensional array of sorters makes poor use of the two-dimensional field of view of an objective lens. Furthermore, to minimize the lateral dimension of an array of sorters on a microfluidic chip is challenging, since space is used by the input and output manifolds, the hydrodynamic focusing region, and the actuator, all of which must be parallelized. In a parallelized microfluidic sorter, all of the individual sorters must work simultaneously at high fidelity, otherwise the purity and yield of the sorted particle population worsen significantly.

<CIT> relates to a microfluidic switching system. <CIT> relates to a microfluidic chip having a plurality of microfluidic flow channels. <CIT> relates to a flow cell sorting system, a focus detection method, as well as a fluidic chip. <CIT> relates to compositions and methods for the collection of rare cells using an interspersed microstructure design.

It is provided a single-junction sorter for a microfluidic particle sorter, the single-junction sorter comprising: an input channel, configured to receive a fluid containing particles; an output sort channel and an output waste channel, each connected to the input channel for receiving the fluid therefrom; an optical reader arranged to optically measure the particles using a laser; a control system arranged to evaluate the optical measurement and to decide to sort or reject a particle; a thermal vapour bubble generator, operable by the control system to selectively displace the fluid around a particle to be sorted and thereby to create a transient flow of the fluid in the input channel; and a vortex element, configured to cause a vortex in the transient flow in order to direct the particle to be sorted into the output sort channel.

The vortex element causes a vortex to be created in the transient flow, which is provided by actuation of the bubble generator. The resultant vortex travels downstream with the particle to be sorted and causes a displacement (i.e. laterally of the flow axis) of the particle toward and into the output sort channel. This displacement is larger than the displacement that would be caused by the actuation of the bubble generator in the absence of the vortex element, and the vortex element therefore obviates the need for a bubble generator provided in a side channel. This advantageously allows for single-junction sorters according to the present invention to be efficiently parallelized on a chip.

As used herein, the word "particle" encompasses biological cells, solid beads, and liquid droplets of one liquid phase in a carrier fluid (such as aqueous droplets in a non-aqueous carrier fluid). Liquid droplets may themselves contain further particles.

As used herein, the word "fluid" encompasses both aqueous and non-aqueous fluids, typically in the liquid or gas phase. For the purposes of the present invention, such a fluid typically contains particles, although fluids not containing particles may also be used.

The skilled person will understand that the terms "particle" and "fluid" are not limited to the above definitions should also be interpreted according to their understood meanings in the art.

Throughout this specification, the terms "output sort channel", "sort output channel", and "sort outlet" are used interchangeably. Similarly, "output waste channel" should be read as interchangeable with "waste output channel" and "waste outlet".

The vortex element may comprise a protrusion in the input channel. The vortex element may comprise a turn in the input channel. The vortex element may comprise a recess in the input channel. The vortex element may be between the bubble generator and the output sort channel. It will be understood that the vortex element may take any shape, form or geometry which is suitable to provide a vorticial flow for directing the selected particle to the output sort channel.

The bubble generator may comprise a microheater. In this case, the fluid may be any liquid that is sufficiently volatile for the microheater to generate a bubble, such as water, an aqueous solution, or a non-aqueous carrier medium.

The single-junction sorter may be configured, in the non-operation of the bubble generator and thereby absence of the said transient flow, to direct the particles into the output waste channel.

The single-junction sorter may comprise an inertial focuser configured to centralise the particles in the fluid along a centre of the input channel. The inertial focuser may comprise a serpentine channel. The input channel may comprise the inertial focuser.

Debris may accumulate during operation of a single-junction sorter according to an embodiment of the first aspect of the invention. In order to address this issue, a single-junction sorter may comprise a valve configured to close to prevent the fluid passing through the output sort channel in order to disrupt the flow of the fluid and thereby direct accumulated debris towards the output waste channel.

As used herein, the word "valve" encompasses conventional flow control devices, such as a normally-open solenoid valve, as well as flow restrictors capable of selectively substantially stopping the flow in the sort output channel of the disclosed embodiments.

The valve could be substituted for any sort of flow restriction device, flow restrictor, closure mechanism/means, flow diverting mechanism/means or blocking mechanism/means that is capable of selectively substantially stopping the flow in the support output channel in order to direct debris into the waste channel. Furthermore, it is not necessary for the channel to be completely blocked, so long as the flow is sufficiently restricted to disrupt the flow of the fluid and direct accumulated debris towards the output waste channel.

According to another aspect there is provided a microfluidic particle sorter, comprising an array of single-junction sorters each as described herein above.

The microfluidic particle sorter may comprise an array of microlenses, each microlens being aligned with a respective one of the array of single-junction sorters.

In the microfluidic particle sorter: the input channels of the single-junction sorters may be connected to a common inlet via an inlet manifold; the output waste channels of the single-junction sorters may be connected to a common waste outlet via a waste manifold; and the output sort channels of the single-junction sorters may be connected to a common sort outlet via a sort manifold.

The microfluidic particle sorter may comprise an objective lens arrangement including one or more objective lenses. The objective lens arrangement may be configured to deliver light to and collect light from every single-junction sorter of the array of single-junction sorters for the purpose of characterizing the particles in the fluid. Thus, the light for control of sorting and particle characterization is delivered and collected through at least one objective lens, covering the whole area of the two-dimensional array of single-junction sorters, as the objective lens arrangement is configured to illuminate the whole area of the two-dimensional array.

According to an aspect of the invention there is provided a method of sorting particles using a single-junction sorter as described herein above, the method comprising: providing the input channel with a flow of fluid containing particles; operating the optical reader and the laser to optically measure the particles; operating the control system to evaluate the optical measurement and to decide to sort or reject a particle; and operating the thermal vapour bubble generator by the control system in order to selectively displace the fluid around a particle to be sorted, thereby to create a transient flow of the fluid in the input channel which encounters the vortex element, so as to cause a vortex in the transient flow in order to direct the particle to be sorted into the output sort channel.

Embodiments will now be described, by way of example, with reference to the accompanying figures in which:.

The sorter is embodied by a microfluidic chip pictured in <FIG>, having an inlet port <NUM>, and two outlet ports: a waste outlet <NUM> and a sort outlet <NUM>. Downstream of the inlet port is first the inlet manifold <NUM>, followed by the inertial focusing region <NUM>, then the two-dimensional array of single-junction sorters <NUM>, then the waste and sort manifolds (<NUM> and <NUM>, overlaid in the diagram), which connect to the waste and sort outlets. On the edge of the chip is an electrical connector <NUM>.

The chip construction is detailed in <FIG>, and consists of several layers of different functions. The first layer <NUM> is a glass sheet of thickness <NUM>, which seals the chip on the lower face and provides the substrate for the deposition of thin film features that make the thermal vapour bubble actuator and electrical contacts (described below). The second layer <NUM> consists of a micromoulded sheet of polydimethylsilicone (PDMS) with a thickness of <NUM>, and contains a set of microchannels described below. The third layer <NUM> consists of a micromoulded layer of cyclic olefin copolymer (COC) of thickness <NUM>, and contains microchannels on one side with a depth of <NUM>, and through-layer vias with a length of <NUM>. The fourth layer <NUM> consists of micromoulded COC of thickness <NUM>, and seals the chip on the upper face. The layers are bonded together using organosilane surface functionalisation, plasma treatment, thermal fusion and alignment/bonding equipment known in the art.

<FIG> shows the design of the first layer <NUM>. The thermal vapour bubble actuators comprise a two-dimensional four-by-four square array of thin film metal resistors <NUM>, each henceforth referred to as a microheater. Each microheater is connected by conduction tracks <NUM> to a contact pad <NUM>, and on the other side to a common ground pad <NUM>.

<FIG> shows the design of the second layer <NUM>. The input manifold <NUM> splits into an array of <NUM> channels of width <NUM> and pitch <NUM>. Each of these channels is the input channel <NUM> of a single-junction sorter. The input channel comprises an inertial focusing section which consists of a symmetric serpentine channel <NUM>. The input channel then connects to a sorter junction <NUM>. The sorter junctions are placed on a two-dimensional four-by-four square array, which has a pitch of <NUM> in both directions, so that each sorter junction aligns with a microheater in the first layer (<NUM>, <FIG>). (The exact alignment is detailed below. ) At the sorter junction, the input channel splits into a waste channel of width <NUM> and a sort channel of width <NUM>. The waste channel <NUM> continues along the chip, where it is part of an array of <NUM> parallel waste channels that join the waste manifold <NUM>. The sort channel <NUM> reaches an end point in the second layer, where it continues through a via into the third layer described below.

<FIG> shows the design of the third layer <NUM>, and presents a perspective view (<FIG>) as well as a drawing of the upper face (<FIG>) and lower face (<FIG>). The third layer provides the continuation of the sort channels. The sort channel end points in the second layer are aligned with a two-dimensional four-by-four square array of through-layer via holes <NUM> each of diameter <NUM>. The sort channels continue from the lower face through these vias to an array of <NUM> parallel microchannels <NUM> on the upper face, each of width <NUM>. The sort channels then join the sort manifold <NUM>. The third layer also provides a via for the input port <NUM> and a via for the waste output <NUM> that are aligned respectively with the end points of the input and waste manifolds in the second layer.

<FIG> shows the design of the fourth layer <NUM>, and presents a drawing of the upper face (<FIG>) and a perspective view (<FIG>). The fourth layer seals the microchannels of the third layer. It also provides the input port <NUM>, the waste output port <NUM>, and the sort output port <NUM>. These ports consist of through-layer vias, which are aligned with vias <NUM> and <NUM> and the end point of the sort manifold <NUM> respectively.

Details of the thermal bubble actuators are presented in <FIG>, showing a drawing of the plan view (<FIG>) and a schematic of the cross sectional profile (<FIG>). The microheater <NUM> comprises a connector section <NUM>, which overlaps with the conduction tracks <NUM>, and a square section <NUM> of dimension <NUM> x <NUM>, which is the active part of the microheater since it does not overlap with any other conductor. The thermal bubble actuator comprises several layers fabricated by thin film deposition techniques on top of the glass substrate <NUM>. These are in order: a passivation layer <NUM> of thickness <NUM> composed of silicon nitride, a resistor <NUM> of thickness <NUM> composed of titanium, a second passivation layer <NUM> of thickness <NUM> composed of silicon nitride, and an anti-cavitation layer <NUM> of thickness <NUM> composed of tantalum. The conduction tracks are composed of <NUM> of nickel-chromium and <NUM> of gold.

The thermal bubble actuators are connected via their conduction tracks to the electrical connector <NUM>: the design of this is shown in <FIG>.

The details of the individual single-junction sorter are shown in <FIG> and <FIG>. <FIG> shows the sorter junction <NUM>, where the input channel <NUM> divides into the sort channel <NUM> and the waste channel <NUM>. A recess <NUM> of width <NUM> in the side of the input channel is aligned with the active part of the microheater <NUM>, and positioned <NUM> upstream of the junction. <FIG> shows the inertial focuser <NUM>, which consists of a symmetric serpentine channel. Each inertial focuser consists of <NUM> circular arcs of alternating direction, each of radius <NUM> (at the channel centre line) and arc angle <NUM>°.

A second example comprising only one single-junction sorter is shown in <FIG>. The inlet manifold <NUM> is connected to the inlet channel <NUM>, which contains the serpentine inertial focusing section <NUM>. The inlet channel then reaches the sort junction <NUM> and splits into the sort channel <NUM> and waste channel <NUM>. These continue to join with the sort manifold <NUM> and waste manifold <NUM> respectively.

A third alternative example of the single-junction sorter is shown in <FIG>. It comprises a main inlet channel <NUM> (dimensions: <NUM> width, <NUM> height), with a microheater <NUM> (dimensions: <NUM> length, <NUM> width) situated in a recess <NUM> off the left wall (viewed looking downwards onto the chip, as in the figure). After the recess, the channel makes a <NUM>° right turn <NUM>, continues for <NUM>, then makes a <NUM>° left turn <NUM>. Immediately following the left turn, there is a second triangular recess on the left wall <NUM> (with dimensions: <NUM> length, <NUM> width), such that an acute angle edge <NUM> is formed between the left turn and second recess. Following the second recess, the channel contains a pinched region <NUM> (<NUM> width), before reaching an opened region <NUM> (<NUM> width), then splitting into two symmetric <NUM> width channels. The left channel is the sort channel <NUM> and the right channel is the waste channel <NUM>.

The operation of the particle sorter is as follows.

The input particle suspension, which may be, for example, an aqueous suspension of lymphocytes of typical diameter <NUM>, at a density of up to around <NUM>×<NUM><NUM> cells/mL, is supplied to the input port <NUM> at a rate of approximately <NUM>/min. The input manifold <NUM> splits the suspension evenly into the <NUM> input channels <NUM>.

The inertial focuser <NUM> causes the particles to align accurately in the centre of the input channel. It is designed to provide flow conditions as follows for a centre streamline flow velocity that is preferably between <NUM>/s and <NUM>/s, more preferably <NUM>/s. For lymphocytes in aqueous suspension, the Dean number of this flow is approximately <NUM>, the channel Reynolds number is around <NUM>, and the particle Reynolds number is in the range <NUM> - <NUM>. We have found experimentally that representative particles in such an inertial focuser spontaneously focus into the centre of the channel. Further embodiments may employ any kind of particle focuser as an alternative to the inertial focuser <NUM>. Several kinds of particle focuser are known in the art that are able to accurately align particles with a streamline in a microfluidic channel, for example sheath flow or hydrodynamic focussing, acoustic focussing and dielectrophoretic focussing.

The particle is measured optically by a laser which is focused at <NUM> just upstream of the microheater <NUM>. The optical measurements typically include fluorescence, forward scattering and back scattering of light, and the optical reader apparatus for their measurement is described below. The preferred embodiment has a single laser focus per single-junction sorter. However in alternative embodiments, separate laser foci may be provided in close proximity upstream of the microheater, e.g. at <NUM> and <NUM>. A control system evaluates the optical measurements in real time and decides on whether to sort or reject each individual particle before it reaches the microheater.

If the decision is to reject the particle, then it carries on in its streamline, which passes into the waste channel <NUM>. However, if the decision is to sort the particle, then the thermal vapour bubble actuator is activated, causing the particle to pass into the sort channel <NUM>. The actuation operates as follows: an electrical pulse of voltage <NUM> V and duration <NUM> is applied between the contact pad <NUM> and ground pad <NUM>, so that an electrical current flows and dissipates a controlled amount of energy at the microheater. The liquid in the channel adjacent to the microheater is rapidly heated and goes through a phase transition from liquid to gas, forming a microscopic vapour bubble that expands and collapses in around <NUM>. Thus the microheater actuates a transient displacement of the liquid around the particle.

This displacement increases dues to the fluid's own inertia as the displaced fluid moves downstream, so that when the particle reaches the sorter junction <NUM>, its lateral displacement is around <NUM>, which is sufficiently large to carry the particle into the sort channel instead of the waste channel.

The waste manifold <NUM> and sort manifold <NUM> collect the outputs of the <NUM> single-junction sorters, and carry them to the waste and sort output ports <NUM> and <NUM>. The optical reader apparatus for measurement of fluorescence, forward scatter and back scatter measurements, is detailed in <FIG>. A laser source of light <NUM> is used for fluorescence excitation. The beam passes through a two-dimensional four-by-four microlens array <NUM> which forms a two-dimensional pattern of dots which is then imaged onto the chip by lenses <NUM> and <NUM>. Polarizing beam splitter <NUM> transmits only one polarization of the light which then reflects from the long-pass dichroic mirror <NUM>. Lens <NUM> focuses the light simultaneously onto the four-by-four array of single-junction sorters of the microfluidic chip <NUM>. (Each focus is made at position <NUM> on the individual single-junction sorter, as described above. ) Back-scattered light that is reflected back from the microfluidic chip is collected by the lens <NUM>, after which it is reflected by the mirror <NUM>; then it enters the beam splitter <NUM>. The polarization orthogonal to the illuminating beam is then reflected and cleaned by polarizer <NUM>, then focused by lens <NUM> and detected as a set of individual spots by a two-dimensional four-by-four array photodetector <NUM>.

While fluorescence detection may be collected according to alternative examples in epi- and through modes, epifluorescence detection is provided in this embodiment. The lens <NUM> collects light from both back-scatter and fluorescence. The long-pass dichroic mirror <NUM> transmits fluorescence light (which has a longer wavelength than the illumination light). This light then passes through a series of fluorescence detection modules <NUM>. Each module is designed to detect wavelengths within a specified band, and transmit longer wavelengths to the next module. Each fluorescence detection module <NUM> has a long-pass dichroic mirror <NUM>, band-pass optical filter <NUM>, focusing lens <NUM> and a two-dimensional four-by-four array of photodetectors <NUM>. Several different spectral ranges can be detected simultaneously by stacking modules with the correct choice of long-pass and band-pass filters, as is known in the art.

Forward-scattered light from the microfluidic chip is collected and collimated by lens <NUM>, then reflected by long-pass dichroic mirror <NUM>, after which it is filtered by polarizer <NUM> to eliminate the directly transmitted light. The forward-scattered light then passes through dark field mask <NUM> which blocks directly transmitted light and selects a band of angles for forward scatter detection. The forward-scattered light is then focused by lens <NUM> and detected with a two-dimensional four-by-four array of photodetectors <NUM>.

In addition to the back scatter, forward scatter and fluorescence measurements, imaging of the microfluidic chip is provided, to allow for focusing and alignment of the illumination source onto the chip. The transmission imaging uses a second collimated light source <NUM> which has a wavelength longer than those measured by the fluorescence detection modules. This light propagates through all the dichroic mirrors <NUM>, lens <NUM>, the microfluidic chip <NUM>, lens <NUM>, and dichroic mirror <NUM>. There is then an additional band-pass filter <NUM> to remove stray light, then lens <NUM> focuses the light onto the camera <NUM>. The light source <NUM> provides constant illumination or short pulses triggered from particle detection events to allow monitoring and control over the sorting procedure.

In a further example, the microfluidic chip is integrated with a two-dimensional microlens array attached to the glass substrate side opposite to the microchannels. Each lens is aligned with the laser focus point <NUM> on each a single-junction sorter. The microlenses serve to increase the efficiency of fluorescence collection from each single-junction sorter.

The sorter's control system is detailed in <FIG> and described as follows. The control system has a signal processing block <NUM> per single-junction sorter, thus making sixteen processing blocks for the four-by-four parallel microfluidic particle sorter. Each signal processing block has six analogue inputs for the four fluorescence channels, forward scatter and back scatter signals. A multi-channel analogue-to-digital converter (ADC) digitizes the signals and transmits them across a digital interface to a field programmable gate array (FPGA). The FPGA performs a peak detection and characterization algorithm, and makes a decision whether to activate the thermal bubble actuator for that channel to divert a cell. Peak detection and characterization algorithms are known in the field of cytometry and not further described here. The actuation signal is passed to a block of parallel drive transistors <NUM>.

In each signal processing block, an external memory is interfaced with a soft processor in the FPGA, and allows data from the peak characterization to be stored until they are required, such as at the end of a run to collect the cells' peak data for analysis. When these data are required, the control processor requests and uploads the data from each block sequentially. Additionally the control processor is used to send data to the FPGA such as thresholds and parameters for the peak-detection algorithm, parameters of the sorting pulse and commands to control the sorting process.

The effect of the thermal bubble actuation is amplified by the geometry of the single-junction sorter, which is shown by fluid flow simulations depicted in <FIG>. Here we use the geometry of the third embodiment of the single-junction sorter above to demonstrate the fluid flow (refer to <FIG> for the geometric features). Due to the substantial inertia of the fluid (channel Reynolds number of around <NUM>), the right turn <NUM> and left turn <NUM> cause a greater flow into the waste channel <NUM> than the sort channel <NUM>, so that in the absence of a thermal bubble actuation, particles that approach the junction on the centre streamline will leave through the waste channel. However, when the thermal vapour bubble is actuated, the growth and collapse of the bubble rapidly displaces the fluid, first away from the microheater and then towards the microheater. As the bubble grows, the transient flow causes a vortex to form near the wall of the second recess <NUM>. At this point in time, the vortex is away from the main flow and has little interaction with the particles. However, when the bubble collapses, the transient flow causes a 'sorting vortex' <NUM> to form around the acute angle edge <NUM>, which subsequently moves downsteam with the main flow. Because the sorting vortex moves downstream with the particle to be sorted, it causes a much larger lateral displacement of a particle than the direct displacement of the particle caused by the thermal vapour bubble alone. Trajectories of a sorted particle <NUM> and an unsorted particle <NUM> are shown.

Many alternative examples also create such a sorting vortex, for example where a recess, bend or edge is placed in the single-junction sorter upstream of the sorting junction.

Further alternative examples of the single-junction sorter are shown in <FIG>, to allow for multi-way sorting. The embodiments shown in <FIG> provide for <NUM>- and <NUM>-way sorting, respectively Each of these embodiments comprises a main inlet channel <NUM>, a microheater <NUM> situated in a first recess <NUM> off the left wall (viewed looking downwards onto the chip, as in the figure). After the first recess <NUM>, there is a second recess <NUM> on the left wall, such that an acute angle edge <NUM> is formed between the two recesses <NUM> and <NUM>. Following the second recess <NUM>, the channel contains a straight region <NUM>, before splitting into several symmetric channels <NUM> and <NUM>. The central channel <NUM> is the waste channel, and either side are separate sort channels <NUM>. Any number of sort outputs could be provided in alternative embodiments. In these alternative embodiments, the waste channel <NUM> will typically be provided such that the fluid is substantially directed towards this waste channel <NUM> during equilibrium flow of the fluid.

In operation, a single thermal bubble actuation is employed to displace a particle into any one of the multi-way sort outputs by the following method. The sorting vortex <NUM> is characterised by a flow profile that varies in position with respect to the flow path: a particle ahead of the centre of the vortex is displaced towards the left, while a particle behind the centre of the vortex is displaced towards the right. The total displacement of a particle depends on the distance from the vortex. Thus, by careful timing of the actuation with respect to the particle position, the total displacement is calibrated to match the positions of the respective output channels. The control system is then programmed to give the actuation pulse at a set of time delays that correspond to each of the multi-way sort outputs.

In operation, for many types of particle suspensions, there is a tendency for debris to accumulate at the sort junction and clog or block the sorter. According to a further embodiment, a valve, such as a normally-open solenoid valve, is placed at (or downstream of) the sort outlet. This valve is capable of stopping the flow in the sort output channel. The technique to unclog the junction is to temporarily close this valve, which causes the flow to change around the sort junction, thus sweeping any debris into the waste channel. Typically the valve is closed for between <NUM> and <NUM> seconds, more typically for around <NUM> second, to have the unclogging effect. The valve can be actuated periodically or whenever debris is detected on the junction by using the camera.

In the case of the multi-way sorting examples (<FIG>), the unclogging mechanism comprises a separate valve on each of the sort outputs. In operation, one or more sort outputs are temporarily closed to have the unclogging effect.

A valve could also be provided on the waste outlet, in addition to or as an alternative to the valve provided on the sort outlet, such that debris is directed towards one or more of the sort outputs when this valve is closed.

Herein it is described a microfluidic particle sorter that is capable of sorting fragile particles (such as biological cells, beads, or droplets containing further particles) at a much higher sort rate than was hitherto possible. The invention achieves a high sort rate by providing a single-junction sorter that is suitable to be parallelized on a microfluidic chip. The single-junction sorters may be arranged on the chip in a two-dimensional array, which allows an efficient use of the field of view of the objective lens. This two-dimensional array is enabled by the design of the single-junction sorter, which allows a dense packing on the chip. Each single-junction sorter provides a bubble generator (e.g. a thermal vapour bubble generator) without a side channel, and a bifurcation of the stream into sort and waste channels. The geometry of the single-junction sorter is chosen so that the actuation of the thermal vapour bubble creates a 'sorting vortex', which travels downstream with the particle to be sorted, and thus causes a much larger lateral displacement of a particle than the direct displacement of the particle caused by a thermal vapour bubble alone.

Claim 1:
A single-junction sorter for a microfluidic particle sorter, the single-junction sorter comprising:
an input channel (<NUM>), configured to receive a fluid containing particles; and
an output sort channel (<NUM>) and an output waste channel (<NUM>), each connected to the input channel (<NUM>) for receiving the fluid therefrom;
and characterized by:
an optical reader arranged to optically measure the particles using a laser;
a control system arranged to evaluate the optical measurement and to decide to sort or reject a particle;
a thermal vapour bubble generator, operable by the control system to selectively displace the fluid around a particle to be sorted and thereby to create a transient flow of the fluid in the input channel (<NUM>); and a vortex element (<NUM>, <NUM>), configured to cause a vortex in the transient flow in order to direct the particle to be sorted into the output sort channel (<NUM>).