Cell analysis and sorting

Abstract

A system (100) and associated method for analysing and / or sorting biological material. The system (100) comprises: an electrospray apparatus configured to receive a fluid sample and generate a stream of droplets (116), the fluid sample comprising biological material suspended in a fluid; a biological identification apparatus configured to classify each of the droplets in the stream (116) based on the biological material contained within each droplet; a plurality of collection receptacles (140) configured to receive the stream of droplets (116); a selection apparatus (130) configured to vary which collection receptacle of the plurality of collection receptacles (140) receives the stream of droplets (116); and a controller (126) configured to operate the selection apparatus (130) such that each droplet of the stream (116) is received by one of the collection receptacles (140) based on the classification determined by the biological identification apparatus.

Description

[0001] CELL ANALYSIS AND SORTING

[0002] FIELD OF THE INVENTION

[0003] The present invention relates to systems and methods for cell analysis and sorting, particularly fluorescent-activated cell sorting (FACS).

[0004] BACKGROUND

[0005] Analysis of biological materials and sorting of these into different aliquots is an important aspect of many scientific and industrial processes . Medical and pharmaceutical techniques may require different biological materials within a heterogenous sample (such as samples of animal / plant cells or cell clusters) to be analysed individually and to be separated from one another. Once the aliquots are separated from one another, further analysis or research can be performed on / with the separated samples.

[0006] Flow cytometry is a process in which a flow or stream of cells (or other particles or biological material) is analysed. A property of each of the cells is determined as it flows through a cytometer, thereby allowing the cells to be classified and sorted based on the determined property. One form of flow cytometry is fluorescent-activated cell sorting (FACS). In FACS, a fluorescent dye is applied to the cells, the dye being configured to couple to specific sites on cells of interest. An optical detector can be used to capture the fluorescence of these ‘biomarkers’ in the flow. Based on the optical signal that is captured by the detector, each of the cells in the flow can be classified and then sorted accordingly.

[0007] However, such systems require a fluid to generate, direct and contain the flow through the cytometer. A jet of air may be used to spray a stream of atomised droplets. In many systems, an additional sheath flow may need to be provided in order to encapsulate the stream of droplets. These air-dispersed systems may not be suitable for all applications. In particular, the components of the system associated with providing a suitable and reliable flow (for example, pumps, compressors, etc.) may greatly increase the size, cost and complexity of systems and methods for cell sorting.

[0008] Therefore, further developments to cell analysis / sorting systems and methods are desirable in order to overcome deficiencies associated with the current state of the art. SUMMARY

[0009] According to a first aspect of the invention, there is provided a system for analysing and / or sorting biological material. The system comprises: an electrospray apparatus configured to receive a fluid sample and generate a stream of droplets, the fluid sample comprising biological material suspended in a fluid; a biological identification apparatus configured to classify each of the droplets in the stream based on the biological material contained within each droplet; a plurality of collection receptacles configured to receive the stream of droplets; a selection apparatus configured to vary which collection receptacle of the plurality of collection receptacles receives the stream of droplets; and a controller configured to operate the selection apparatus such that each droplet of the stream is received by one of the collection receptacles based on the classification determined by the biological identification apparatus .

[0010] Using the electrospray apparatus to generate a stream or ‘jet’ of droplets for sorting may provide numerous advantages over systems of the prior art that rely on air-dispersed droplets, particularly systems that rely upon a sheath flow.

[0011] The fluid suspending the biological material may comprise or consist of a biocompatible fluid. For example, the suspending fluid may comprise water, consist of water or be substantially water-based. The suspending fluid may comprise phosphate buffer saline, glycerol, polyethylene oxide and / or polyethylene glycol, for example. Fluids that have a relatively high conductivity may be avoided, due to their interaction with the electrospray apparatus.

[0012] The electrospray apparatus may enable the system to be more compact, as components required to provide a jet of air (e.g., compressors, sheath fluid inlets and outlets, conduits, etc.) may be eliminated. Furthermore, the collection receptacles may be positioned closer to an outlet of the electrospray apparatus compared to systems that use a jet of air, thereby allowing the ‘length’ of the system to be reduced. Whereas a jet of air may disrupt the collection receptacles (e.g., causing splashing in the receptacles), the electrospray stream may cause reduced or no disruption.

[0013] The use of the electrospray apparatus may also result in a system that is more reliable, easier to maintain, and / or cheaper, as the stream of droplets is produced via electrostatic forces rather than relying upon moving parts (e.g., vibrators, compressors or pumps). The electrospray apparatus may comprise an electrically charged needle, the electrically charged needle imparting a charge to the fluid sample such that a fluid sample cone is formed at an exit orifice of the needle. The needle may comprise a conductive material, such that the fluid sample may be charged by the needle. The needle may be made substantially from a conducting material (e.g., the needle may be made from an electrically conductive metal). The needle may be made substantially from a nonconducting material. A conductive, charged probe may be provided within the needle for charging the fluid sample. Portions of the needle (for example, the exit orifice of the needle and / or the portion of the needle at the tip) may be coated with a conductive material for charging the fluid sample.

[0014] The needle may be a tubular elongate member made of a conductive material. A voltage may be applied to the needle so that the needle is electrically charged. Alternatively, the voltage may be applied directly to the fluid sample itself, for example at an inlet to the needle. The voltage may be supplied by any suitable means, such as a high voltage power supply. The fluid sample may be supplied to a first end of the needle. For example, an input conduit may be fluidly coupled to the first end of the needle. The fluid sample may flow through the needle to a second end (the exit orifice).

[0015] Above a threshold voltage, a Taylor cone of the fluid sample may form at the exit orifice. A cone -jet may form at the tip of the Taylor cone. The cone -jet may break up under surface tension / the electric field, thereby generating a stream of fine droplets.

[0016] The exit orifice of the needle may have a diameter of between 10 pm and 5000 pm. The diameter of the exit orifice may be greater than 150 pm or greater than 250 pm or greater than 350 pm. The diameter of the exit office may be between 70 pm and 2000 pm, or between 150 pm and 3000 pm, or between 150 pm and 1000 pm, or between 250 pm and 750 pm or between 400 pm and 600 pm. The diameter of the exit orifice required may be dependent upon the properties of the suspending fluid and the biological material. The diameter may be at least 5 times, 10 times or 20 times a characteristic size of the biological material (e.g. a diameter of a cell type to be analysed / sorted) .

[0017] Use of an electrospray needle may allow for a wider range of biological materials to be processed by the sorting system. A diameter of approximately 500 pm, for example (e.g., between 150 pm and 1000 pm, or between 250 pm and 750 pm or between 400 pm and 600 pm), will allow a wide range of biological material to pass through - it is large enough to allow for relatively large multicellular clusters to flow - while still enabling droplets to be produced containing single cells . The generation of the jet / droplets may be controlled by the voltage that is applied to the needle, meaning that the same electrospray apparatus can be used for very small cells (e.g., sperm cells) and relatively large cell clusters (e.g., embryos) without having to change physical components of the system. By contrast, air dispersed methods typically require the exit orifice of the needle (or an orifice of a separate jetting nozzle) to be varied in orde r to produce droplets of different sizes and to accommodate different sized cells. The exit orifice used for a sperm cell may not be physically large enough to be used with embryos, for example.

[0018] Using an electrospray needle with an exit office of at least 150 pm or at least 250 pm or at least 350 pm may reduce ‘sorter induced cell stress’ on the sample. A diameter of approximately 500 pm may reduce the amount of shear stress applied to the sample / cells at the exit orifice. Using electrospraying apparatus with parameters like those discussed above may enable single cell droplets to be produced with a thin film of liquid, while reducing damage to the sample / cells.

[0019] The electrospray apparatus may further comprise a grounding ring situated proximate to the exit orifice of the needle.

[0020] The grounding ring may be situated at a distance of 10 to 200 mm from the exit orifice of the needle and configured to generate an electric field that applies an accelerating force to the stream of droplets. The distance may be dependent upon the suspending fluid used and may be adjusted accordingly in order to achieve a desired jet. The potential difference between the electrospray needle (which imparts a charge to the fluid sample) and the grounding ring, may result in a high velocity stream or jet of droplets. The grounding may comprise or be incorporated into the selection apparatus. Where the selection apparatus comprises charged deflection plates, the plates may act as a grounding ring in addition to deflecting the stream of droplets.

[0021] A flow rate of the fluid sample from the needle and / or the distance between the needle and the grounding ring / selection apparatus may be configured to achieve sufficient deflection and produce desirable droplet sizes (discussed further below). A relatively low flow rate may reduce the length of the jet before it breaks up into a stream of droplets, and vice versa. The grounding ring / selection apparatus may be positioned to account for this parameter. For example, the grounding ring and / or selection apparatus may be situated at the point where the jet breaks down into droplets, or at least 10 mm or at least 50 mm or at least 100 mm from said point.

[0022] The electrospray apparatus may be configured to produce droplets with a diameter of less than 50 pm, or less than 20 pm, or less than 10 pm.

[0023] The electrospray apparatus may be configured to produce smaller droplets than air-dispersed methods. The electrospray apparatus (parameters of which are discussed below), may allow droplets with a diameter of less than 1 pm. The droplets may have a diameter of 50 nm, 100 nm, or 200 nm, for example. By contrast, air-dispersed methods may only be capable of producing droplets with a diameter of a few micrometres (for example, approximately >10pm), as they are inherently limited by physical aspects of the apparatus. The smaller droplets enabled by the electrospray apparatus may enable more accurate, more reliable, or faster identification / classification of the biological material contained therein, particularly for FACS apparatuses. The smaller droplet sizes may mean that light scattered from the biological material is more easily transmitted to the detector due to the reduced amount of fluid through which the light must travel.

[0024] The size of droplets produced (or the average size of the droplets produced) may be at least as large as a characteristic size of the biological material in the sample. Electrospraying may enable small droplets (e.g., droplets with a diameter of less than 10 pm or less than 5 pm) to be produced using relatively large bore needles (e.g., having an exit orifice of 500 pm or more). This may enable the advantages associated with small droplets to be realised (e.g., thinner layers of fluid surrounding each cell) without causing excessive shear stress to be applied to the cells.

[0025] The system may be configured to cause the stream of droplets to comprise one or more drops with an individual cell encapsulated by a relatively thin liquid layer.

[0026] In certain embodiments, the stream of droplets may comprise one or more drops with an individual cell encapsulated by a relatively thin liquid layer. For example, the liquid layer encapsulating the cell may be less than 0.5 pm, or less than 1 pm, or less than 5 pm, or less than 10 pm.

[0027] More generally, the median diameter of the stream of droplets may be less than 20 pm larger (or 1 pm, or 2 pm, or 10 pm larger) than a median cell diameter of the sample.

[0028] The parameters of the system and the electrospraying apparatus (properties of the suspending fluid, flow rate, needle orifice, needle voltage, etc.) may be configured to produce droplets that enable such a thin fluid layer. As discussed above, the electrospraying apparatus may produce a Taylor cone and associated jet. The jet of fluid may have a diameter approximately equal to the diameter of cells in the fluid sample. The cells within the cone of material may flow gently into the jet to form a ‘string’ of cells. Upon breakdown of the jet into a stream of droplets, at least some of the droplets may thus contain only a single cell encapsulated in a thin layer of liquid.

[0029] The droplets produced by electrospraying may enable a significant proportion (for example, at least 60%, at least 70%, at least 80% or at least 90%) of the droplets produced to contain just a single cell / piece of biological material. For example, at least 60% of the droplets may encapsulate a single cell, approximately 30% of droplets may encapsulate two or more cells, and approximately 10% of droplets may contain no cells.

[0030] The system may be configured to produce a stream of droplets at a rate of at least 300,000 per second.

[0031] The electrospray apparatus may also be configured to provide a greater droplet production rate than air-dispersed methods. The electrospray apparatus may allow for droplet production rates of at least 300,000, or 400,000 or 500,000 per second. By contrast, air-dispersed methods are typically limited to approximately 200,000 droplets per second, again due to physical limitations of the apparatus. Unlike air spraying, where droplets originate from the exit orifice of the needle, droplets originate from a jet at the tip of a Taylor cone in electrospraying. The diameter of the jet (and thus the droplets produced) may be much smaller than the exit orifice of the needle, which may enable a greater droplet production rate. The voltage applied to the needle may be more straightforward to control than analogous droplet-production parameters in air spraying (e.g., air flow and air pressure drop at the exit orifice). The electrospray apparatus may therefore provide for shorter sample processing times.

[0032] The system may comprise a voltage source configured to apply a voltage of at least 4 kV to the needle.

[0033] The voltage source may be configured to apply a voltage of between 1 kV and 30 kV to the needle.

[0034] A voltage of at least 4 kV may be applied to the needle (e.g., approximately 6 kV). The voltage may be in the range of 1 to 25 kV or 4 kV to 8 kV. The voltage may be varied in order to control a flow rate of the stream of droplets. A voltage of 6 kV may result in a flow rate of at least 1 x IO-10m3s-1(e.g., approximately 1 x 10’9m3s-1), which may be a desirable flow rate in order to achieve a suitably fast processing time while still being detectable by the biological identification apparatus. The flow rate may be varied between approximately 1 x 1015and 1 x 10’5m3s-1.

[0035] The biological identification apparatus may comprise a fluorescent-activated cell sorting (FACS) apparatus. FACS is a widely used flow cytometry technique that may be used to identify different biological materials within a flow or a stream of a sample. The biological identification apparatus may be a commercial, off-the-shelf, FACS or analyser.

[0036] The FACS apparatus may comprise a light source configured to generate a probe beam, and a detector. The stream of droplets may pass through the probe beam, thereby generating scattered light that is received by the detector. The scattered light comprises inelastically scattered light (including resulting from the Raman effect and by fluorescence) and elastically scattered light (Rayleigh / Mie scattering).

[0037] The light source may be a laser and the probe beam may be a laser beam , for example. The wavelength of light used may be dependent on the fluorescent dye and / or the FACS system used. The wavelength may correspond to a commercially available FACS system and associated off-the-shelf dye(s), e.g., 405 nm (violet), 488 nm (blue), or 561 nm (yellow). Multiple lasers may be provided so that laser beams of different wavelengths may be used to interrogate the fluid sample. This may allow for analysis of a wider range of biological material. Other optical elements (such as mirrors, lenses or collimators) may be provided in order to direct and widen the probe beam so that the stream of droplets passes through the probe beam. As the probe beam encounters droplets, light may be scattered from the biological material contained therein, thereby generating scattered light. The scattered light may therefore contain information regarding properties of the biological material in the droplet being analysed.

[0038] The detector may be any suitable detector configured to receive the scattered light. The detector may, for example, comprise a CMOS or a CCD sensor. The detector may comprise a spectrogram configured generate a fluorescence spectrum based upon the scattered light. The spectrogram may comprise one or more filters or beam splitters such that different wavelengths may be separated and analysed. Different biological materials may fluoresce at different frequencies, therefore allowing the biological material(s) with the fluid sample to be identified based off the detected scattered light.

[0039] The detector may be positioned in line with the probe beam (i.e., directly in front of the light source), such that the scattered light corresponds to forward scatter light from the stream of droplets. Alternatively, the detector may be off-axis of the probe beam (for example, perpendicular to the probe beam), such that the scattered light corresponds to side scatter or back scatter light from the stream of droplets.

[0040] The FACS apparatus may comprise a processor configured to receive a signal from the detector and classify the biological material within the droplet being analysed. The processor may be configured to compare the received signal to a lookup table of known values for biological materials of interest.

[0041] The fluid sample may comprise a fluorescent dye. The biological material may be dyed prior to being put into the suspending fluid (i.e., the cells may be dyed but the fluid may be substantially free of dye). Classifying the droplets may comprise detecting a biomarker of the biological material contained within each droplet. In FACS, one or more fluorescent dyes (for example, a fluorescent antibody) may be applied to the sample, the dye being configured to bind to specific sites on biological materials of interest. Biological materials that bind to these dyes may fluoresce, thereby providing a ‘biomarker’ (i.e., a label which signifies the nature of the biological material) for that biological material. Droplets containing said biological material can therefore be identified and classified appropriately based off of the perceived fluorescence. Multiple dyes / antibodies may be provided, thereby providing labelling of multiple biological materials. Multiple biological materials within a heterogeneous sample may be identified and sorted.

[0042] Although the biological identification apparatus has been described as a FACS apparatus, alternative identification methods (including other flow cytometry methods) be used. For example, biological materials may be labelled by using radioactive isotopes, thereby providing other suitable biomarkers.

[0043] The selection apparatus may comprise electrostatically charged deflection plates configured to selectively deflect the stream of droplets via an electric field. A parallel pair of charged plates may be provided, each connected to a power source, thus generating a substantially linear electric field therebetween. Using an electric field to deflect the stream of droplets may be advantageous as no moving parts are required . This may allow for faster deflection variation, as the deflection is controllable solely by varying a voltage applied to the plates . A charging ring may also be provided, the charging ring being situated upstream of the deflecting plates . The stream of droplets may pass through the charging ring, thereby becoming charged and thus capable of being deflected by the electric field. Alternatively, the stream of droplets may already be sufficiently charged from the electrospray apparatus such that they can be deflected by the electric field.

[0044] The stream of droplets enters a gap between the deflection plates at a distance of between 10 and 200 mm, or between 10 mm and 100 mm, from the exit orifice. The deflection plates may be between 5 and 100 mm apart, or between 10 mm and 50 mm apart. The pair of deflection plates may be arranged as an inverted V shape. The gap between the plates at the apex (the end nearest the needle) may be between 5 mm and 10 mm. The gap between the plates at the base (the end furthest from the needle) may be between 50 mm and 100 mm). The deflection plates may be provided with a potential difference of between 1 and 30 kV. The potential difference provided will depend upon the desired deflection, as well as the geometry of the plates and the properties of the fluid sample. The selection apparatus may comprise an air nozzle configured to selectively deflect the stream of droplets by providing a stream of air. The air nozzle may be situated near the stream of droplets and configured to provide short, focussed ‘puffs’ of air that deflect the droplets. The nozzle may be configured to provide the puff of air perpendicularly to the stream of droplets. The nozzle may be configured to provide the puff when the droplet is inline with the nozzle.

[0045] The stream of droplets may enter a volume affected by the air nozzle at a distance of between 10 and 200 mm, or between 10 mm and 100 mm, from the exit orifice.

[0046] The selection apparatus may comprise a translation stage configured to selectively move the plurality of collection receptacles relative to the stream of droplets. The translation stage may be driven by a motor, stepper motor or an electromagnetic actuator, for example. The translation stage may be moved so that different collection receptacles are positioned under the stream of droplets.

[0047] Use of an air nozzle or translation stage may be advantageous as these selection methods do not rely on electrostatic forces that may be impacted by the electrospray apparatus.

[0048] In all of the selection apparatuses discussed above, the deflection may occur in only one axis (i.e., in a direction perpendicular to the stream of droplets) or in multiple directions (i.e., in a plane that is orthogonal to the stream of droplets). For example, the electric field potential or the velocity of the air stream may be varied to provide differing amounts of deflection in the x axis. Alternatively, the selection apparatus may comprise two perpendicular pairs of charged plates or a plurality of air nozzles to provide deflection in the x axis and the y axis. The collection receptacles may be arranged accordingly, such that different aliquots of the sample are deflected into different receptacles.

[0049] The system may further comprise a mixing device configured to disperse the biological material in the suspending fluid such that the fluid sample is substantially homogenous. The mixing device may comprise a magnetic stirrer, for example . The magnetic stirrer may be operated at a speed of 35 revs / min. The magnetic stirrer may be operated at speeds ranging from approximately 12 to 125 revs / min. Mixing the fluid sample so that it is substantial homogenous may allow for consistent detection / classification of the biological material, as each of the droplets in the stream may be substantially similar in composition.

[0050] The fluid sample may comprise a biological material concentration of between 1 x 105to 5 x 107parts per ml The concentration of biological material (e.g., cells) in the suspending fluid may be approximately 1 x 107to 2 x 107cells / ml. The concentration may be in the range of approximately I x l05to 5 x l07cells / ml. The magnetic stirrer may be operated and / or the concentration may be configured so that a suitable homogenous fluid sample is obtained. This may allow a stream of droplets to be generated wherein each droplet (or at least a substantial proportion of the droplets, for example, 60%, 70%, 80% or 90%) contain only one unit of biological material, e.g., one cell. This may allow for more effective sorting, as only one cell is to be classified in each droplet.

[0051] The biological material may comprise one or more cells or cell clusters. A cell may be taken to mean any individual biological cell. The cells may comprise, for example, sperm or egg cells from animals, other types of animal cells, or plant cells. The cells may comprise single-celled organisms, such as bacteria, archaea or protozoa. ‘Cell clusters’ may be taken to mean any multicellular biological structure. The cell clusters may comprise, for example, fertilised embryos, blastocysts or multicellular microbiological organisms.

[0052] The cell sorting system may be used to sort a sample of cells or cell clusters, the cells / cell clusters having different properties (e.g., different biological properties that give rise to different biomarkers). In some examples the cells may be of the same type, but have different properties. For example, the biological material may comprise a sample of sperm cells. The sperm cells may be separated by the sorting system into sperm cells having an X chromosome (female) or Y chromosome (male).

[0053] The biological material may comprise a plurality of different cell types and / or clusters of cell types. The biological material may comprise a sample containing multiple different types of cells (e.g., being a heterogenous sample). The sorting system may then be used to separate the cells / clusters into different aliquots. This may be used to separate multiple cell types of interest or to obtain a homogeneous sample of a single cell type (with all other cells / clusters being discarded). It will be appreciated that the specific parameters (needle voltage, exit orifice size, sample concentration, stirring rate, etc.) discussed above are only an example configuration. Given the number of different variables in the system (including the biological material that is being sorted), different combinations of the parameters that have been discussed may be used, though certain combinations may be preferable. The operation of the system may be optimised by the user based on these exemplary parameters.

[0054] According to a second aspect of the present invention, there is provided a method of analysing and / or sorting biological material. The method comprises: generating a stream of droplets from a fluid sample using an electrospray apparatus, the fluid sample comprising biological material suspended in a fluid; classifying each of the droplets in the stream based on biological material contained within each of the droplets; and sorting each of the droplets into a plurality of collection receptacles based on the determined classification of the droplet.

[0055] The method may be implemented using a system according to the first aspect of the present invention.

[0056] Classifying each of the droplets in the stream may comprise directing the stream through a fluorescent-activated cell sorting (FACS) apparatus. Classifying each of the droplets in the stream may comprise: directing the stream through an optical probe beam, thereby generating a scattered light; and receiving the scattered light at a detector. A controller (which may be any suitable processor or computer) may be provided for automatically classifying each of the droplets based on the scattered light received at the detector. The controller may be electronically coupled to a selection apparatus such that the stream of particles can be directed into one of the collection receptacles based on the classification. In this way, different aliquots (which may correspond to different cell types or cells with different properties), may be automatically collected in each of the collection receptacles. The method may further comprise applying a fluorescent dye to the fluid sample, wherein classifying the droplets comprises detecting a fluorescent biomarker of the biological material contained within each droplet.

[0057] The method may further comprise mixing the sample fluid such that the biological material is dispersed substantially homogenously throughout the suspending fluid. Sorting each of the droplets may comprise applying an electric field to the stream of droplets, the electric field being transverse to the direction of the stream such that the stream of droplets is selectively deflected.

[0058] Sorting each of the droplets may comprise applying a jet of air to the stream of droplets, the jet of air being transverse to the direction of the stream such that the stream of droplets is selectively deflected.

[0059] Sorting each of the droplets may comprise selectively moving the plurality of collection receptacles transversely to the direction of the stream, such that the collection receptacle in the path of the stream of droplets is varied.

[0060] DETAILED DESCRIPTION

[0061] Embodiments of the invention will be described, purely by way of example, with reference to the accompanying drawings, in which:

[0062] Figure 1 shows a schematic diagram of a system for sorting biological material;

[0063] Figure 2 shows a schematic diagram of an electrospray apparatus;

[0064] Figure 3 shows a schematic diagram of an electrostatic-based selection apparatus;

[0065] Figure 4 shows a schematic diagram of an air-based selection apparatus;

[0066] Figure 5 shows a schematic diagram of a translation -based selection apparatus; and

[0067] Figure 6 shows high speed images of a stream of droplets subjected to various deflection forces.

[0068] Like features of the various figures shall be denoted by the same reference numerals.

[0069] Referring to Figure 1, a system 100 for sorting biological material is shown. The system comprises an electrospray apparatus. The electrospray apparatus comprises a needle 110 electrically coupled to a power supply 112 such that a voltage is applied to the needle 110. A fluid sample containing biological material (for example, a suspension of cells in a suspending liquid) may be provided to the needle 110. An input conduit may be fluidly coupled to an end of the needle 110. The needle 110 may have a generally tubular body (e.g. with a circular shape in cross section) . The power supply 112 is configured such that a sufficiently high voltage is applied the needle 110, so that electrospraying can be performed. A cone 114 of the fluid sample forms at an exit orifice 111 of the needle 110, thereby producing a jet of fluid that results in a stream of droplets 116a (i.e., the electrospray apparatus produces a Taylor cone).

[0070] The stream of droplets 116a is propelled forwards by the electric field of the electrospray apparatus to an interrogation region 121 of a biological identification apparatus. FACS may be used for identification / classification of the biological material in the stream of droplets 116a, as shown in Figure 1. The interrogation region 121 comprises the region or plane in which a probe beam 124a passes through the stream of droplets 116a.

[0071] The biological identification apparatus comprises a light source 120 configured to generate the probe beam 124a. The light source 120 may be a laser, or a plurality of lasers. The probe beam 124a interacts with a droplet in the stream 116a, thereby generating scattered light 124b. The scattered light beam may be at a non-zero angle to the probe beam - e.g., at 90 degrees. In the case of FACS, the scattered light 124b comprises fluorescence produced by a fluorescent markers that may be present in at least some of the cells, thereby indicating the presence of a particular biomarker of the biological material within that droplet. The biological identification apparatus further comprises a detector 122 configured to receive the scattered light 124b. The apparatus 100 may comprise a one or more filter configured to filter particular wavelengths of interest, for example to enhance a fluorescence signal .

[0072] After passing through the interrogation region 121, the stream of droplets 116a continue to a selection apparatus 130. The selection apparatus shall be described in further detail below. In the system 100 of Figure 1, the selection apparatus 130 is configured to deflect the stream of droplets 116a, thereby producing a deflected stream 116b.

[0073] The system 100 further comprises a controller 126. The controller 126 is configured to receive a detection signal from the detector 122. The detection signal comprises information regarding the biological material in each of the droplets that pass through the interrogation region 121. The detection signal could correspond to an intensity or a spectrum of the scattered light 124b received at the detector 122. Based on the detection signal, the controller 126 may classify each of the droplets according to the detected biological material in each droplet. For example, a fluorescence at a particular wavelength may indicate a biomarker associated with a particular cell type. Therefore, droplets which produce such fluorescence (as received by the detector 122) may be accordingly classified as containing that cell type.

[0074] The selection apparatus 130 is coupled to the controller 126. The controller 126 provides a control signal to the selection apparatus 130 that varies the amount and / or direction of the deflection that is applied to the deflected stream 116b by the selection apparatus 130. The system 100 further comprises a plurality of collection receptacles 140. The deflected stream 116b can be deflected so that droplets are collected by the collection receptacles 140. The controller 126 and selection apparatus 130 may be configured to operate so that each droplet of the stream can be individually deflected into a desired one of the collection receptacles 140. In doing so, the biological material in the fluid sample can be automatically sorted into different aliquots.

[0075] Referring to Figure 2, a sample input apparatus for the cell sorting system 100 is shown. The system 100 comprises an electrospray needle 110 that is configured to produce a Taylor cone of sample material 114 at the exit orifice 111 of the needle 110. The electrospray apparatus may also comprise a grounding ring 113. The grounding ring 113 may generate an electric field potential between the needle 110 and the grounding ring 113. The liquid is charged by the high electrical voltage applied to the needle 110. This electric field accelerates charged droplets breaking free at the tip of the Taylor cone 114 towards the plane of the grounding ring . Thus, the grounding ring 113 may assist in generating the stream of droplets 116a or directing the stream to the interrogation region.

[0076] The system 100 further comprises a sample reservoir 150. The sample reservoir 150 may comprise any suitable receptacle for holding a volume of sample fluid 151. The sample reservoir may be provided with a mixing apparatus 152 for mixing the fluid sample 151. This may ensure that the biological material, the suspending fluid, and any identification means (for example, dyes) are mixed before being passed to the electrospray apparatus. The mixing apparatus 152 may comprise a magnetic stirrer.

[0077] An input conduit 154 is fluidly coupled to the sample reservoir 150 and the electrospray needle 110. The fluid sample may be pumped from the sample reservoir 150 to the needle 110 so that it can be electrosprayed. A mechanical pump may be provided for pumping the sample fluid through the input conduit 154. The sample reservoir 150 may comprise an air inlet 156. Pressurised air may be injected into the inlet 156 of the sample reservoir 150, thereby forcing the sample fluid 151 through the input conduit 154 via pneumatic pressure. The rate at which the sample fluid is provided to the needle (which may be dependent on the pressure provided) may be the primary determinant of fluid flow through the needle. The pressure may be adjusted based upon the biological material, the viscosity of the fluid, and a desired droplet production rate, for example. The air pressure applied may be between 0.1 kPa and 50 kPa, or between 1 kPa and 25 kPa, or between 1 kPa and 10 kPa.

[0078] Referring to Figure 3, an example of a selection apparatus 130 is shown that uses an electric field E to selectively deflect droplets from a stream of droplets 116a. The selection apparatus 130 may comprise a charging ring 131 configured to impart an electric charge to the stream of droplets 116a. Alternatively, the stream of droplets 116a may already be charged by the electrospray apparatus.

[0079] The stream of droplets 116a then passes between a pair of electrically charged plates 132. A potential difference is set up between the plates 132 so that an electric field E is formed. The electric field may be substantially linear between the plates 132. The electric field provides an electrostatic force to the stream of droplets 116a, resulting in the stream being deflected. The potential difference / electric field between the plates can be controlled so that the deflected stream 116b is directed towards one of a plurality of collection receptacles 140a-c. Each of the droplets may be directed to a specific collection receptacle based on a determined characteristic of the biological material within that droplet. For example, when used to sort sperm cells, droplets containing an X chromosome sperm cell may be deflected to a receptacle 140a, droplets containing a Y chromosome sperm cell may be deflected to another receptacle 140c, while all other droplets may be collected in a waste receptacle 140b. Referring to Figure 4, an example of a selection apparatus 130 is shown that uses a stream of air to selectively deflect a stream of droplets 116a. The selection apparatus comprises an air nozzle 134 (for example a nozzle, jet, pump, etc.) that is configured to provide a stream of air 135 to the stream of droplets 116a. The stream of air 135 may consist of a short blast of air to each droplet in the stream, thereby deflecting the droplets. The velocity, duration and / or direction of the blast may be controlled so that the deflected stream 116b is directed towards one of a plurality of collection receptacles 140a-c. Further air nozzles 134 may be provided to deflect the stream in multiple directions, including deflection in two axes.

[0080] Referring to Figure 5, an example of a selection apparatus is shown that uses a translation stage 136. Unlike the selection apparatuses of Figures 3 and 4, where the stream of droplets 116a is deflected, the translation stage 136 moves the collection receptacles 140a-c relative to the undeflected stream of droplets 116a. Nonetheless, the stream of droplets 116a is still directed into one of the collection receptacles 140a-c based on control of the translation stage 136. The translation stage 136 may comprise any suitable means for moving the receptacles and may allow for translation in two axes.

[0081] Referring to Figure 6, high speed images show different levels of deflection as a stream of droplets 116a passes through a pair of charged plates. The direction of the deflected stream 116b can be controlled by varying the electric field generated between the plates. Frame a) shows a high negative deflection; frame b) shows a low negative deflection; frame c) shows no deflection (i.e., no voltage is applied to the plates) ; frame d) shows a low positive deflection; and frame e) shows a high positive deflection. The five configurations shown in Figure 6 could be used to defect the droplets into five different collection receptacles, thereby allowing five aliquots be collected. Shown in frame c) in the region 117 is the point at which the jet of fluid propelled from the Taylor cone breaks up into discrete droplets.

[0082] Although specific examples have been described, the skilled person will appreciate that variations are possible, within the scope of the invention, which should be determined with reference to the accompanying claims.

Claims

CLAIMS1. A system for sorting biological material, the system comprising: an electrospray apparatus configured to receive a fluid sample and generate a stream of droplets, the fluid sample comprising biological material suspended in a fluid; a biological identification apparatus configured to classify each of the droplets in the stream based on the biological material contained within each droplet; a plurality of collection receptacles configured to receive the stream of droplets; a selection apparatus configured to vary which collection receptacle of the plurality of collection receptacles receives the stream of droplets; and a controller configured to operate the selection apparatus such that each droplet of the stream is received by one of the collection receptacles based on the classification determined by the biological identification apparatus .

2. The system of claim 1, wherein the electrospray apparatus comprises an electrically charged needle, the electrically charged needle imparting a charge to the fluid sample such that a fluid sample cone is formed at an exit orifice of the needle.

3. The system of claim 1, wherein the exit orifice of the needle has a diameter of between 2 mm and 4 mm.

4. The system of claims 2 or claim 3, wherein the electrospray apparatus further comprises a grounding ring situated proximate to the exit orifice of the needle.

5. The system of claim 4, wherein the grounding ring is situated at a distance of 10 to 200 mm from the exit orifice of the needle and configured to generate an electric field that applies an accelerating force to the stream of droplets.

6. The system of any preceding claim, wherein the electrospray apparatus is configured to produce droplets with a diameter of less than 10 pm.

7. The system of any preceding claim, wherein the system is configured to cause the stream of droplets to comprise one or more drops with an individual cell encapsulated by a relatively thin liquid layer.

8. The system of any preceding claim, configured to produce a stream of droplets at a rate of at least 300,000 per second.

9. The system of any preceding claim, comprising a voltage source configured to apply a voltage of at least 4 kV to the needle.

10. The system of claim 8, wherein the voltage source is configured to apply a voltage of between 4 kV and 8 kV to the needle.

11. The system of any preceding claim, wherein the biological identification apparatus comprises a fluorescent-activated cell sorting (FACS) apparatus.

12. The system of claim 12, wherein the FACS apparatus comprises: a light source configured to generate a probe beam; and a detector, wherein the stream of droplets passes through the probe beam, thereby generating a scattered light that is received by the detector.

13. The system of any preceding claim, wherein the selection apparatus comprises deflection plates configured to selectively deflect the stream of droplets via an electric field.

14. The system of claim 13, wherein the stream of droplets enters a gap between the deflection plates at a distance of between 10 and 200 mm from the exit orifice.

15. The system of any of claims 1 to 12, wherein the selection apparatus comprises an air nozzle configured to selectively deflect the stream of droplets by providing a stream of air.

16. The system of claim 15, wherein the stream of droplets enters a volume affected by the air nozzle at a distance of between 10 and 200 mm from the exit orifice.

17. The system of any of claims 1 to 12, wherein the selection apparatus comprises a translation stage configured to selectively move the plurality of collection receptacles relative to the stream of droplets.

18. The system of any preceding claim, further comprising a mixing device configured to disperse the biological material in the suspending fluid such that the fluid sample is substantially homogenous.

19. The system of any preceding claim, wherein the fluid sample comprises a biological material concentration of between 1 x 105to 5 x 107parts per ml.

20. A method of sorting biological material, the method comprising: generating a stream of droplets from a fluid sample using an electrospray apparatus, the fluid sample comprising biological material suspended in a fluid; classifying each of the droplets in the stream based on biological material contained within each of the droplets; and sorting each of the droplets into a plurality of collection receptacles based on the determined classification of the droplet.

21. The method of claim 20, wherein classifying each of the droplets in the stream comprises directing the stream through a fluorescent-activated cell sorting (FACS) apparatus.

22. The method of any of claims 21 to 22, further comprising mixing the sample fluid such that the biological material is dispersed substantially homogenously throughout the suspending fluid.

23. The method of any of claims 20 to 23, wherein sorting each of the droplets comprises selectively applying an electric field to the stream of droplets, the electric field being transverse to the direction of the stream such that the stream of droplets is selectively deflected.

24. The method of any of claims 20 to 23, wherein sorting each of the droplets comprises selectively applying a jet of air to the stream of droplets, the jet of air being transverse to the direction of the stream such that the stream of droplets is selectively deflected.

25. The method of any of claims 20 to 23, wherein sorting each of the droplets comprises selectively moving the plurality of collection receptacles transversely to thedirection of the stream, such that the collection receptacle in the path of the stream of droplets is varied.

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