阅读说明：本技术 微流体路由中的流控制 (Flow control in microfluidic routing ) 是由 刘诚迅 于 2018-12-20 设计创作，主要内容包括：一种用于检测、分选、提纯和/或表征液体样本中的感兴趣对象的方法(1)。该方法包括在微流体路由系统(100)的准备模块(120)中准备(3)液体样本以供处理,其中所述准备包括将所述液体样运送通过微流体通道,以及将所准备的样本从所述准备模块(120)的出口转发(4)到路由模块(105)的入口中。这一转发包括将微流体流耦合在所述出口与所述入口之间,以在所述出口处被动地缓冲对抗和/或主动地补偿所准备的样本的流率变化,以及在所述路由模块中将感兴趣对象从微流体流中转出(5)。转发所述样本包括在所述准备和/或路由模块和/或它们之间的流连接中感测(6)所述样本的流特性,并将所感测到的流特性纳入考虑通过闭环流控制来控制(7)流控制元件以补偿流率变化。(A method (1) for detecting, sorting, purifying and/or characterizing an object of interest in a liquid sample. The method comprises preparing (3) a liquid sample for processing in a preparation module (120) of a microfluidic routing system (100), wherein the preparing comprises transporting the liquid sample through a microfluidic channel, and forwarding (4) the prepared sample from an outlet of the preparation module (120) into an inlet of a routing module (105). This forwarding includes coupling the microfluidic flow between the outlet and the inlet to passively buffer against and/or actively compensate for flow rate variations of the prepared sample at the outlet, and diverting (5) the object of interest from the microfluidic flow in the routing module. Forwarding the sample comprises sensing (6) flow characteristics of the sample in the preparation and/or routing modules and/or flow connections therebetween, and controlling (7) a flow control element to compensate for flow rate variations by closed loop flow control taking the sensed flow characteristics into account.)

1. A method (1) for detecting, sorting, purifying and/or characterizing an object of interest in a liquid sample, the method comprising:

-preparing (3), in a preparation module (120) of a microfluidic routing system (100), a liquid sample in a microfluidic flow comprising an object of interest for processing, wherein the preparing comprises transporting the liquid sample from at least one inlet of the preparation module through at least one microfluidic channel to at least one outlet of the preparation module,

-forwarding (4) the prepared liquid sample in a microfluidic flow from the at least one outlet of the preparation module (120) into at least one inlet of a routing module (105) of the microfluidic routing system, the forwarding comprising coupling the microfluidic flow between the at least one outlet of the preparation module and the at least one inlet of the routing module to passively buffer against and/or actively compensate for flow rate variations of the prepared liquid sample in the microfluidic flow at the at least one outlet of the preparation module, and

-transferring (5), in the routing module of the microfluidic routing system, an object of interest from the prepared microfluidic flow of the liquid sample,

wherein the forwarding (4) of the liquid sample comprises sensing (6) flow characteristics of the liquid sample in the microfluidic flow in a flow connection between the preparation module (120) and/or the routing module (105) and/or the preparation module (120) and the routing module (105) and controlling (7) at least one flow control element taking the sensed flow characteristics into account to compensate for flow rate variations of the prepared liquid sample in the microfluidic flow,

wherein the control (7) comprises a closed loop flow control for compensating for a deviation of the sensed flow characteristic from a predetermined target value of the flow characteristic.

2. -the method according to claim 1, wherein sensing (6) the flow characteristics comprises, in the routing module (105), measuring the flow rate (Qr) of a liquid sample prepared in a microfluidic flow.

3. -the method according to claim 2, comprising injecting (2) the liquid sample or a further liquid into the preparation module (120) at a first pressure (Ps), and wherein the controlling (7) comprises adjusting the first pressure (Ps) by taking into account the deviation of the flow rate (Qr) from the predetermined target value.

4. -the method according to any of the preceding claims, wherein the controlling (7) comprises activating the at least one flow control element in response to the sensed flow characteristics to activate a fluid source in a stage or module of the microfluidic routing system (100).

5. -the method according to any of the preceding claims, wherein the roll-out (5) of the object of interest comprises injecting a further liquid at a second pressure (Pr) into the microfluidic flow of the prepared liquid sample in the routing module, and wherein the controlling (7) comprises controlling the second pressure (Pr).

6. -the method according to any of the preceding claims, wherein the forwarding (4) of the liquid sample comprises injecting an auxiliary flow of a further liquid at a third pressure (Pa) into the microfluidic flow between the at least one outlet of the preparation module (120) and the at least one inlet of the routing module (105), and wherein the controlling (7) comprises adjusting the third pressure (Pa) by taking into account the deviation of the flow rate (Qr) from the predetermined target value.

7. -the method according to any one of the preceding claims, wherein the forwarding (4) of the liquid sample from the at least one outlet of the preparation module (120) to the at least one inlet of the routing module (105) comprises coupling the microfluidic flow between the at least one outlet of the preparation module and the at least one inlet of the routing module through a fluidic resistor (115) having a flow resistance (Rc) to passively oppose changes in the flow resistance of the preparation module and/or routing module, wherein the flow resistance (Rc) of the fluidic resistor (115) is at least equal to a predetermined value corresponding to expected changes and/or fluctuations in the flow resistance of the preparation module and/or routing module.

8. -the method according to any of the preceding claims, wherein said detecting comprises detecting at least one characteristic feature of said object of interest in an optical detection signal, and wherein said transferring of said object of interest out of the microfluidic flow comprises calculating a routing signal in response to said detecting of said at least one characteristic feature and controlling an actuation element by means of the calculated routing signal to transfer the detected object of interest out of the main component of the microfluidic flow.

9. -a microfluidic flow for preparing a fluid impregnated with an object of interest and a microfluidic routing device (110) for routing the object of interest in the microfluidic flow, the device comprising:

-a sample preparation module (120) adapted to prepare a liquid sample in a microfluidic flow comprising the object of interest for processing, the preparation module (120) comprising at least one microfluidic channel for transporting the liquid sample connecting at least one inlet of the preparation module to at least one outlet of the preparation module;

-a routing module (105) adapted to divert the object of interest out of the prepared microfluidic flow of the liquid sample;

-a microfluidic connection for interconnecting the preparation module and the routing module for forwarding the prepared liquid sample from the at least one outlet of the preparation module (120) to at least one inlet of the routing module (105) in a microfluidic flow,

wherein the preparation module (120) and/or the routing module (105) and/or the microfluidic connection are adapted to sense (6) a flow characteristic of the liquid sample in the microfluidic flow to take the sensed flow characteristic into account for controlling at least one flow control element by closed loop flow control to compensate for a deviation of the sensed flow characteristic from a predetermined target value of the flow characteristic.

10. -the microfluidic routing device of claim 9, wherein the routing module (105) comprises an actuation element for diverting a detected object of interest out of the main component of the microfluidic flow in response to a routing signal.

11. -the microfluidic routing device of claim 9 or claim 10, wherein the microfluidic connection comprises a fluidic resistor (115) having a flow resistance and adapted to passively buffer against flow rate variations of the prepared liquid sample in the microfluidic flow at the at least one outlet of the preparation module.

12. -microfluidic routing device according to any one of claims 9 to 11, comprising an inlet junction (125) for the routing module (105) adapted to inject a secondary liquid flow into the microfluidic flow between the at least one outlet of the preparation module and the at least one inlet of the routing module.

13. -a microfluidic routing system (100) comprising a microfluidic routing device (110) according to any of claims 9 to 12 and a meter device (160),

wherein the microfluidic routing system comprises at least one sensor (95) for sensing the flow characteristic of a liquid sample in a microfluidic flow in the preparation module (120) and/or the routing module (105) and/or a microfluidic connection,

wherein the microfluidic routing system comprises a first fluid actuator for injecting the liquid sample into the preparation module (120) at a first pressure (Ps), and/or a second fluid actuator for injecting a liquid into a microfluidic flow of the prepared liquid sample at a second pressure (Pr) at the routing module (105), and/or a third fluid actuator for injecting an auxiliary fluid flow into the microfluidic flow between the at least one outlet of the preparation module (120) and the at least one inlet of the routing module (105) at a third pressure (Pa),

wherein the meter device (160) comprises a controller for controlling the first pressure (Ps) and/or the second pressure (Pr) and/or the third pressure (Pa),

wherein the controller is adapted to perform closed loop flow control to compensate for a deviation of the sensed flow characteristic from a predetermined target value of the flow characteristic.

14. -the microfluidic routing system of claim 13, wherein the controller is adapted to adjust the first pressure (Ps) and/or the third pressure (Pa) by taking into account a deviation indicative of a flow rate (Qr) of a flow into or through the routing module from the predetermined value.

15. -a diagnostic device comprising a microfluidic routing device according to any one of claims 9 to 12 and/or a microfluidic routing system according to claim 13 or claim 14.

Technical Field

The present invention relates to the field of microfluidic devices. More particularly, it relates to a microfluidic device for preparing a microfluidic flow of fluid in which an object of interest is immersed and for routing an object of interest in the microfluidic flow, a related cartridge, a related system and a related method.

Background

Microfluidic routing devices are useful for a variety of applications, such as in cell routing systems for detecting, sorting, and/or characterizing biological entities of interest (e.g., target cells). Such cell routing systems can be used as general or special clinical tools, for example for quantification of target cell types and cell characterization. For example, the number of cells of a given target type detected may be an important clinical marker for treatment follow-up, such as follow-up of cancer metastasis and/or minimal residual disease. Exemplary applications of the cell routing system include analyzing blood samples for chronic lymphocytic leukemia diagnosis or follow-up or circulating tumor cell monitoring, analyzing bone marrow samples for myeloma diagnosis or follow-up, analyzing lymph node biopsies for hodgkin's disease diagnosis or follow-up, analyzing urine samples and/or sample enrichment for sequencing. The routing system may also be used for detection and purification of entities of interest immersed in the microfluidic flow. For example, a biological entity of interest (e.g., a target cell) can be introduced into a microfluidic flow as a component of a mixture of multiple entities (e.g., a mixture of different cell types obtained from crude samples). Isolation of the target entity from the background entity may be preferred or required for downstream analysis, e.g., specific analysis of target cells, such as cell culture, immunocytochemistry, DNA and/or RAN fluorescence in situ hybridization, and/or next generation sequencing analysis. Such approaches may find application in companion diagnostics, cell therapy, and pathology research. For example, T cells may be selected from a blood sample for use in cell therapy, or specific sperm cells may be selected from a semen sample for use in sperm separation.

It is known in the art that processes for sample preparation, sorting of entities immersed in a fluid, and processing of such entities may be implemented in microfluidic chips. For example, various sample preparation and particle sorting methods have been described in microfluidic chip technology.

In process flows known in the art, the fluid sample may be prepared by manual or automated methods. Such preparation methods may include one or more concentration steps, dyeing steps, and/or washing steps. The prepared sample may then be introduced into a sorting module for separating out objects of interest, such as biological cells or other biological entities of interest, from the fluid sample.

Disclosure of Invention

It is an object of embodiments of the present invention to provide a good and efficient apparatus and method for detecting, sorting, purifying and/or characterizing objects of interest in a liquid sample using an integrated microfluidic system.

The above object is achieved by a method and device according to the present invention.

An advantage of embodiments of the present invention is that objects of interest in a microfluidic flow, such as biological particles in a fluid sample, may be sorted, analyzed, separated, or otherwise processed in a simple, fast, and affordable apparatus.

An advantage of embodiments of the present invention is that a fluid sample preparation phase (e.g. a sample pre-processing phase) and a sample processing phase (e.g. a fluid processing phase in which objects of interest in a fluid are processed and/or sorted and/or analyzed) can be easily co-integrated in a device.

An advantage of embodiments of the present invention is that automated, efficient transfer of objects of interest in a fluidic sample (e.g., biological particles in a microfluidic flow) may be transferred between different portions of a microfluidic chip, e.g., between different modules, e.g., between fluid preparation, fluid processing, fluid sorting, and/or post-processing stages, with low risk of sample loss, such as due to adhesion of objects of interest to the walls of the tube in the microfluidic flow during transfer. An advantage of embodiments of the present invention is that different stages in a microfluidic flow processing system (e.g., stages for preparing a fluid sample having objects of interest immersed therein, routing the fluid sample, sorting objects of interest in the fluid sample, and/or post-processing the sorted objects of interest) may be directly connected along one (or at least one) flow path.

An advantage of embodiments of the present invention is that the effect of the operation of a stage in a microfluidic flow processing system on the flow characteristics at the interface between the stage and a subsequent stage (e.g., this effect may or may not be predictable) can be easily detected and/or compensated for.

An advantage of embodiments of the present disclosure is that the effects of flow fluctuations and/or deviations can be decoupled between the sample preparation module (e.g., sample preparation unit) and the sorting module (e.g., sorting unit) such that independent fluidic operations can be achieved inside each module (e.g., each unit) while the modules (e.g., units) can still be highly co-integrated.

An advantage of embodiments of the present invention is to provide robust performance of each module (e.g., each cell) in a microfluidic system against interface fluctuations in the operation of the system.

An advantage of embodiments of the present invention is that each module (e.g., each cell) can be modified, selected, and/or reconfigured in designing a microfluidic integrated system without strongly affecting the selection or parameters of other modules (e.g., other cells). Thus, interchangeability and flexibility of each module (e.g., each unit) may be provided during the device development phase.

In a first aspect, the present invention relates to a method for detecting, sorting, purifying and/or characterizing an object of interest in a liquid sample. The method includes preparing a liquid sample in a microfluidic flow including an object of interest for processing in a preparation module (e.g., a preparation unit) of a microfluidic routing system. The preparing includes transporting the liquid sample from the at least one inlet of the preparation module to the at least one outlet of the preparation module through the at least one microfluidic channel. The method also includes forwarding the prepared liquid sample in the microfluidic flow from the at least one outlet of the preparation module to at least one inlet of a routing module (e.g., a routing unit) of the microfluidic routing system. This forwarding includes coupling the microfluid between the at least one outlet of the preparation module and the at least one inlet of the routing module to passively buffer against and/or actively compensate for flow rate variations of the prepared liquid sample in the microfluidic flow at the at least one outlet of the preparation module. The method also includes diverting the object of interest from the prepared microfluidic flow of the liquid sample in a routing module of the microfluidic routing system, such as to detect, sort, purify, and/or characterize the object of interest, for example.

The forwarding of the liquid sample comprises: sensing a flow characteristic of the liquid sample in the microfluidic flow in the preparation module and/or the routing module and/or a flow connection between the preparation module and the routing module, and controlling the at least one flow control element to compensate for a change in the flow rate of the prepared liquid sample in the microfluidic flow taking the sensed flow characteristic into account. This control includes closed loop flow control to compensate for deviations of the sensed flow characteristic from a predetermined target value of the flow characteristic.

Methods according to embodiments of the invention may further include introducing a liquid sample including the object of interest into the microfluidic routing system.

In a method according to embodiments of the invention, the introducing step may comprise injecting the liquid sample into the preparation module at a first pressure.

In a method according to embodiments of the invention, the step of transferring out the object of interest may comprise injecting a further liquid (e.g. the same carrier liquid as used to carry the object of interest in the liquid sample) at a second pressure into the microfluidic flow of the prepared liquid sample in the routing module.

In a method according to embodiments of the invention, the step of forwarding the liquid sample may comprise injecting an auxiliary flow of a further liquid (e.g. the same carrier liquid as used to carry the object of interest in the liquid sample) into the microfluidic flow at a third pressure between the at least one outlet of the preparation module and the at least one inlet of the routing module.

Methods according to embodiments of the invention may include controlling the first pressure and/or the second pressure and/or the third pressure.

Methods according to embodiments of the invention may include monitoring and/or measuring the first pressure and/or the second pressure and/or the third pressure, for example, using at least one pressure gauge.

Methods according to embodiments of the invention may include monitoring and/or measuring a first flow rate indicative of flow through the preparation module, and/or a second flow rate indicative of flow through the routing module, and/or a third flow rate indicative of the auxiliary flow, for example using at least one flow meter, an injected acoustic flow sensor, and/or a thermal flow sensor.

In the method according to embodiments of the present invention, the preparing step may include a sample concentrating step, a mixing step, a diluting step, an agitating step, a staining step, a cell lysing step, and/or a cell dissociating step.

In a method according to embodiments of the invention, said transferring of said object of interest out of the microfluidic flow may comprise detecting an object of interest of objects of interest in the microfluidic flow of the prepared liquid sample.

In methods according to embodiments of the invention, the detecting may comprise obtaining an optical detection signal, such as a fluorescence, bright-field, dark-field and/or scatter signal and/or an image (e.g. a microscopic image, a holographic image and/or a diffraction image) of the object.

In a method according to embodiments of the invention, the detecting may comprise detecting at least one characteristic feature of an object of interest in the optical detection signal.

In a method according to embodiments of the invention, said diverting of said object of interest from the microfluidic flow may comprise calculating a routing signal in response to said detecting of said at least one characteristic feature, and controlling the actuation element by means of the calculated routing signal to divert the detected object of interest from the main component of the microfluidic flow.

In methods according to embodiments of the invention, controlling may include activating the at least one flow control element in response to the sensed flow characteristic, e.g., to activate a stage or module, e.g., to activate a fluid source in the stage or module when a moving fluid front is detected upstream of the stage or module.

In methods according to embodiments of the invention, sensing a flow characteristic may include measuring a flow rate, detecting a fluid presence, and/or detecting a fluid optical parameter. For example, the flow characteristic may be flow front detection, wherein the presence of a fluid carrying the object of interest is detected at the sensing location due to a change in light transmission, light reflection or similar optical properties.

In a method according to embodiments of the invention, the step of sensing a flow characteristic may comprise measuring a flow rate of a prepared liquid sample in the microfluidic flow in the routing module.

The method according to embodiments of the invention may comprise injecting the liquid sample (or the further liquid) into the preparation module at a first pressure, wherein the controlling step comprises adjusting the first pressure by taking into account deviations of the flow rate from a predetermined target value.

In a method according to embodiments of the invention, the transferring out of the object of interest may comprise injecting a further liquid into the microfluidic flow of the prepared liquid sample at a second pressure in the routing module, and the controlling step may comprise controlling the second pressure.

In a method according to embodiments of the invention, the controlling step may comprise controlling at least one further pressure of a further fluid source in a stage forming part of the preparation module.

In a method according to embodiments of the invention, the forwarding of the liquid sample may comprise injecting a further auxiliary flow of liquid into the microfluidic flow at a third pressure between the at least one outlet of the preparation module and the at least one inlet of the routing module, wherein the controlling step comprises adjusting the third pressure by taking into account a deviation of the flow rate from a predetermined target value.

In a method according to embodiments of the invention, forwarding the liquid sample from the at least one outlet of the preparation module to the at least one inlet of the routing module may comprise coupling the microfluidic flow between the at least one outlet of the preparation module and the at least one inlet of the routing module through a fluidic resistor having a flow resistance to passively buffer against flow rate variations of the prepared liquid sample in the microfluidic flow at the at least one outlet of the preparation module.

In a method according to embodiments of the invention, the flow resistance of the fluidic resistor may be at least equal to a predetermined value corresponding to an expected variation or fluctuation of the flow resistance of the preparation module and/or the routing module.

In a second aspect, the present invention relates to a microfluidic flow for preparing a fluid impregnated with an object of interest and a microfluidic routing device for routing the object of interest in the microfluidic flow, the device comprising:

a sample preparation module (e.g. a sample preparation unit) adapted to prepare a liquid sample in a microfluidic flow comprising the object of interest for processing, the preparation module comprising at least one microfluidic channel for transporting the liquid sample connecting at least one inlet of the preparation module to at least one outlet of the preparation module;

a routing module (e.g. a routing unit) for diverting the object of interest from the prepared microfluidic flow of the liquid sample;

-a microfluidic connection for interconnecting the preparation module and the routing module for forwarding the prepared liquid sample from the at least one outlet of the preparation module to at least one inlet of the routing module in a microfluidic flow.

The preparation module and/or the routing module and/or the microfluidic connection are adapted to sense a flow characteristic of the liquid sample in the microfluidic flow, e.g. such that the flow characteristic is sensible by an internal or external sensor element to take the sensed flow characteristic into account for controlling the at least one flow control element by closed loop flow control to compensate for a deviation of the sensed flow characteristic from a predetermined target value of the flow characteristic.

The apparatus may comprise said at least one flow control element which may be adapted to receive a control signal for controlling said at least one flow control element by closed loop flow control taking sensed flow characteristics into account to compensate for deviations of the sensed flow characteristics from predetermined target values of the flow characteristics.

In a microfluidic routing device according to embodiments of the present invention, the routing module may comprise an actuation element (e.g. different from the at least one flow control element) for diverting the detected object of interest out of the main component of the microfluidic flow in response to the routing signal.

In a microfluidic routing device according to embodiments of the present invention, the microfluidic connection may comprise a fluidic resistor having a flow resistance and adapted to passively buffer against flow rate variations of the prepared liquid sample in the microfluidic flow at the at least one outlet of the preparation module.

Microfluidic routing devices according to embodiments of the present invention may comprise an inlet junction for a routing module, wherein the inlet junction is adapted to inject a secondary liquid flow into the microfluidic flow between the at least one outlet of the preparation module and the at least one inlet of the routing module.

In a third aspect, the invention relates to a microfluidic routing system comprising a microfluidic routing device according to embodiments of the second aspect of the invention and a meter device. The microfluidic routing system comprises at least one sensor for sensing a flow characteristic of a liquid sample in the microfluidic flow in the preparation module and/or the routing module and/or the microfluidic connection. The microfluidic routing system comprises a first fluid actuator for injecting a liquid sample into the preparation module at a first pressure, and/or a second fluid actuator for injecting a liquid into the microfluidic flow of the prepared liquid sample at a second pressure at the routing module, and/or a third fluid actuator for injecting an auxiliary fluid flow into the microfluidic flow between the at least one outlet of the preparation module and the at least one inlet of the routing module at a third pressure, and/or at least one further fluid actuator for injecting at least one further liquid into the preparation module at least one further pressure. The meter device comprises a controller for controlling the first pressure and/or the second pressure and/or the third pressure and/or the at least one further pressure.

The controller is adapted to perform closed loop flow control to compensate for deviations of the sensed flow characteristic from a predetermined target value of the flow characteristic.

In a microfluidic routing system according to embodiments of the present invention, the controller may be adapted to adjust the first pressure and/or the third pressure by taking into account a deviation indicative of a flow rate of the flow into or through the routing module relative to a predetermined target value.

In a further aspect, the present invention relates to a diagnostic device comprising a microfluidic routing device according to embodiments of the present invention and/or a microfluidic routing system according to embodiments of the present invention. The diagnostic device may be adapted to detect, sort and/or characterize a biological entity of interest, such as a target cell. The diagnostic device may be adapted for use as a general or special clinical tool, e.g. for quantification of target cell types and cell characterization.

The diagnostic device may be adapted to analyze a body tissue sample and/or a body fluid sample, e.g. a blood sample, a saliva sample, a urine sample, a semen sample, a lymph fluid sample, a stool sample, a bone marrow sample and/or another sample obtained from a human or animal body.

Particular and preferred aspects of the invention are set out in the accompanying independent and dependent claims. Features from dependent claims may be combined with features of the independent claims and features of other dependent claims as appropriate and not merely as explicitly set out in such claims.

These and other aspects of the invention will be apparent from and elucidated with reference to the embodiment(s) described hereinafter.

Drawings

FIG. 1 illustrates an exemplary method according to various embodiments of the invention.

Fig. 2 illustrates a preparation module passively coupled to a routing module via a flow resistor, according to embodiments of the invention.

Fig. 3 illustrates a closed-loop flow control approach for adjusting the first pressure taking into account flow rate measurements according to embodiments of the invention.

Fig. 4 illustrates an auxiliary flow source for stabilizing flow rates at an inlet of a routing module, in accordance with various embodiments of the invention.

FIG. 5 illustrates a closed-loop flow control approach for adjusting the secondary flow pressure taking into account flow rate measurements according to various embodiments of the invention.

Fig. 6 shows the use of light barriers for flow front detection, illustrating embodiments of the present invention.

Fig. 7 shows detection signals of an optical barrier for flow front detection illustrating embodiments of the present invention.

Fig. 8 illustrates a microfluidic routing system according to various embodiments of the present invention.

Fig. 9 illustrates an exemplary microfluidic routing device according to various embodiments of the present invention.

FIG. 10 illustrates flow rates and timing diagrams of an exemplary device according to various embodiments of the invention.

The drawings are only schematic and are non-limiting. In the drawings, the size of some of the elements may be exaggerated and not drawn on scale for illustrative purposes.

Any reference signs in the claims shall not be construed as limiting the scope.

The same reference numbers in different drawings identify the same or similar elements.

Detailed Description

The present invention will be described with respect to particular embodiments and with reference to certain drawings but the invention is not limited thereto but only by the claims. The drawings described are only schematic and are non-limiting. In the drawings, the size of some of the elements may be exaggerated and not drawn on scale for illustrative purposes. The dimensions and relative dimensions do not correspond to actual reductions to the invention.

Moreover, the terms first, second, and the like in the description and in the claims, are used for distinguishing between similar elements and not necessarily for describing a sequential order temporally, spatially, in ranking, or in any other manner. It is to be understood that the terms so used are interchangeable under appropriate circumstances and that the embodiments of the invention described herein are capable of operation in other sequences than described or illustrated herein.

Furthermore, the terms top, bottom, and the like in the description and the claims are used for descriptive purposes and not necessarily for describing relative positions. It is to be understood that the terms so used are interchangeable under appropriate circumstances and that the embodiments of the invention described herein are capable of operation in other orientations than described or illustrated herein.

It is to be noticed that the term 'comprising', used in the claims, should not be interpreted as being limitative to the means listed thereafter; it does not exclude other elements or steps. Accordingly, the terms are to be interpreted as specifying the presence of the stated features, integers, steps or components as referred to, but do not preclude the presence or addition of one or more other features, integers, steps or components, or groups thereof. Thus, the scope of the expression "an apparatus comprising means a and B" should not be limited to an apparatus consisting of only components a and B. This means that for the present invention, the only relevant components of the device are a and B.

Reference throughout this specification to "one embodiment" or "an embodiment" means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. Thus, appearances of the phrases "in one embodiment" or "in an embodiment" in various places throughout this specification are not necessarily all referring to the same embodiment, but may. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments as will be apparent to one of ordinary skill in the art in view of the present disclosure.

Similarly, it should be appreciated that in the description of exemplary embodiments of the invention, various features of the invention are sometimes grouped together in a single embodiment, figure, or description thereof for the purpose of streamlining the disclosure and aiding in the understanding of one or more of the various inventive aspects. This method of disclosure, however, is not to be interpreted as reflecting an intention that the claimed invention requires more features than are expressly recited in each claim. Rather, as the following claims reflect, inventive aspects lie in less than all features of a single foregoing disclosed embodiment. Thus, the claims following the detailed description are hereby expressly incorporated into this detailed description, with each claim standing on its own as a separate embodiment of this invention.

Furthermore, although some embodiments described herein include some but not other features included in other embodiments, combinations of features of different embodiments are intended to be within the scope of the invention and form different embodiments, as will be understood by those skilled in the art. For example, in the appended claims, any of the claimed embodiments can be used in any combination.

In the description provided herein, numerous specific details are set forth. It is understood, however, that embodiments of the invention may be practiced without these specific details. In other instances, well-known methods, structures and techniques have not been shown in detail in order not to obscure an understanding of this description.

References to 'modules' in this description refer to functional units, for example units that can be easily integrated in more complex systems, such as cassettes.

In a first aspect, the present invention relates to a method for detecting, sorting, purifying and/or characterizing an object of interest in a liquid sample. The method includes preparing a liquid sample in a microfluidic flow including an object of interest for processing in a preparation module of a microfluidic routing system. The step of preparing the liquid sample comprises transporting the liquid sample from the at least one inlet of the preparation module through the at least one microfluidic channel to the at least one outlet of the preparation module.

The method also includes forwarding the prepared liquid sample in the microfluidic flow from the at least one outlet of the preparation module to at least one inlet of a routing module of the microfluidic routing system, wherein this forwarding includes coupling the microfluidic flow between the at least one outlet of the preparation module and the at least one inlet of the routing module to passively buffer against and/or actively compensate for flow rate variations of the prepared liquid sample in the microfluidic flow at the at least one outlet of the preparation module.

The method further includes diverting the object of interest from the prepared microfluidic flow of the liquid sample in a routing module of the microfluidic routing system.

Forwarding the liquid sample comprises sensing flow characteristics of the liquid sample in the microfluidic flow in the preparation module and/or the routing module and/or a flow connection between the preparation module and the routing module and taking the sensed flow characteristics into account for controlling the at least one flow control element to compensate for variations in the flow rate of the prepared liquid sample in the microfluidic flow. This control includes closed loop flow control to compensate for deviations of the sensed flow characteristic from a predetermined target value of the flow characteristic.

Referring to fig. 1, an exemplary method 1 according to an embodiment of the invention is shown. Method 1 is a method for detecting, sorting, purifying, and/or characterizing an object of interest in a liquid sample (e.g., an entity of interest in a liquid sample). For example, the object of interest may comprise a biological entity of interest in a liquid sample (such as a body fluid sample). For example, the method may be a method for detecting and purifying an object of interest immersed in a microfluidic flow. For example, the method may be a method for biological cell sorting and/or biological cell routing for clinical use.

For example, method 1 may include introducing 2 a liquid sample including an object of interest into a microfluidic routing system 100, e.g., as illustrated in fig. 8. The microfluidic routing system may include (e.g., may include at least) a preparation module 120 and a routing module 105. The microfluidic routing system may include a microfluidic connection between a preparation module and a routing module, such as fluidic resistor 115. By integrating the preparation module and the routing module in a single integrated microfluidic routing device, the loss of (some of) the objects of interest in the liquid sample during transfer and/or manipulation between different phases (e.g. between the preparation phase and the routing phase) may be kept advantageously low or even substantially prevented.

A microfluidic routing system (e.g., a cell routing system) may include a microfluidic routing device 110, for example, in the form of a cartridge. This microfluidic routing device 110 may have an integrated sample preparation module 120 and an integrated routing module 105, such as a cell routing module, for example for biological cell sorting. The microfluidic routing device 110 may include microfluidic, electrical, and/or optical connections 111 between modules and/or other components in the cartridge.

The preparation module (e.g., for biological cell concentration) and the routing module (e.g., for biological cell routing) may be interconnected by a microfluidic device (e.g., one or more microfluidic channels) in an integrated cartridge. For example, the microfluidic device (e.g., the at least one interconnected microfluidic channel) may have a minimum cross-sectional dimension (e.g., channel width, height, and/or diameter) in the range of 1.1 times (e.g., 1.5 times) the size of the object of interest (e.g., target cell) to about 2 mm. For example, the cross-sectional dimension may be in the range of 30 μm to 1mm, for example in the range of 50 μm to 500 μm. For example, this may be a suitable range for biological entities of interest (such as cells, which are typically less than 50 μm). In particular, the preparation module and the routing module may be interconnected without any intermediate storage banks, containers and/or buffers (e.g., in the general sense with respect to flow from the preparation module to the routing module) having a cross-sectional dimension greater than or equal to 5mm (e.g., greater than or equal to 2mm, such as greater than or equal to 1 mm).

The microfluidic routing system may also include a meter in which the routing device may be loaded for analysis and/or processing of the sample. The microfluidic routing device may be disposable, e.g. may be a disposable cartridge. The microfluidic routing device may include functional components for physically processing the sample, such as sample and/or reagent tanks, cell preparation components, cell routing elements, and/or post-sort processing components. The meter may include all peripheral hardware and/or software for facilitating operation of the microfluidic routing device, e.g., for system level control and providing a user interface to actuate and control fluidics in the routing device, for light detection of light sources and signal processing for fluorescence detection and/or cell imaging, for controlling and driving electrical components in a cartridge of the routing device, and/or for post-sort cell distribution (e.g., transfer of sorted cells to vials or glass slides in a sorted cell suspension). In particular, the hardware of the meter may advantageously be designed to avoid physical contact with the meter, for example to improve sample processing accuracy with a minimal likelihood of sample contamination.

Introducing 2 a liquid sample comprising the object of interest into the microfluidic system may comprise injecting the liquid sample into the preparation module at a pressure Ps. The pressure Ps may be a predetermined pressure, or may be a controllable pressure. For example, in embodiments according to the invention, the method may include controlling the pressure Ps, e.g., as described further below. The method may also include monitoring (e.g., measuring) the pressure Ps, for example, using a pressure gauge.

The method includes preparing a liquid sample for processing in a preparation module 120 of the microfluidic routing system 100. The liquid sample is in a microfluidic flow and includes an object of interest.

The step of preparing 3 a liquid sample comprises transporting (e.g., continuously transporting) the liquid sample from at least one inlet of the preparation module 120 through at least one microfluidic channel to at least one outlet of the preparation module 120.

Preparing 3 the liquid sample may comprise concentrating the objects in the fluid sample, e.g. to increase the concentration of objects of interest in the fluid sample, e.g. by reducing the volume of the sample without significantly reducing the number of objects of interest therein, or at least keeping the loss of objects of interest within acceptable limits.

The volumetric flow rate of the liquid sample in the microfluidic flow may differ by a factor of at least 5 between the at least one outlet of the preparation module and the at least one outlet of the preparation module. For example, the (total) volumetric flow rate at the (or all) outlets of the preparation module (whether outlets for waste treatment) may be significantly lower than the (total) volumetric flow rate at the (or all) inlets of the preparation module. More generally, there may be a large difference between the volumetric flow rate through the at least one inlet and the volumetric flow rate through the at least one outlet, for example such that one of them is significantly larger than the other. An advantage of embodiments of the present invention is that such large differences between inflow and outflow can be effectively and efficiently handled in a continuous or stepwise continuous flow system.

Furthermore, the at least one outlet of the preparation module may be connected to the at least one input of the routing module for forwarding the prepared liquid sample from the preparation module to the routing module in the microfluidic flow. Although the volumetric flow in the at least one outlet of the preparation module may be significantly lower than the volumetric flow injected into the preparation module, as discussed above, e.g. due to a substantial part being removed as waste liquid, the flow obtained from the at least one outlet of the preparation module may merge with at least one further fluid flow (e.g. a fluid used as a carrier and/or a directional flow control medium) in the routing module. Thus, the routing module may also involve a large relative difference in volumetric flow between the total flow through the routing module and the prepared liquid sample received from the preparation module. It is noted that such variations in the magnitude of the primary flow of interest throughout the preparation module and the routing module may require an advantageously accurate and efficient way of synchronizing the stages in a serial flow arrangement, such as provided by embodiments of the present invention.

The method may also include monitoring (e.g., measuring), e.g., at the at least one outlet of the preparation module, a flow rate Qs indicative of flow through the preparation module. For example, monitoring the flow rate Qs may include measuring the flow rate using a flow meter (e.g., an acoustic flow sensor and/or a thermal flow sensor).

The method may also include monitoring (e.g., measuring), e.g., at the at least one inlet of the routing module, a flow rate Qr indicative of flow through the routing module. For example, monitoring the flow rate Qr may include measuring the flow rate using a flow meter (e.g., an acoustic flow sensor and/or a thermal flow sensor). Alternatively, the flow rate Qr may be estimated from the detected speed and/or frequency of detection of objects detected in the flow in the routing module.

The liquid sample may be manipulated in the step of preparing the liquid sample, for example by a sample concentration step, a mixing step, a dilution step and/or an agitation (no mixing) step. A liquid sample, which may include a biological object of interest, such as a biological cell, may be manipulated in the step of preparing the liquid sample by, for example, a staining step (e.g., for microscopic imaging and/or fluorescence characterization), a cell lysis step, and/or a cell dissociation step. The step of preparing the liquid may include any suitable combination of these operations and/or other similar operations known in the art. Suitable liquid sample preparation and the steps and/or parameters of the routing operations referred to below may be substantially determined by the properties of the sample liquid, the properties of the object of interest, the properties of the intended application of the method, e.g. as may be determined in practice by a person skilled in the art without inventive effort based on application requirements.

The method further includes forwarding 4 (e.g., continuously forwarding) the prepared liquid sample in the microfluidic flow from the at least one outlet of the preparation module 120, e.g., via a microfluidic connection between the modules, to at least one inlet of the routing module 105 of the microfluidic routing system 100.

For example, by providing a direct connection for maintaining a continuous flow of samples between the sample preparation module and the routing module, the loss of objects of interest (e.g. biological particles such as cells) may advantageously be reduced, e.g. kept low.

The step of forwarding 4 the liquid sample from the at least one outlet of the preparation module to the at least one inlet of the routing module comprises coupling (e.g., by a microfluidic flow connection) the microfluidic flow between the at least one outlet of the preparation module and the at least one inlet of the routing module to passively buffer against and/or actively compensate for flow rate variations of the prepared liquid sample in the microfluidic flow at the at least one outlet of the preparation module. Thus, a less variable and/or more stable flow rate of the prepared liquid sample in the microfluidic flow may be provided at the at least one inlet of the routing module.

Thus, an 'intelligent' connection may be provided between the preparation module and the routing module such that changes in flow parameters in one module do not have an effect on the operation of the other module, for example to the extent that such changes are within a predetermined reasonable margin that can be expected in the normal operation of the device, and to the extent that the effect of one module on the other module is significantly reduced such that the normal operation of that module can be expected to be within an operational tolerance margin.

For example, without such coupling to dampen and/or actively compensate for changes in pressure, flow rate, flow resistance, and/or other fluid dynamic parameters, such fluctuating flow conditions in one module may adversely affect the proper operation of another module.

The method further comprises diverting 5 the object of interest from the prepared microfluidic flow of the liquid sample in a routing module of the microfluidic routing system.

For example, the object of interest may be diverted 5 from the prepared microfluidic flow of the liquid sample to sort out the object of interest from another type of object or types of objects present in the prepared microfluidic flow of the liquid sample.

Roll-out 5 of the object of interest may comprise detecting the object of interest in the prepared microfluidic flow of the liquid sample, e.g. at one or more predetermined positions in the routing module. This detection may include optical detection, such as fluorescence signal detection. For example, the biological entity may be stained with a fluorescent dye during the preparation step, and the characteristic of the object may be detected optically as the stained object of interest moves through the routing module. The optical detection may also include another type of optical detection, such as a bright field signal, a dark field signal, and/or a scatter signal. Optical inspection may also include inspecting an image of the object, such as a conventional image, e.g., a microscopic image, a holographic image, or a diffractive image. The detecting step may include detecting at least one characteristic feature of the object of interest in the optical detection signal (e.g., fluorescence signal, optical signal, scatter signal, and/or image).

Transferring 5 the object of interest may comprise calculating a routing signal in response to detecting the object of interest (e.g. when the at least one characteristic feature of the object of interest is detected in the optical detection signal). For example, the signal may be calculated to control the actuation element at a suitable time, for example after a suitable delay taking into account the movement of the object from the detection position to the position where it is to be turned out of the main component of the microfluidic flow by means of the actuation element. Furthermore, the signal may be calculated to control the actuation element for a suitable duration, e.g. taking into account the flow rate, size, mass and/or volume of the object of interest. Furthermore, the signal may be calculated to control the actuation element with a suitable strength, e.g. the force exerted by the actuation element on the object of interest may depend on the signal strength of the control signal provided to the actuation element.

Diverting 5 the object of interest may comprise controlling the actuation element by means of the calculated control signal to divert the detected object of interest from the main component of the microfluidic flow.

Roll-out 5 of the object of interest may comprise injecting (e.g. and mixing) liquid into the microfluidic flow of the prepared liquid sample in the routing module. For example, the liquid may be injected with a pressing force Pr. The pressure Pr may be a predetermined pressure, or may be a controllable pressure. For example, in embodiments according to the invention, the method may include controlling the pressure Pr, e.g., as described further below. The method may also include monitoring (e.g., measuring) the pressure Pr, for example, using a pressure gauge.

The liquid (e.g., a carrier fluid, such as water or another suitable solvent) may be added at or near the inlet of the routing module.

The liquid (e.g., a carrier fluid such as water or another suitable solvent) may also be added near the middle or near the tail of the routing module (with respect to the direction of flow).

The step of forwarding 4 the liquid sample from the at least one outlet of the preparation module to the at least one inlet of the routing module may comprise coupling the microfluidic flow between the at least one outlet of the preparation module and the at least one inlet of the routing module through a passive fluidic resistor 115 to passively buffer against flow rate variations of the prepared liquid sample in the microfluidic flow at the at least one outlet of the preparation module. In particular, the passive fluidic resistor may add a substantially constant reference flow resistance to the total flow resistance (e.g., serially flow connected) to dampen large relative changes in the opposing total flow resistance (i.e., stabilize the total flow resistance).

For example, such passive fluidic resistors may form microfluidic connections between modules to isolate the effect of one module on another with respect to flow rate variations.

The passive fluidic resistor may include at least one microfluidic channel having a cross-sectional dimension (e.g., channel width, height, and/or diameter) in a range of 1.1 times (e.g., 1.5 times) the size of an object of interest (e.g., target cell) to about 2 mm. For example, the cross-sectional dimension may be in the range of 10 μm to 1 mm. For example, the passive fluidic resistor may be configured according to the range of flow resistances to be achieved for a particular embodiment, e.g. by selecting an appropriate diameter of the at least one microfluidic channel. Furthermore, the length of the at least one microfluidic channel may be selected to more accurately achieve a desired predetermined flow resistance value. The passive fluidic resistor may be tightly integrated in the preparation module and/or the routing module or as a separate component functionally coupled between the preparation module and the routing module, e.g. in a cassette comprising the preparation module and the routing module.

In particular, the preparation module and the routing module may be interconnected without any intermediate storage banks, containers and/or buffers (e.g., in the general sense with respect to flow from the preparation module to the routing module) having a cross-sectional dimension greater than or equal to 5mm (e.g., greater than or equal to 2mm, such as greater than or equal to 1 mm).

The flow resistance of the fluidic resistor may be at least equal to a predetermined value corresponding to an expected variation or fluctuation of the flow resistance of the preparation module and/or the routing module.

Referring to fig. 2, the priming module 120 may have a flow resistance Rs, such as between the at least one inlet and the at least one outlet of the priming module. Also, the routing module 105 may have a flow resistance Rr.

The flow resistances Rs, Rr of the preparation and routing modules are subject to variations and/or fluctuations, for example randomly distributed and/or unstable variations and/or fluctuations. Furthermore, the pressure at which the sample fluid is injected into the preparation module may also be subject to such variations and/or fluctuations. Such variations and/or fluctuations may be caused, for example, by partial channel blockage and/or unclogging, the presence and/or removal of air bubbles, differences in joint tightness, or variations between different sample preparation steps. For example, in continuous sample preparation, the processing element may remain inactive later until the moving fluid front reaches the processing element. Thus, the flow characteristics may change as such initially inactive elements begin to participate in the processing of the continuous flow. Predicting such variations and/or fluctuations is difficult or even impossible. Embodiments of the present invention may provide advantageous apparatus and methods for achieving stability (e.g., within operational tolerances) of flow rates Qr in a routing module (e.g., at an inlet of a routing module).

The flow connection between the preparation module 120 and the routing module 105 may be formed by a fluidic resistor 115 having a fluidic resistance Rc. Since the preparation module 120 and the routing module 105 are directly connected, for example via the fluidic resistor 115, a serial connection is formed. Thus, the flow rate Qs at the outlet(s) of preparation module 120 may be equal to the flow rate Qr at the inlet(s) of routing module 105.

The total flow resistance between the inlet(s) of the preparation module 120 and the outlet(s) of the routing module 105 is thus R_{General assembly}＝Rs+Rc+Rr。

By providing a relatively large flow resistance Rc between the preparation module and the routing module, fluctuations and/or deviations of the flow resistances Rs, Rr (relative to a reference value) of the preparation module and the routing module may be stabilized, for example such that the influence of changes in the flow resistances Δ Rs, Δ Rr of the preparation module and the routing module on the flow rate Qs, Qr may be kept low, for example may be reduced, for example may be minimized.

The flow resistance value Rc may be at least equal to (e.g., preferably 2, 3, 5, or 10 times larger than) the variation and/or fluctuation Δ Rs, Δ Rr of the flow resistance of the preparation module and routing module.

The pressure Pr of the liquid (e.g., the second carrier liquid) injected into the microfluidic flow in the routing module may be determined by Pr ═ Ps-Qs (Rs + Rc), e.g., when the liquid is injected at or near the inlet(s) of the routing module.

The pressure Pr may be determined by Pr ═ Ps-Qs — (Rs + Rc +0.5 × (Rr)), for example when liquid is injected at or near the middle of the routing module.

The step of forwarding 4 the liquid sample from the at least one outlet of the preparation module to the at least one inlet of the routing module may comprise sensing 6 flow characteristics of the liquid sample in the microfluidic flow, e.g. using a flow sensor, in the preparation module and/or the routing module and/or in a flow connection between said modules, and controlling 7 the at least one flow control element taking the sensed flow characteristics into account to compensate for flow rate variations of the prepared liquid sample in the microfluidic flow, e.g. such flow rate variations at the at least one outlet of the preparation module.

In embodiments of the invention, the flow characteristic of the liquid sample may be a flow rate measured at the sensing location. However, embodiments of the invention are not limited thereto. For example, the flow characteristic may be a fluid presence detection or a fluid optical parameter detection.

Flow characteristics (e.g., flow rate, fluid presence, or another parameter indicative of flow) may be determined by optical measurements, electrical measurements (such as impedance measurements), or detection of another physical property related to the flow. For example, the impedance between at least two electrodes (when present in the microfluidic channel) in electrical contact with the fluid, e.g., at least two electrodes in or on a wall of the microfluidic channel, may be measured. For example, when the fluid is at a sensing location in the channel, an impedance change, such as a drop, may be detected.

For example, the flow characteristic may be flow front detection, wherein the presence of a fluid carrying the object of interest is detected at the sensing location due to a change in light transmission, light reflection or similar optical properties. For example, the flow characteristic may be such a flow front detected by the light barrier, as illustrated in fig. 6. In this example, the moving front 201 of liquid in the microfluidic channel (e.g., defined between the cover glass or film 202 and the plastic cassette 203) may be detected by a change in signal detected by the photodetector 204 resulting from a change in transmission of light emitted by the laser diode 205 through the microfluidic channel, such as exemplified in fig. 7.

The controlling step may comprise controlling the pressure Ps, the pressure Pr, the pressure Pa and/or at least one further pressure Px (e.g. of a further liquid source in a stage forming part of the preparation module).

The controlling step may include activating the at least one flow control element in response to the sensed flow characteristic, such as activating a stage or module, such as activating a fluid source in the stage or module when a moving fluid front is detected upstream of the stage or module, such as to synchronize a stage with fluid movement throughout the module.

For example, forwarding 4 the liquid sample may comprise controlling the at least one flow control element in a closed loop flow control approach to compensate for a deviation of the sensed flow characteristic from a predetermined target value of the flow characteristic.

For example, the at least one flow control element may comprise an actuation element, such as a microfluidic valve or a microfluidic switch.

For example, the at least one flow control element may comprise a flow source, such as a second flow source. For example, the at least one flow control element may comprise a controllable fluid pump.

Referring to fig. 3, a flow characteristic may be measured 6, such as a flow rate Qr. The flow rate may be measured in the routing module 105, for example at or near the inlet(s) of the routing module. However, for example, where the connection between the sample preparation module and the routing module is formed by a passive fluidic resistor, such that the preparation module, fluidic resistor and routing module form a serial fluidic connection, the flow rate Qs-Qr may be measured at any suitable point along the serial flow path.

Furthermore, at least one flow control element may be controlled 7 taking into account the sensed flow characteristics to compensate for variations in the flow rate of the prepared liquid sample in the microfluidic flow.

This control 7 may, for example, comprise the determination of the new pressure Ps to be applied, for example by determining in a time step n_nTo adjust the pressure at which the liquid sample is injected into the preparation moduleForce Ps.

For example, the pressure Ps_nMay be different from the pressure applied in the previous time step by a difference Δ Ps ═ Ps_n-Ps_n-1. For example, the difference may be determined by calculating a value that depends on the difference between the measured flow characteristic and a predetermined target value of the flow characteristic (e.g., the difference between the measured flow rate Qr and the set point Qr). For example, the flow rate Qr measured in time step n_nAnd the set point Qr deg. can be used as an argument for calculating the function F of the pressure difference deltaps. The function F may be a linear scaling function, e.g. the difference Qr-Qr may be multiplied by a predetermined scaling factor. The function F may also be a non-linear function, such as a monotonically increasing or increasing function. The function F may be unbiased, e.g. a zero argument may be mapped to a zero function value. The function F may be antisymmetric, for example such that F (-x) — F (x). For example, the function F may be implemented by a Proportional Integral Derivative (PID) method, such as by a PID controller.

Alternatively or additionally, the pressure Pr of the liquid (e.g. the second carrier liquid) injecting the microfluidic flow in the routing module may be controlled 7.

For example, the pressure Pr may be determined, for example, by Pr ═ Ps-Qs (Rs + Rc) when the liquid is injected at or near the inlet(s) of the routing module, or may be determined, for example, by Pr ═ Ps-Qs (Rs + Rc +0.5 Rr) when the liquid is injected at or near the middle of the routing module.

At the actual Qr (e.g., the value Qr of Qr in time step n)_n) When deviating from the set point Qr, for example, regardless of the cause of the deviation, the closed-loop flow rate control algorithm may adjust the pressure (e.g., pressure Ps) to increase or decrease the flow rate Qs Qr.

Thus, the output pressure P0 of the routing module may be advantageously maintained at or near the reference pressure by methods according to embodiments of the invention.

Referring to fig. 4 and 5, in a method according to embodiments of the invention, the step of forwarding 4 the liquid sample from the at least one outlet of the preparation module to the at least one inlet of the routing module may comprise injecting 8 a secondary liquid flow (e.g. a carrier fluid, such as water or another suitable solvent) into the microfluidic flow between the at least one outlet of the preparation module and the at least one inlet of the routing module. For example, at the inlet junction 125 of the routing module, a microfluidic flow of the prepared liquid sample carrying the object of interest to be routed may be merged with the auxiliary flow.

Injecting 8 the auxiliary flow may comprise injecting the liquid with a pressure Pa into a junction between the preparation module and the routing module, for example between the flow resistor and the routing module. The pressure Pa may be a predetermined pressure, or may be a controllable pressure. For example, in embodiments according to the invention, the method may comprise controlling the pressure Pa, for example as described further below. The method may also include monitoring (e.g., measuring) the pressure Pa, for example, using a pressure gauge.

The method may also include monitoring (e.g., measuring) a flow rate Qa indicative of the secondary flow. For example, monitoring the flow rate Qa may include measuring the flow rate using a flow meter (e.g., an acoustic flow sensor and/or a thermal flow sensor).

In a method according to embodiments of the invention, the step of forwarding 4 the liquid sample from the at least one outlet of the preparation module to the at least one inlet of the routing module may further comprise monitoring (e.g. measuring) the pressure Pj at the inlet(s) of the routing module, e.g. using a pressure gauge, e.g. by means of a pressure gauge. For example, the pressure Pj may be measured between the entry junction 125 and the routing module 105.

In a method according to embodiments of the present invention, the step of preparing 3 a liquid sample in a microfluidic flow comprising an object of interest may comprise a plurality (e.g. a series) of different preparation steps. Furthermore, the flow rate through the preparation module may be non-uniform over time, or may even stop occasionally, for example due to flow stoppage between different preparation steps. In such situations, or in other use cases where the flow rate Qs is not substantially constant over time or experiences pauses from time to time, maintaining a stable flow rate Qs may be particularly difficult. However, by controlling the secondary flow, a steady flow rate Qr through the routing module may be advantageously maintained.

Taking sensed flow characteristics into account to compensate for preparation in microfluidic flowThe flow rate of the liquid sample is varied and at least one flow control element can be controlled 7. This controlling 7 may comprise adjusting a pressure Pa at which the secondary flow is injected 8 into the microfluidic flow between the at least one outlet of the preparation module and the at least one inlet of the routing module. For example, at each time step n, a new pressure Pa may be applied_n。

For example, in a closed-loop control algorithm, the pressure Pa may be controlled. For example, the pressures Pa and Ps may be controlled. The pressures Ps, Pa, and Pr may have an effect on the flow rates Qs, Qa, and Qr.

Pressure Pa_n+1 may be different from the pressure Pa applied in the previous time step_nThe difference is delta Pa ═ Pa_n+1-Pa_n. For example, the difference Δ Pa may be determined by calculating a value that depends on the difference between the measured flow characteristic and a predetermined target value of the flow characteristic (e.g., the difference between the measured flow rate Qr and the set point Qr). For example, the flow rate Qr measured in time step n_nAnd the set point Qr deg. can be used as an argument for the function Fa for calculating the pressure difference Δ Pa. The function Fa may be a linear scaling function, e.g. the difference Qr-Qr may be multiplied by a predetermined scaling factor. The function Fa may also be a non-linear function, such as a monotonically increasing or increasing function. The function Fa may be unbiased, e.g., a zero argument may be mapped to a zero function value. The function Fa may be antisymmetric, e.g., such that Fa (-x) is-Fa (x).

In addition, the pressure Ps at which the liquid sample is injected into the preparation module can also be controlled 7.

Pressure Ps_n+1 may be different from the pressure Ps applied in the previous time step_nThe difference is Δ Ps ═ Ps_n+1-Ps_n. For example, the difference Δ Ps may be determined by calculating a value that depends on the difference between the measured flow characteristic and a predetermined target value of the flow characteristic (e.g., the difference between the measured flow rate Qr and the set point Qr). For example, the flow rate Qr measured in time step n_nAnd the set point Qr deg. can be used as an argument for the function Fs for calculating the pressure difference deltaps. The function Fs may be a linear scaling function, e.g. the difference Qr-Qr may be multiplied by a predetermined scaling factor. The function Fs may also be a non-linear function, such as a monotonically increasing or increasing function. The function Fs may be unbiasedFor example, a zero argument may be mapped to a zero function value. The function Fs may be antisymmetric, e.g. such that Fs (-x) — Fs (x).

For example, a closed-loop-control algorithm (e.g., a PID algorithm) according to embodiments of the invention may take into account the following system of equations:

Qr＝Qs+Qa

Qs＝(Ps–Pj)/(Rs+Rc)

Qa＝(Pa–Pj)/Ra

and Qr-P0/Rr when no additional pressure source is injecting liquid at pressure Pr into the routing module, or Qr-Pr/Rr when additional pressure Pr in the routing module is taken into account. The flow resistance Rr may act on the microfluidic flow in the routing module depending on where the additional pressure acts on the microfluidic flow in the routing module, for example with reference to the flow resistance of the microfluidic flow path from the inlet(s) of the routing module to the point of sample fluid in the routing module where the auxiliary fluid is incorporated into the microfluidic flow with pressure Pr in a second aspect, the invention also relates to a microfluidic routing device 110 (e.g. in the form of a cartridge, such as a disposable cartridge) for preparing a microfluidic flow of fluid saturated with an object of interest and for routing the object of interest in the microfluidic flow; for example for detecting, sorting, purifying and/or characterizing objects of interest in a liquid sample, i.e. entities of interest in a liquid sample. For example, the object of interest may comprise a biological entity of interest in a liquid sample (such as a body fluid sample). For example, the microfluidic device 110 may include an enclosure that encloses the below-referenced components of the device to form a cartridge. The apparatus 110 may be adapted to detect and purify an object of interest immersed in the microfluidic flow. For example, the apparatus may be an apparatus for biological cell sorting and/or biological cell routing in clinical applications.

The microfluidic routing device 110 includes an integrated sample preparation module 120 and an integrated routing module 105, such as a cell routing module, for example, for biological cell sorting. In particular, the preparation module 120 and the routing module 105 may be integrated in a single package, e.g., collectively on a single substrate.

The preparation module 120 is adapted to prepare a liquid sample in a microfluidic flow comprising an object of interest for processing (e.g. intended processing for detecting, sorting, purifying and/or characterizing the object of interest). The preparation module 120 comprises at least one microfluidic channel for transporting the liquid sample connecting at least one inlet of the preparation module to at least one outlet of the preparation module.

The preparation module and/or the routing module and/or the microfluidic connection are adapted to sense a flow characteristic of the liquid sample in the microfluidic flow, e.g. by means of an internal or external sensor element, to take the sensed flow characteristic into account for controlling the at least one flow control element by means of closed loop flow control to compensate for a deviation of the sensed flow characteristic from a predetermined target value of the flow characteristic.

In use, the volumetric flow rate of the liquid sample in the microfluidic flow may differ significantly between the at least one outlet of the preparation module and the at least one outlet of the preparation module, for example such that there may be a large difference in flow characteristics between the at least one input flow and the at least one output flow. As already mentioned above, the routing module may also involve a large difference in scale between the flow containing the entity of interest received from the preparation module and the total flow through the routing module (e.g., including the second flow for dilution, the flow carrying and/or directing the entity of interest).

The preparation module 120 may include a plurality of preparation stages, such as a sample concentration stage, a mixing stage, a dilution stage, an agitation stage, a staining stage, a cell lysis stage, and/or a cell dissociation stage.

The routing module 105 is adapted to divert objects of interest out of the prepared microfluidic flow of the liquid sample. The routing module 105 may be adapted to facilitate the detection of objects of interest in the microfluidic flow of the prepared liquid sample in the routing module by a detector of the system according to embodiments of the third aspect of the invention. For example, the routing module 105 may have components that are substantially transparent to allow optical detection through the routing module. The routing module 105 may comprise an actuation element for receiving a routing signal and for diverting a detected object of interest out of the main component of the microfluidic flow in response to the routing signal.

The preparation module (e.g., for biological cell concentration) and the routing module (e.g., for biological cell routing) are interconnected, e.g., by microfluidic connections, e.g., by one or more microfluidic channels, to forward the prepared liquid sample from the at least one outlet of the preparation module 120 to the at least one inlet of the routing module 105 in a microfluidic flow. The microfluidic flow between the at least one outlet of the preparation module and the at least one inlet of the routing module may be coupled through this microfluidic connection to passively buffer against and/or actively compensate in operation for flow rate variations of the prepared liquid sample in the microfluidic flow at the at least one outlet of the preparation module.

By integrating the preparation module and the routing module in a single integrated microfluidic routing device, the loss of (some of) the objects of interest in the liquid sample during transfer and/or manipulation between different phases (e.g. between the preparation phase and the routing phase) may be kept advantageously low or may even be substantially prevented.

Furthermore, tandem operations (e.g., pipelining) may advantageously provide low overall sorting times. In-stream concentration and mixing in the preparation module may also reduce the number of valves on the cartridge and thus may reduce costs and may avoid or reduce clogging.

The microfluidic connection (e.g., at least one interconnected microfluidic channel) may have a minimum cross-sectional dimension (e.g., channel width, height, and/or diameter) in the range of 1.1 times (e.g., 1.5 times) the size of the object of interest (e.g., target cell) to about 2 mm. In particular, the preparation module and the routing module may be interconnected without any intermediate storage banks, containers and/or buffers (e.g., in the general sense with respect to flow from the preparation module to the routing module) having a cross-sectional dimension greater than or equal to 5mm (e.g., greater than or equal to 2mm, such as greater than or equal to 1 mm).

The microfluidic connection may comprise a fluidic resistor 115 between the preparation module and the routing module for coupling the microfluidic flow between the at least one outlet of the preparation module and the at least one inlet of the routing module. The flow resistor 115 may have a flow resistance Rc. The fluidic resistor may be adapted to passively buffer against flow rate variations of the prepared liquid sample in the microfluidic flow at the at least one outlet of the preparation module.

The priming module 120 may have a flow resistance Rs, for example, between the at least one inlet and the at least one outlet of the priming module. Also, the routing module 105 may have a flow resistance Rr.

The flow resistance Rc of the fluidic resistor may be at least equal to a predetermined value corresponding to an expected variation or fluctuation of the flow resistance of the preparation module and/or the routing module.

The flow resistance value Rc may be at least equal to (e.g., preferably 2, 3, 5, or 10 times larger than) the variation and/or fluctuation Δ Rs, Δ Rr of the flow resistance of the preparation module and routing module. Thus, due to the significantly large flow resistance value Rc, the relative (e.g., percentage) flow rate fluctuations due to Δ Rs and/or Δ Rr may be maintained at acceptable levels.

For example, referring to the exemplary embodiment shown in fig. 2, if Pr is 0, Qr is Qs is Ps/(Rs + Rc + Rr). Then, Rc may be selected such that (Δ Rs + Δ Rr)/(Rs + Rr + Rc) < m, where m is a predetermined maximum flow rate fluctuation percentage. For example, Rc may be selected to be Rc > (Δ Rs + Δ Rr)/m-Rs-Rr. In embodiments where additional flow parameters (e.g., Ps, Pa, and/or Qa, as schematically illustrated in fig. 4) are to be taken into account, similar expressions may be readily obtained to estimate the appropriate flow resistance Rc.

The microfluidic routing device 110 may comprise an inlet junction 125 for the routing module 105 for injecting a secondary liquid flow into the microfluidic flow between the at least one outlet of the preparation module and the at least one inlet of the routing module, e.g. such that a microfluidic flow of a prepared liquid sample carrying an object of interest to be routed may be merged with the secondary flow before entering the routing module. For example, in operation of the device, at the inlet junction 125, a microfluidic flow of the prepared liquid sample carrying the object of interest to be routed may be merged with the auxiliary flow.

In a third aspect, the present invention relates to a microfluidic routing system 100, such as a cell routing system. Referring to fig. 8, a microfluidic routing system 100 includes a microfluidic device 110 according to embodiments of the second aspect of the present invention. The microfluidic routing system also includes a meter device 160. The microfluidic routing device 110 may be loaded into a meter device 160 for analysis and/or processing of a sample. The microfluidic routing device 110 may be disposable, for example, may be a disposable cartridge. The microfluidic routing device may include functional components for physically processing the sample, such as sample and/or reagent tanks, cell preparation components, cell routing elements, and/or post-sort processing components. The meter device 160 may include all peripheral hardware and/or software for facilitating operation of the microfluidic routing device, e.g., for system level control and providing a user interface to actuate and control fluidics in the routing device, for light detection of light sources and signal processing for fluorescence detection and/or cell imaging, for controlling and driving electrical components in a cartridge of the routing device, and/or for post-sort cell distribution (e.g., transfer of sorted cells to vials or glass slides in a sorted cell suspension). In particular, the hardware of the meter may advantageously be designed to avoid physical contact with the meter, for example to improve sample processing accuracy with a minimal likelihood of sample contamination.

The system may comprise a detector for detecting an object of interest in a microfluidic flow of the prepared liquid sample in the routing module.

The detector may comprise an optical detector, for example for obtaining optical detection signals, such as fluorescence, bright-field, dark-field and/or scatter signals and/or images of the object (e.g. microscopic images, holographic images and/or diffraction images).

The system may comprise a processor for determining at least one characteristic feature of the object of interest in the optical detection signal.

The processor may be adapted to calculate a routing signal in response to detecting the at least one characteristic feature and to control the actuation element of the routing module by means of the calculated routing signal to divert the detected object of interest out of the main component of the microfluidic flow.

The system may be adapted to calculate a routing signal in response to detecting the at least one characteristic feature, wherein the routing signal is adapted to control the actuation element at a suitable time, e.g. after a suitable delay taking into account a movement of the object from the detection position to a position where it is to be turned out of the main component of the microfluidic flow by means of the actuation element. Furthermore, the signal may be calculated to control the actuation element for a suitable duration, for example taking into account the flow rate, size, mass and/or volume of the object of interest. Furthermore, the signal may be calculated to control the actuation element with a suitable strength, e.g. the force exerted by the actuation element on the object of interest may depend on the signal strength of the control signal provided to the actuation element.

The microfluidic routing system may include a first fluidic actuator, such as a fluid pump, for injecting the liquid sample into the preparation module 120 at a first pressure Ps.

The microfluidic routing system may comprise a second fluid actuator, e.g. a fluid pump, for injecting liquid into the microfluidic flow of the prepared liquid sample at a second pressure Pr in the routing module 105.

The microfluidic routing system may include a third fluid actuator, such as a fluid pump, for injecting a secondary fluid flow into the microfluidic flow at a third pressure Pa (e.g., via inlet junction 125) between the at least one outlet of preparation module 120 and the at least one inlet of routing module 105.

The first, second and/or third fluidic actuators may form part of the microfluidic device 110 or may be partially implemented in the microfluidic device. For example, the fluid handling components of the fluid actuators may preferably be implemented in the microfluidic device, while the actuation and/or power components of the fluid actuators may be implemented in the microfluidic device 110 or in the meter device 160.

The microfluidic routing system may comprise at least one pressure detector, such as a pressure gauge, for monitoring and/or measuring the first pressure Ps, the second pressure Pr and/or the third pressure Pa and/or the fourth pressure Pj at the inlet(s) of the routing module. For example, a fourth pressure Pj may be measured between the entry junction 125 and the routing module 105.

The microfluidic routing system may comprise at least one sensor 95 for sensing a flow characteristic of the liquid sample in the microfluidic flow in the preparation module 120 and/or the routing module 105 and/or in the microfluidic connection between the preparation module and the routing module 105.

The at least one sensor 95 may comprise a flow rate sensor, a fluid presence detector, or a fluid optical parameter detector. For example, the flow characteristic may be flow front detection, wherein the presence of a fluid carrying the object of interest is detected at the sensing location due to a change in light transmission, light reflection or similar optical properties. For example, the sensor may comprise a light barrier, as illustrated in fig. 6.

The at least one sensor may comprise at least one flow detector for monitoring and/or measuring a flow rate Qs indicative of flow through the preparation module, and/or a flow rate Qr indicative of flow into or through the routing module, and/or a flow rate Qa indicative of the auxiliary flow.

The at least one flow detector may comprise a flow meter, such as an acoustic flow sensor and/or a thermal flow sensor. Alternatively, the flow rate Qr may be estimated by the processor of the system from the detected velocity and/or detected frequency of objects detected in the flow in the routing module.

The meter apparatus 160 may include a controller for controlling the first pressure Ps, the second pressure Pr, and/or the third pressure Pa.

The controller may also be adapted to control at least one fourth pressure Px (e.g. of a further fluid source in a stage forming part of the preparation module), e.g. injection, as schematically illustrated in the example of fig. 9.

For example, the controller may be adapted to control at least one flow control element, e.g. an actuation element, e.g. a microfluidic valve or a microfluidic switch, to control the first, second, third and/or further pressure.

For example, the at least one flow control element may comprise a flow source, such as a second flow source. For example, the at least one flow control element may comprise a controllable fluid pump. For example, the at least one flow control element may comprise a first fluid actuator, a second fluid actuator and/or a third fluid actuator and/or a further fluid actuator.

The controller may be adapted (e.g., configured, e.g., programmed) to perform closed loop flow control to compensate for deviations in the sensed flow rate from predetermined target values of the flow characteristics.

Alternatively or additionally, the controller may be adapted (e.g., configured, e.g., programmed) to activate the at least one flow control element in response to the sensed flow characteristic, e.g., to activate a stage or module, e.g., to activate a fluid source in the stage or module when a moving fluid front is detected upstream of the stage or module.

The controller may be configured to adjust the first pressure Ps by taking into account deviations indicative of the flow rate Qr of the flow into or through the routing module from a predetermined target value.

For example, the controller may be adapted to control the second pressure Pr.

For example, the controller may be adapted to adjust the third pressure Pa by taking into account a deviation of the flow rate Qr from a predetermined target value.

The system (e.g., microfluidic routing device 110) may include at least one post-processing module 130, e.g., a post-sort processing component, e.g., for manipulating a fluid sample, e.g., a selected portion of a fluid sample.

For example, such post-processing may include a review unit 140 for reviewing selected portions (e.g., sorted objects). The review unit may be adapted to image or facilitate imaging of these objects. The review unit may be adapted to reroute the object.

The post-processing module may comprise a distribution unit 150 for distributing the object in a suitable output form, e.g. after the object has been selected by the routing device.

Fig. 9 illustrates an exemplary microfluidic routing device 110 according to various embodiments of the present invention. For example, such devices may be implemented on advantageously limited footprint, e.g., 10cm x 6cm chips. It will be understood by those skilled in the art that the dimensions indicated on the drawings and provided in the present description are exemplary only and are not intended to limit the invention in any way.

A liquid sample comprising an object of interest may be provided via a reservoir or inlet 181. For example, the device may be adapted to process 2mL samples. The sample preparation module may include multiple stages.

In the first stage 82, the sample may be concentrated. During this stage, a significant portion of the liquid may be separated as waste liquid 83, e.g., up to about 2 mL. The concentration may be performed, for example, by acoustic focusing. For example, the piezoelectric disc 84 may apply vibrational energy to the device 110 in the first stage 82. In the second stage 85, the object of interest in the sample may be stained. For example, 50 μ Ι _ of dye (which can be added to the microfluidic flow) from reservoir 86. In a third stage 87, the sample fluid may be washed, for example by a concentration step, for example using another piezoelectric disc for acoustic focusing, followed by a mixing step in which 80 μ Ι _ of buffer from a reservoir 89 is blended using a serpentine mixer 88. The fourth stage 90 may be by final concentration. For example, acoustic focusing may again be used to further concentrate the sample fluid. The microfluidic flow obtained at the outlet of the fourth stage 90 may then be fed into the inlet of the routing module 105. In this routing module, another buffer fluid (e.g., 3mL of buffer from reservoir 91) may be mixed into the sample fluid. The output of the routing module 105 may, for example, provide objects of interest that are routed out of the sample (e.g., collected in the repository 92, e.g., in a 0.5mL collection container) and a by-product of the routing stage (e.g., in the 3mL waste repository 93).

Exemplary flow rates throughout this exemplary apparatus are illustrated in fig. 10. Fig. 10 also shows a timing diagram to illustrate the overall dwell time for the entire sample to be processed through each stage. The various values indicated in the drawing are exemplary only and are in no way intended to limit the invention.