APPARATUS AND METHOD FOR ISOLATING SINGLE PARTICLES FROM A PARTICLE SUSPENSION

Abstract

A particle isolation apparatus 100 for isolating particles from a suspension sample, includes a droplet dispenser device 10 for collecting the suspension sample from a carrier substrate 20 and for dispensing droplets onto a target substrate 30, a mechanical pump device 40 being coupled with the droplet dispenser device 10 for loading a dilution liquid into the droplet dispenser device 10 and for aspirating a first portion of the suspension sample into the droplet dispenser device 10, and a syphon pump device 50 being coupled with the droplet dispenser device 10 and being arranged for aspirating a second portion of the suspension sample into the droplet dispenser device 10. Preferably, the droplet dispenser device 10 is configured for dispensing single particle droplets on the target substrate 30. Furthermore, a method of isolating particles from a suspension sample is described.

Claims

1. A particle isolation apparatus, configured for isolating particles from a suspension sample, comprising: a droplet dispenser device configured for collecting the suspension sample from a carrier substrate and for dispensing droplets onto a target substrate; a mechanical pump device coupled with the droplet dispenser device for loading a dilution liquid into the droplet dispenser device and for aspirating a first portion of the suspension sample into the droplet dispenser device; and a syphon pump device being coupled with the droplet dispenser device and being arranged for aspirating a second portion of the suspension sample into the droplet dispenser device.

2. The particle isolation apparatus according to claim 1, wherein the mechanical pump device comprises a syringe pump coupled with the droplet dispenser device.

3. The particle isolation apparatus according to claim 1, wherein the syphon pump device comprises a system liquid reservoir arranged at a level lower than the carrier substrate and coupled with the droplet dispenser device.

4. The particle isolation apparatus according to claim 1, further comprising a valve device configured for controlling liquid connections of at least one of the mechanical pump device and the syphon pump device with the droplet dispenser device.

5. The particle isolation apparatus according to claim 4, wherein the valve device comprises a three-way valve configured for providing a liquid connection between the mechanical pump device and the droplet dispenser device or between the syphon pump device and the droplet dispenser device.

6. The particle isolation apparatus according to claim 1, wherein the carrier substrate has a hydrophobic surface.

7. The particle isolation apparatus according to claim 1, wherein the droplet dispenser device is configured for dispensing single particle droplets on the target substrate.

8. The particle isolation apparatus according to claim 1, further comprising a recovery vessel being arranged for re-capturing droplets including multiple particles.

9. The particle isolation apparatus according to claim 1, wherein the syphon pump device and the carrier substrate are configured for collecting the suspension sample in a residue-free manner.

10. A method of isolating particles from a suspension sample, comprising the steps of: providing the suspension sample on a carrier substrate; loading a dilution liquid to a droplet dispenser device; loading the suspension sample in the droplet dispenser device, so that the suspension sample is diluted in the dilution liquid, including a first aspiration phase of aspirating a first portion of the suspension sample into the droplet dispenser device by action of a mechanical pump device and a second aspiration phase of aspirating a second portion of the suspension sample into the droplet dispenser device by action of a syphon pump device, and dispensing droplets with the droplet dispenser device on a target substrate, wherein the droplets include less than one particle per droplet in a temporal average.

11. The method according to claim 10, comprising the further step of loading a further quantity of the dilution liquid in the droplet dispenser device after loading the suspension sample.

12. The method according to claim 10, wherein the particles comprise at least one of biological cells, cell aggregates, cell components, biological macromolecules and micro-particles functionalized with biomolecules.

13. The method according to claim 10, wherein the dilution liquid is a physiological buffer solution.

14. The method according to claim 10, further comprising at least one of the features: the mechanical pump device comprises a syringe pump being coupled with the droplet dispenser device; the syphon pump device comprises a system liquid reservoir arranged at a level lower than the carrier substrate and coupled with the droplet dispenser device; liquid connections of at least one of the mechanical pump device and the syphon pump device with the droplet dispenser device are controlled with a valve device, and dispensed droplets including multiple particles are re-captured with a recovery vessel.

15. The method according to claim 14, wherein the liquid connections of at least one of the mechanical pump device and the syphon pump device with the droplet dispenser device are controlled with a three-way valve.

16. The method according to claim 10, wherein the suspension sample is collected from the carrier substrate in a residue-free manner.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Further advantages and details of the invention are described in the following with reference to the attached drawings, which show in:

[0028] FIG. 1: a schematic illustration of a preferred embodiment of the particle isolation apparatus according to the invention;

[0029] FIG. 2: a flow chart illustrating features of preferred embodiments of a method of particle isolation according to the invention;

[0030] FIGS. 3 to 7: schematic illustrations of the particle isolation apparatus according to FIG. 1 in different operational phases of the particle isolation according to the invention; and

[0031] FIG. 8: a schematic illustration of recapturing multiple particle droplets.

DESCRIPTION OF PREFERRED EMBODIMENTS

[0032] Features of preferred embodiments of the invention are described in the following with exemplary reference to a particle isolation apparatus comprising a droplet dispenser device with one single piezoelectric droplet dispenser. The invention is not restricted to this embodiment. An array of droplet dispensers can be used, having advantages for implementing a parallel operation with multiple suspension samples. Furthermore, other types of droplet dispensers can be used instead of the piezoelectric droplet dispenser. Furthermore, the implementation of the invention is not restricted to the particular configuration of the pump and valve devices in the figures. In particular, the syringe pump can be replaced by another mechanical pump device, and the bottle-shaped system liquid reservoir of the syphon pump device can be replaced by another reservoir type.

[0033] The embodiments of the invention are described with exemplary reference to the processing of biological particles, in particular biological cells. The invention is not restricted to this application. Non-biological particles can be processed with the inventive technique as well, e.g. for collecting particles from a sample in nano-techniques or environmental techniques, i.e. for analyzing purposes.

[0034] The invention is substantially described with reference to the dilution of the suspension sample in the droplet dispenser. The subsequent single particle droplet dispensing operation of the droplet dispenser device is not described as it is known per se. US 2017/0274689 A1 and EP No. 17189875.2 (not published on the priority date of the present specification) are introduced into the present specification by reference, in particular with regard to the control of the droplet dispenser device for obtaining dispensing of droplets including single particles.

[0035] According to FIG. 1, an embodiment of the inventive particle isolation apparatus 100 comprises a droplet dispenser device 10, a mechanical pump device 40, a syphon pump device 50 and a valve device 60. The droplet dispenser device 10 is i.e. a dispenser system of the type sciFLEXARRAYER (manufacturer: Scienion AG, Germany). The mechanical pump device 40 comprises a syringe pump 41 with a pump actuator 42. The syringe pump 41 has a working volume of e. g. some ml. Advantageously, the components 40, 50 and 60 can be integrated into an operation platform of the droplet dispenser device 10.

[0036] The droplet dispenser device 10 comprises a dispenser head 11, which is a mechanical support for holding at least one droplet dispenser 12 with vertical orientation. The dispenser head 11 is connected with a driving device (not shown), being capable of moving the dispenser head 11 in all three spatial directions. The droplet dispenser 12 comprises a first (lower) end with the droplet dispenser tip 13 (nozzle tip 13) and an opposite (upper) end with the upper droplet dispenser junction 14. Between both ends, a dispenser reservoir with a volume of about 60 l and a piezoelectric actuator unit for dispensing droplets with the droplet dispenser 12 are provided. The upper droplet dispenser junction 14 is connected via a system liquid line 15 with the first port of a three-way valve 61 of the valve device 60. The system liquid line 15 is e.g. a flexible tubing.

[0037] The droplet dispenser device 10 is provided with a dispenser control device 16, being configured for controlling the operation of the droplet dispenser 12. Furthermore, the dispenser control device 16 is coupled with the pump actuator 42 for controlling the operation of the syringe pump and with a valve actuator 62 for switching the liquid connections of the three-way valve 61.

[0038] With a second port of the tree-way valve 61, the syringe pump 41 of the mechanical pump device 40 is connected. As an alternative to the direct connection (as shown in FIG. 1), a coupling via a syringe pump line (not shown) can be provided.

[0039] With the third port of the three-way valve 61, the syphon pump device 50 is connected. The syphon pump device 50 comprises a system liquid reservoir 51 connected via the syphon line 52 with the third port of the three-way valve 61. The system liquid reservoir 51 includes a system liquid 53, e. g. water, into which the syphon line 52 is immersed. The system liquid reservoir 51 is a bottle, which is arranged such that the system liquid 53 is positioned below the carrier substrate 20. Accordingly, with a continuous liquid connection between a sample droplet on the carrier substrate 20 (see below) via the droplet dispenser 12, the system liquid line 15, the three-way valve 61 and the syphon line 52 to the system liquid 53, a negative pressure is applied to the sample droplet. The height H of the system liquid 53 below the surface of the carrier substrate 20 is selected in a range of e.g. 2 cm to 10 cm.

[0040] Furthermore, FIG. 1 shows the carrier substrate 20 with a hydrophobic substrate surface 21, a buffer tube 22 and a target substrate 30. The carrier and target substrates 20, 30 are shown as plane substrate plates (provided e.g. by sample pick up slides). Alternatively, well structures, e.g. a microtiter plate or a nanotiter plate, can be used as a substrate. Both of the carrier and target substrates 20, 30 can be provided by a common substrate.

[0041] In the following, the inventive method of isolating particles from a sample suspension is described with reference to the flow chart of FIG. 2, wherein some of the operational phases of the flow chart are shown in the remaining FIGS. 3 to 8.

[0042] According to FIG. 2, the illustrated embodiment of the inventive method firstly includes preparing steps S1 to S3. Firstly, the droplet dispenser 10 is moved to the buffer tube 22 (see FIG. 1), so that the droplet dispenser tip 13 (nozzle tip) is introduced into the buffer liquid within the buffer tube 22 (step S1).

[0043] Subsequently, a dilution liquid 3 (sample buffer) is loaded from the buffer tube 22 into the droplet dispenser 12 as shown in FIG. 3 (step S2). For loading the sample buffer, the three-way valve 61 is set such that the syringe pump 41 is connected via the system liquid line 15 with the droplet dispenser 12. By the action of the syringe pump 41, the sample buffer is aspirated as the dilution liquid 3 into the droplet dispenser 12. With a practical example, 20 l of the sample buffer solution are aspirated. Advantageously, this assures that the cells accommodated from the sample suspension do not experience an osmotic shock. Furthermore, the buffer tube 22 can be used for aspirating further sample buffer to the droplet dispenser 12 after collecting the sample (formation of a buffer zone, see below). The buffer solution comprises e. g. a solvent that stabilizes the cells or particle types or a chemically similar liquid, preferably, phosphate buffered saline and/or fetal bovine serum (PBS and FBS).

[0044] Subsequently, a droplet of a suspension sample 2 is loaded to the carrier substrate 20 and the dispenser 12 is adjusted above the droplet of the sample suspension 2 as shown in the enlarged section of FIG. 4 (step S3). The droplet of the suspension sample 2 on the carrier substrate 20 may include a plurality of particles 1, like biological cells, e. g. 100 cells or more, which are to be isolated from each other. Alternatively, the droplet may include only a few cells to be isolated from each other, or even only one cell to be recovered and transferred to the target substrate. For example, the droplet of the suspension sample 2 comprises e.g. 1 l to 10 l of a cell suspension containing rare cells with a density of about 1 to 30 cells per l. Loading the droplet of the suspension sample onto the carrier substrate 20 can be conducted with another droplet dispenser (not shown) provided at the dispenser head 11, or with another liquid droplet handling technique, like i.e. a pipette or an opening of a microfluidics channel.

[0045] Subsequently, the droplet dispenser 12 is moved towards the carrier substrate 20 such that the nozzle tip 13 is introduced into the droplet of the suspension sample 2 as shown in FIG. 5 (step S4). According to FIG. 5 (in particular the enlarged section of FIG. 5), the nozzle tip 13 is arranged with a distance from the substrate surface 21 of the carrier substrate 20, wherein the distance is in a range from 100 m to 250 m. The nozzle tip 13 contacts the droplet of the suspension sample 2. Depending on the size of the droplet, the nozzle tip 13 can be submerged into the droplet down to the distance D of 100 m.

[0046] Subsequently, the first aspiration phase is conducted, wherein a first portion 4 (see arrow) is collected via the nozzle tip 13 into the droplet dispenser 12 by the action of the syringe pump 41. The syringe pump 41 is controlled such as a predetermined volume of e.g. 2 l is aspirated as the first portion 4 into the droplet dispenser (step S5).

[0047] Subsequently, the operation of the syringe pump 41 is stopped, and the three-way valve 61 is switched such that the first port is connected with the third port of the three-way valve 61. Accordingly, a continuous liquid connection between the syphon pump device 50 and the droplet dispenser 12 is provided (see FIG. 6). In this situation, a second portion 5 of the droplet of the suspension sample 2 is collected in a second aspiration phase by the effect of the syphon pump device 50 (step S6). Step S6 includes passive aspiration by gravity syphon to pickup remaining liquid, e. g. 2 l. This may take 10 sec to 2 min, depending on syphon height and remaining volume. Advantageously, due to high surface tension of the system liquid and a small orifice size at the nozzle tip 13, capillary action keeps nozzle tip 13 from aspirating air. During this second aspiration phase, the complete or nearly the complete droplet of the suspension sample 2 is collected into the droplet dispenser 12.

[0048] FIG. 7 shows the situation when the sample aspiration is complete. The carrier substrate 20 is empty, and the droplet of the suspension sample is completely accommodated by the droplet dispenser 12. The complete aspiration of the sample is supported by the hydrophobic surface 21 of the carrier substrate 20. Furthermore, it is preferred that the sample has a high surface tension, which is fulfilled by most of the available buffer solutions.

[0049] Subsequently, the droplet dispenser 12 is moved to the buffer tube 22 (see FIG. 1), in order to aspirate another quantity of buffer liquid through the nozzle tip 13 for creating a buffer zone within the lower end of the droplet dispenser 12 (step S8). Provision of the buffer zone in step S8 has the advantage that the suspension sample within the droplet dispenser 12 is not negatively affected by a subsequent washing step S9. The buffer liquid is aspirated into the droplet dispenser 12 by the action of the syphon pump device 50. Formation of the buffer zone may have a duration of 1 to 20 seconds.

[0050] With a next step (step S9), the droplet dispenser is moved to a wash station (not shown) for cleaning an outside surface of the nozzle 13. Subsequently, the dispenser head 11 is moved to a camera device 70 to begin with the single cell dispensing operation (step S10). With the camera device 70 and the calculation unit of the dispenser control device 16 (see FIG. 1), a distribution of the cells within the droplet dispenser 12 is evaluated. When a single cell condition of the droplet dispenser 12 is detected, e.g. as described in US 2017/0274689 A1 and EP No. 17189875.2 (not published on the priority date of the present specification) or in the references cited therein, single cell droplets are deposited on the target substrate 30 (see FIG. 1) or alternatively, if multiple cell droplets are detected, they are recaptured by a recovery tube 23 as shown in FIG. 8 (step S11).

[0051] With more details, after the sample is loaded into the droplet dispenser 12, cell detection is performed at the camera device 70. When the droplet dispenser 12 is positioned at the camera device 70, all droplets ejected from this position are captured in the recovery tube 23 located below the nozzle 13. If a single cell condition is detected, e.g. if there is one single cell in an ejection zone (corresponding to a volume ejected with one dispenser operation) and no cell is located in a sedimentation zone above the ejection zone, the droplet dispenser 12 is moved to the target substrate 30 for dispensing the next droplet including the single cell onto the target substrate 30. The sedimentation zone corresponds to a volume from which cells can sediment into the ejection zone during the movement of the dispenser device to the target substrate.

[0052] Subsequently, cell analyses can be conducted with the single cells on the target substrate 30. The cell analyses can include any cell investigation known per se, like e. g. microscopic imaging, optical, in particular fluorescence measurements, mechanical measurements, chemical sensing, etc. Depending on the result of the cell analysis, the cells can be sorted or subjected to further cell processing, like cell cultivation.

[0053] With the inventive particle isolation apparatus 100 and the above method, practical experiments have demonstrated the advantageous capability of recovery of rare cells with a recovery rate in a range from 95% to 99% or above. Initial experiments have been conducted by the inventors using fluorescently marked PBMC samples (including about 70 cells in 5 l suspension liquid, as a model for cells of interest in autoimmune diseases. By spreading 5 l droplets onto a substrate surface and counting the number of particles, the density of about 70 particles per 5 l was confirmed by employing a fluorescence microscope. By applying the inventive single particle isolation, the single particles were deposited as a straight line or a matrix arrangement of particles confirming the single particle isolation capability and high recovery rate of the inventive device. An average single particle recovery of 97% was obtained.

[0054] The features of the invention disclosed in the above description, the drawings and the claims can be of significance both individually as well as in combination or sub-combination for the realization of the invention in its various embodiments.