Patent Application
20240424493
The present invention relates to microfluidics, in particular to generating microdroplets encapsulating samples such as microparticles, cells, or any other deformable samples and sorting the microdroplets using droplet-based microfluidic devices encapsulating samples in an efficient manner.
Precision biological cell handling tools are needed in research and production to enable efficient manipulation of cells or other samples. Current methods for single cell encapsulations in droplets, for instance, are inefficient and waste costly materials. Particularly, the separation of cells from a carrier fluid into microdroplets not much larger than the cell itself, allows a simplified further processing of the cells in succeeding microfluidic modules configured to manipulate discrete droplets.
In general, droplet-based microfluidic devices can be configured to perform a variety of operations such as transportation of droplets, storage of droplets, mixing of droplets, analysis of droplets and the like. For example, these devices can be used as microreactors to encapsulate various biological entities for biomedicine and biotechnology applications.
Droplet-based single cell assays are based on the ability to encapsulate and confine single cells in individual droplets and e.g. enable high efficiency genome-wide expression profiling. For instance, encapsulation of one-cell-one-bead in droplets is an essential operation for high throughput screening of single cells and droplet sequencing for e.g. transcriptomic analyses.
Matula, Kinga, Francesca Rivello, and Wilhelm T S Huck. “Droplet Microfluidics: Single-Cell Analysis Using Droplet Microfluidics (Adv. Biosys. 1/2020).” Advanced Biosystems 4.1 (2020): 2070012 review the various droplet microfluidic strategies reported in the literature, with a focus on approaches to targeted-and whole-genome analysis in droplet-encapsulated single cells, as well as whole-transcriptome profiling techniques. Si Da Ling, Yuhao Geng, An Chen, Yanan Du, and Jianhong Xu, “Enhanced single-cell encapsulation in microfluidic devices: From droplet generation to single-cell analysis”, Biomicrofluidics 14, 061508 (2020), https://doi.org/10.1063/5.0018785 explore the field of single-cell encapsulation and analysis, and provide an overview of the droplet formation mechanism, fabrication methods of microchips, and several of passive and active encapsulation techniques to enhance single-cell encapsulation. However, as most of the techniques for passive single cell encapsulations in droplets are performed randomly under the regime of Poisson statistics, the number of cells inside each droplet is generally unknown, raising major concerns for downstream analysis, including e.g. quantification, screening, merging for one-bead-one-cell and/or one-cellA-one-cellB encapsulation. Moreover, potential harm toward encapsulated cells, meticulous manipulation of flows or very long operation time may further happen during implementation of such passive methods. Accordingly, one major challenge in performing droplet sequencing is achieving high efficiency for one-droplet-one-cell or one-cell-one-bead encapsulation, while preferably operating with high-throughtput and safeguard of the sample.
It is an object of the present invention to provide a microfluidic device for encapsulating deformable samples such as cells, microparticles of the like in microdroplets which can be easily separated from empty droplets, in such way to deterministically obtain single deformable samples in each droplet, i.e. sample droplets. In an additional aspect, the device and method according to the invention can be suitably used for producing child droplets, i.e. sampleless droplets, deriving from the breakup of a parent droplet, that is, for splitting a droplet into different sizes of child droplets.
It is a further object of the present invention to provide a microfluidic device which allows to separate sample droplets including deformable samples from empty droplets in a reproducible manner.
This object has been achieved by the microfluidic device according to claim 1, the microfluidic system and a method for operating the microfluidic device according to the further independent claim.
Further embodiments are indicated in the dependent subclaims.
According to a first aspect of the present invention, a microfluidic device is provided comprising a droplet-splitting junction. The droplet-splitting junction comprises:
Furthermore, an aspect ratio may be defined as λ=hi/wi said aspect ratio is λ>1, wherein particularly said aspect ratio is 1<λ<10.
Also, a width ratio may be defined as φ=wi/wo, said width ratio is φ>1, wherein particularly said width ratio is 1<φ<10.
In one embodiment, two outlet channel branches may be provided which extend perpendicularly with respect to said inlet channel.
The passage point may correspond to an intersection P which acts as a droplet-splitting junction for droplets passing the breakup point and which may be located in the center of the outlet channel, thereby separating the outlet channel into the two equal outlet channel branches.
Furthermore, the outlet of each outlet channel branch may be connected with a channel portion with a widening cross-section with an inclination angle of at least one channel wall of between 1 to 30°.
Only one outlet channel branch may be provided, wherein the outlet channel branch is running coaxially to the inlet channel.
The microfluidic device may be comprised in a microfluidic system. The microfluidic system may be operatively connected with a droplet source, wherein the droplet source provides parent droplets of a sample fluid floating in a carrier fluid, wherein the sample fluid and the carrier fluid are non- or minimally mixable, wherein particularly the sample fluid is a watery solution and the carrier fluid is an oil or wherein the sample fluid is an oil and the carrier fluid is a watery solution.
According to a further aspect, a method for operating the above microfluidic device for producing sample droplets is provided, wherein a parent droplet of a sample fluid in a carrier fluid is flown through the inlet channel to the intersection, wherein flow rate, parent droplet length and capillary number are selected to promote a central breakup of the droplet thread at the intersection, wherein flow rate, parent droplet length and capillary number are further selected so that when a deformable sample is included in the parent droplet the central breakup of the droplet thread is delayed so that a breakup occurs instead in at least one of the outlet channel branch downstream the intersection thereby forming a small sample droplet and at least one larger child droplet.
According to a further aspect a method for setting up operation of the above microfluidc device is provided, wherein parent droplets of a sample fluid in a carrier fluid are flown through the inlet channel to the passage point, wherein flowrate, parent droplet length, and capillary number are selected to promote a breakup of the droplet thread at the passage point wherein at least the parent droplet length and capillary number are selected by the steps of:
With the geometric condition the above method allows to find an operating point in terms of droplet length and capillary number which can be varied by the flowrate once the kind of droplet fluid and carrier fluid are chosen.
The capillary number is the total effect of viscosity, surface tension and flowrate. Capillary number (Ca) can be tuned by adjusting the flowrate once the sort of fluids is fixed which determine viscosity and surface tension. The critical capillary number regarding a transition from central breakup to a lateral or step breakup in at least one outlet channel branch (for a given length of droplet) should be fixed as long as the geometry is fixed.
According to a further embodiment, the parent droplet may contain a deformable sample which has a cross-section that corresponds to a critical dimension (smaller value of width and height) of the outlet channel or has a cross-section of between 90% and 200% of the critical dimension of the outlet channel, wherein the sample is compressible under exertion of an external force to a cross-section across the critical dimension of the outlet channel which has a size of 40 to 95% of the cross-section of the non-compressed sample. Basically, the sample may be more rigid or more viscous than the fluid of the droplet.
To operate the above microfluidic device, parent droplets are supplied through the inlet channel which may possibly contain deformable samples, such as biological cells, micro-organisms, microparticles, pollens, deformable beads, or the like. At low to medium capillary numbers, i.e. less than a critical capillary number, the inflowing parent droplets experience a lateral break-up in the outlet channel branches after having passed the intersection at the droplet-splitting junction.
The capillary number (Ca) is defined as a dimensionless quantity representing the relative effect of viscous drag forces versus surface tension forces acting across an interface between two immiscible liquids. The critical capillary numbers depend on the droplet length and the aspect ratio h/wo of the outlet channel branch as well as the width ratio wi/wo of the inlet and outlet channel. The critical capillary number Cacrit is given with respect to a ratio of the droplet length L in the inlet channel and the width wo (normalization) of the outlet channel by L/wo=q/Cacrit, where q may be between 0.1 and 30, preferably between 0.1 and 10. The critical capillary number Cacrit depends on the aspect and the width ratios wherein the larger the aspect ratio or the width ratio are, the higher is the critical the capillary number Cacrit for the same droplet length L.
The lateral break-up is characterized by necking and separation of the droplet downstream the intersection where the parent droplet has been split. To reduce the stress on incoming samples a widening channel portion may be provided with a widening cross-section with an inclination angle of at least one channel wall of between 1 and 30°. In other words, the lateral breakup defines a dividing of the split droplet in each of the outlet channel branches. When the capillary number exceeds the critical capillary number Cacrit which depends on the droplet length L, the droplet will experience a central break-up instead of the lateral breakup.
The central break-up is characterized by necking and separation of the droplet directly at the intersection where the parent droplet is divided into the different outlet channel branches. The central break-up process occurs simultaneously with the development of the lateral break-up process in the background and stops by the preemptive occurrence of the central break-up.
The overall operation regime for the proposed configuration shall be the central break-up regime that is close to the transition into the lateral breakup regime. This transition is caused/determined by a time competition of central breakup regime and the lateral breakup regime. This means that although the break-up fate for any incoming droplet is the central break-up, the underlying lateral break-up process is also nearly accomplished, which is achieved when the process is carried out close to the critical capillary number Cacrit, i.e. within a range of 0 to 50% above the critical capillary number Cacrit.
It has been found that when a parent droplet contains a deformable sample such as a biological cell, the cell is temporarily detained at the intersection due to its larger size than the outlet channel branches. This leads to an increase of pressure at the intersection which delays the central breakup and shifts the breakup mode to the lateral breakup. The lateral breakup dominating the central breakup has the result that a small satellite droplet, a sample droplet, around the intersection is created which is a droplet that confines the sample. Substantially at the same time, two large child droplets are formed from the same parent droplet. They also have a similar size as those which would have been created from a central break-up for any parent droplets not containing samples.
One major advantage of this microfluidic device together with its operating method is that the sample droplets containing samples are much smaller in size than the child droplets which are formed by the lateral break-up. The size difference is essential, as the sample droplets containing the deformable samples can be easily separated from the large child droplets in a succeeding sorting module which may be part of the microfluidic system.
As a precondition, to enable an operation of the microfluidic device close to the transition between lateral break-up and central break-up, the microfluidic device has to be geometrically configured to reliably/robustly work in the lateral break-up regime. It has been found that a droplet splitting junction should have an aspect ratio between height ho and width wo of the outlet channel and a width ratio of the width wi of the inlet channel and the width wo of the outlet channel both larger than 1.
In addition, the relation of
with z>1 should be fulfilled. This relation reflects that when ho>wo is fulfilled, the limiting dimension becomes wo, thus only wo appears in the z criterion.
Geometries satisfying above condition intrinsically provide enough confinement difference and thus instability in the droplet that will allow lateral break-up to occur. Increasing either of the above ratios in the geometry design will enhance the lateral break-up regime. Extremely enhanced ratios may negatively affect the operation method as they cause a too strong lateral break-up regime that is unfavorable for the process. For large width ratios, such as width ratios wi/wo>10, no central break-up regime can be observed preventing the use of the technique. For large-aspect ratios, such as aspect ratios h/wo>10, it has been observed that several lateral break-ups may occur for each incoming parent droplet causing potential problems for size sorting carried out after sample droplet generation.
The performance of the microfluidic device operated according to the above method can be influenced by either increasing the aspect ratio h/wo or the width ratio wi/wo. If one of the aspect ratio and the width ratio is increased, the transition curve is shifted towards higher capillary values which may increase the throughput. As described above, increasing the aspect or width ratio enhances the lateral break-up which significantly alters the relation between the break-up regimes. Basically, the operation state of the microfluidic device which is defined by the geometry of the microfluidic device as well as the operation parameters shall be selected so that the lateral and central break-up will normally occur in a timely close manner, i.e. the time gap needed to be overcome by a deformable sample temporarily stuck at the intersection is as low as possible. In general, enhancing the lateral break-up by increasing the aspect ratio and/or width ratio could possibly improve the throughput and sensitivity of the operation method.
Compared to techniques as known from prior art the proposed microfluidic device allows a robust operation for sample encapsulation where no fine tuning is required after first experimental effort for the mapping. Furthermore, the method allows an easy up-scaling because of the passiveness. Also, as the operation of the microfluidic device does not depend on the sample concentration it can be applied for a wide range of application. Basically, the widening channel portion serves to release the confinement stress for the sample (e.g., cell) to avoid damage and other adverse impacts. Therefore, the inclination angles may be within a high range of between 1-30° for one or multiple channel walls. The length of the outlet channel branch has to be selected sufficiently long for enabling the lateral breakup regime but should not be too long to avoid stress on the sample. Particularly the length N of each outlet channel branch should be related to the parent droplet length L such that 0.2<N/L<2. All combinations of these design parameters have produced the necessary breakup regime competition required by this technique.
In addition, the working principle requires that the sample to be encapsulated is retained temporarily at the intersection and will eventually pass through one of the outlet channel branches. This limits the use of the operating method to deformable samples, including but not limited to biological cells and other soft particles. Accordingly, the outlet width wo should be slightly smaller than the diameter of this sample (in addition to fulfilling the lateral breakup enabling criterion).
In an alternative embodiment, the outlet of each outlet channel branch is connected with a channel portion with a step-like widened cross-section (not with an inclination) which has at least a cross-section of more than 200% of the cross-section of the respective outlet channel branch.
In this second embodiment, the microfluidic device has no slowly widening outlet channel, but an outlet channel that adds at a wider reservoir without slowly widening its cross-section. While basically the geometry parameter and operating conditions remain as explained above, it leads to an operation mode where no lateral break-up occurs, but a step break-up when the droplets enter the reservoir formed by the widened channel portion. The step break-up is defined that instead of a lateral breakup a breakup occurs when the child droplet enters the wider channel portion. In the wider channel portion the child droplet starts to form a bulk that will grow with time with increasing radius. This bulk connects to the main thread of the child droplet in the respective outlet channel branch through a neck near the step-like widening of the outlet channel branch into the channel portion.
Due to the pressure drop between the neck and the bulk, the curvature of the bulk decreases due to the growth of the bulk. This increases the pressure difference so that the droplet fluid flow from the neck to the bulk will increase. When the outlet flow becomes larger than the inlet flow, entering the intersection the neck will become thinner and thinner and eventually leads to the break-up at the step-like widening into the channel portion.
The earlier the bulk starts to form, which can be achieved by a longer droplet or shorter length of the outlet channel branch, step break-up will be more prominent. On the contrary, when the capillary number is large (e.g. above a critical capillary number which may be determined experimentally), especially during central break-up, then the separation fluid, e.g. oil pinches the interface at the intersection and pushes the droplet thread into the outlet channel branches, the flow entering the inlet channel is high, compensating the necking effect. As a result, the process of the step break-up will cease and only central break-up will occur.
At the target flow condition, the parent droplet passing the intersection will experience central break-up, while the flow entering the intersection and the flow after the stepwise widening of the outlet channel is such that the step break-up is almost achieved, but is canceled by the occurrence of a central break-up.
When a supplied parent droplet contains a deformable sample, the sample obstructs the intersection which will see an enormous increase in resistance, thus significantly reducing the flow entering through the intersection first into both outlet channel branches and then in only the one outlet channel branch through which the sample is flowing. When the outlet flow of the obstructed outlet channel branch remains constant, the necking process can re-boost, and step break-up will eventually happen at the respective channel portion through which the deformable sample will pass.
In contrast, the other outlet channel branch which has not been obstructed with the deformable sample, experiences an increasing flow through the intersection compared to cases in which no deformable sample is included in the parent droplet. The necking will still be compensated preventing the step break-up process. Finally, the deformable sample will be encapsulated in a small droplet generated in one of the outlet channel branches due to the central break-up and a child droplet formed by a step break-up on the respective other outlet channel branch. The deformable sample is therefore caught between two empty child droplets originating from the same parent droplet. The size of the sample droplet is only determined by the geometry of the length of the outlet channel branches and the height of the outlet channel branches.
As described above in conjunction with the working scheme between the central breakup and the lateral breakup not all microfluidic devices with a droplet-splitting junction can produce the step breakup regime. However, in contrast to the working principle of the microfluidic device with the slowly widening channel portion, each parent droplet including a sample experiences different breakup schemes consisting of a step breakup and a central breakup. This is because these competing breakup regimes occur geometrically far apart, and achieving one will not necessarily stop the other. In fact, there is never a stand-alone step breakup it is always accompanied with one last breakup close to the intersection: be it a central breakup or a lateral breakup (which one exactly depends on the same physics as described before).
On the contrary, it is possible to have only the central breakup in the scheme—It just requires the flow from the intersection pushes the droplet thread enough such that the step breakup process is suppressed and cannot accomplish before the central breakup finally finishes. In fact, the ideal scheme as explained above is that for all parent droplets the central breakup occurs, but the development of the step breakup is also timely close but interrupted by the central breakup.
Such scheme requires geometric conditions and a specific operating method. It has been found that the geometric conditions require that the outlet channel branches connect to the widened channel portion forming a wider chamber at both ends of the outlet channel branches thereby creating the step. In addition, the aspect ratio h/wo should be large enough.
According to a further embodiment, just one outlet channel branch may be provided, wherein the outlet channel branch is running coaxially to the inlet channel.
According to a further embodiment, the droplet source comprises a droplet generating unit operatively connected with a reservoir and with the inlet of the inlet channel. Particularly, an operatively connected pressure source may be configured to apply pressure inside the microfluidic device to move a parent droplet from the droplet source towards the passage point.
In a further embodiment, the microfluidic system may further comprise merging channels connected with the outlets of the outlet channel branches to merge the droplet flow through the outlets of the outlet channel branches into a merging point. For instance, the merged droplet flow may be fed to a sorting module for sorting the droplets by size to filter out smaller sample droplets generated by the microfluidic device from larger child droplets.
It may be provided that the above microfluidic device or the above system are used for the production of sample droplets as droplets containing a deformable sample included in a parent droplet and deriving from the breakup of a parent droplet into at least one child droplet in at least one outlet channel branch downstream the passage point.
According to a further embodiment, the above microfluidic device or the above system may be used for the production of droplets from a parent droplet and deriving from the breakup of the parent droplet into at least one child droplet in at least one outlet channel branch downstream the passage point.
According to a further aspect, a method for operating the above microfluidic device for producing sample droplets is provided, comprising the step of:
According to an embodiment, the parent droplet may contain a deformable sample which has a cross-section that corresponds to a critical dimension of the outlet channel or has a cross-section of between 90% and 200% of the critical dimension of the outlet channel, wherein the sample is compressible under exertion of an external force to a cross-section across the critical dimension of the outlet channel which has a size of 40 to 95% of the cross-section of the non-compressed sample.
Furthermore, a separation module may be provided, where the separation module comprises:
This separation module can be applied to any microfluidic devices providing both sample droplets and sampleless droplets in a continuous carrier fluid stream, such as microfluidic devices as described above. Instead of from the outlet channel branch droplets can be received from any outlet openings providing such mixture of sample and sampleless droplets.
Moreover, the separation chamber may be directly connected to the receiving inlet and having a first wall extending with a first angle of between 0° and 45° with respect to an axial direction of the at least on outlet channel branch at the outlet opening and an opposing second wall extending with a second angle with respect to an axial direction of the at least on outlet channel branch at the outlet opening which is larger than the first angle.
The separation element may be formed as a structure with separation walls located between the trajectories of the sample droplets and the child droplets, wherein the wall particularly separates different droplet channels for the sample droplets and the sampleless droplets.
For the afore-mentioned embodiments, there is a further task to separate sample droplets from sampleless droplets in a reproducible manner. The droplet separation may be based on a newly found effect which occurs if the outlet channel from which sample droplets and sampleless droplets are released, extends into an asymmetric separation chamber.
The asymmetric chamber is formed in a preferred case with a first wall directly connected with the receiving inlet and having an angle of 0° and a second wall having an angle of 90° with respect to the axial direction. A droplet that relaxes quickly is pushed away from the first wall immediately when entering the receiving inlet because of the remaining contact to the first wall and the release from the channel walls of the respective outlet channel branch. If a droplet is more viscous or if its relaxation from the deformed state in the outlet channel to a substantially circular shape is retarded by any means such as presence of a sample squeezed in the narrow section of the outlet channel, the droplet propagates longer in axial direction so that it will be less deviated.
Hence, the asymmetric geometry of sidewalls of the separation chamber results in the physical turning of all droplets from axial direction to the lateral direction. Stiffer droplets turn later which means that they follow the axial direction for a longer time while softer droplet turn earlier.
As an essence of the above-described effect, while the sample and sampleless droplets moved through the outlet chamber are exerted by the dimensions of the outlet channel, the asymmetric widening of the separation chamber allows to make use of different relaxation behaviors of sample droplets and child droplets.
As am possible explanation of the effect, when the sample droplets and sampleless droplets arrives at an outlet opening at the end of the outlet channel, their deformations are substantially influenced by the presence of a deformable sample in the droplets. Sampleless droplets moving through the outlet channel have concave rear caps with interfaces attached to the walls of the separation chamber, while for sample droplets the deformable sample occupies the rear capped region which helps the rear cap withstand the concaving deformation as this region is more rigid than the corresponding region of the droplet fluid. This results in different droplet attachment behaviors to the part of the walls of the separation chamber directly abutting the outlet of the outlet channel and further influences the following droplet relaxation process and their movements.
Furthermore, what has been found is that in a separation chamber which extends from an outlet opening of the outlet channel and which has an asymmetric cross-section with respect to an axial direction of the outlet channel, the sample droplets have a higher speed vector in the axial direction of the outlet channel compared to sampleless droplets without any deformable samples. This effect also helps separating the trajectories of sample droplets and sampleless droplets.
Due to the asymmetric shape of the separation chamber, there is further a radial flow scheme of the carrier fluid originating from the outlet opening which transports the droplets also in a direction perpendicular to the axial direction of the outlet channel. This results in amplification of the initially formed different trajectories of sample droplets and sampleless droplets in the separation chamber. The separation of the droplets can then be easier achieved by including a separation wall which separates the fluid streams containing the sample droplets and the sampleless droplets, respectively.
Embodiments are described in more detail in conjunction with the accompanying drawings, in which:
The microfluidic system 1 comprises a droplet source 2, a microfluidic device 3, a merging portion 4, and a sorting module 5.
The droplet source 2 includes a reservoir 21 which contains a sample fluid containing samples. The droplet source 2 may include a droplet generating unit 22, such as those described for instance, and without limitation, in Chong, Zhuang Zhi, et al. “Active droplet generation in microfluidics.” Lab on a Chip 16.1 (2016): 35-58, generating parent droplets P of the sample fluid. Parent droplets P are generated in a carrier fluid. The sample fluid and the carrier fluid are non-mixable or minimally mixable, wherein particularly the sample fluid is a watery solution and the carrier fluid an oil or wherein the sample fluid is an oil and the carrier fluid is a watery solution.
The droplet source 2 may comprise an operatively connected pressure source 23 configured to apply a pressure towards the microfluidic device 3 to move parent droplets P from the droplet generating unit 22 towards the microfluidic device 3.
The microfluidic device 3 is configured and operated to produce sample droplets S and child droplets C as described below. The microfluidic device basically comprises a T-junction splitting parent droplets P fed from an inlet channel 31 to two outlet channel branches 32.
The child and sample droplets C, S are produced in the two outlet channels branches and fed to the merging portion 4 where the fluid flows are mixed to merge the two separate flows into one which then is fed to a sorting module 5.
The sorting module 5 may be configured to separate droplets by size. As the sample droplets S are smaller than the child droplets C the sample droplets S containing deformable samples will be directed into one or more sample droplets collection channel and the other child droplets C into a waste collection channel. The sorting module 5 may comprise one or more electrodes that sort the droplets by dielectrophoresis (DEP) so that droplets are manipulated in non-uniform electric fields. The movement of droplets in DEP is based on the difference in polarizability between the droplets and the surrounding medium.
Furthermore, the sorting module 5 may comprise a lateral cavity across the transducer (LCAT) that sorts the droplets exploiting the phenomenon of acoustic microstreaming to manipulate fluid flow. This is e.g. described in US 2014/0011291 A1. Preferably, due to the large size difference of child and sample droplets C, S, sorting can be accomplished in a passive way, using methods like Deterministic lateral displacement (DLD) or Pinched Flow fractionation (PFF). Active sorting involving electric and/or acoustic forces is not necessary.
The microfluidic system 1, therefore, aims to separate deformable samples from threads of parent droplets P flowing through the inlet channel 31 towards the sorting module 5. The parent droplets P are supplied by a droplet source 2 including a droplet generating unit 22 providing parent droplets of a sample fluid in a carrier fluid. However, in alternative embodiments, the droplet source 2 may comprise already formed parent droplets that can be released inside the system by e.g. pumping means well known in the art.
According to a first embodiment, the microfluidic device 2 has a configuration that is shown in more detail in
The deformation of the samples may be so that it can be compressed under exertion of an external force to a cross-section that has a size of 40 to 90% of the cross-section of the non-compressed sample.
As shown in more detail in
The microfluidic device 3 is operated with a low capillary number so that the parent droplets P experience a lateral breakup after passing the intersection as schematically shown in
At low to medium capillary number (Ca), the parent droplets P experience a lateral breakup LB at the T junction. When above the critical capillary number, the droplet breakup fate becomes a central breakup CB. The central break-up defines a state where the droplet which has already extended into the two outlet channel branches experiences a central necking and eventually breaks up at the intersection, thereby forming two separated child droplets. While in the central breakup regime, the lateral breakup process is ongoing in the background and is canceled at the time instant of the preemptive occurrence of the central breakup CB.
By varying the droplet length and capillary number on a T-junctions that fulfill the geometric condition, the critical capillary number for each of the droplet lengths can be determined as in a mapping shown in
In a different representation of
This means, although the breakup regime for any incoming parent droplet P shall be set as the central breakup, the underlying lateral breakup LB process is also nearly accomplished. This is e.g. shown in
As shown in
To enable the lateral breakup regime, any T-junction should have both the aspect ratio (ho/wo) and the width ratio (wi/wo) larger than 1. More specifically, a relation of
should be fulfilled. Defining a z number:
it requires z>1 to have the lateral breakup, which corresponds to the shaded region above the curve in
There are two ways the geometrical parameters can influence performance. One is when either aspect ratio or width ratio (or both) are increased, the transition curve is shifted towards high capillary numbers, as if the power law is timed a larger coefficient. This influences the throughput of the technique, which is increased with higher capillary numbers.
Second, as increasing either ratio would enhance the lateral breakup, such geometrical modification(s) would significantly alter the competition between the two breakup regimes. On an exemplary geometry of wi=12 μm, wo=30 μm, h=37 μm, the time difference between the instant of achieving lateral breakup and the time instant of achieving the central breakup is so large that at any operating point close to the transition region both breakups will not occur at the same moment. However, this can be observed in the geometry of wi=12 μm, wo=30 μm, h=62 μm. It indicates that in the latter case, the time for the lateral and central breakup process to finish is quite close, meaning the time difference needed to be overcome by a cell temporarily obstructing the intersection is reduced. Thus, the sensitivity of cell triggering breakup regime is increased.
Therefore, enhancing the lateral breakup by increasing the aspect and/or width ratio within a defined range, can improve both the throughput and sensitivity of this technique.
The other geometrical parameters of the sidewall angle θ of the channel portion and the length N of the outlet channel branch 32 only have a secondary effect. N has to be sufficiently long for the lateral breakup regime but should not be too long to avoid additional stress on the sample. The angle θ of one side wall with respect to the associated sidewall of the outlet channel branch 32 should be between 1 to 30° while the length N should be 0.2<N/L<2. All combinations have produced the necessary breakup regime competition required by this technique. However, N=25 um may be considered as the lower limit for the sample droplet generation in general applications.
In addition, the working principle requires that the sample to be encapsulated is retained temporarily at the junction and will eventually pass through the outlet channel. This limits the use of the T junction technique to deformable samples. Accordingly, the outlet width wo should be slightly smaller than the cross-section of these samples (in addition to fulfilling the lateral breakup enabling criterion).
As shown in
Hence, the lateral breakup regime as described before will be completely suppressed by the chosen fluidic condition formed by the step-like widening channel portion. The step breakup is defined by a process where a droplet will be broken exactly at the step between the outlet channel branch 32 and the step-like widened channel portion 36 it is coupled with, as illustrated in
At the operational condition, it is the central breakup, as described before, and this step breakup (instead of the lateral breakup) that are dominant and in competition. Here, it can be provided a new mechanism for creating cell-triggered droplet formation, which allows to better control and increase the size of the sample droplets, with preferably higher sensitivity, without departing from the general inventive concept.
The geometrical parameters N, wi, wo and h are defined as described in conjunction with the first embodiment. Instead of a slight expansion, there is now a wide reservoir connected to each end of the outlet channel branches, with an angle θ>80°.
As shown in
At the target flow condition, the parent droplet passing the intersection 33 will experience a central breakup, but due to the balance of the flow condition of the flow in the outlet channel branch, and the flow entering the channel portion 36, the step breakup is almost achieved but is canceled by the timely preceding occurrence of the central breakup. When a parent droplet contains a cell (as deformable sample), the cell obstructs one outlet branch after passing intersection 33, which will see an enormous increase in resistance thus significantly reduced the flow entering one of the outlet channel branches. With the flow exiting the respective outlet channel branch 32 remaining constant, the necking process can reboost, and step breakup will eventually happen in this outlet channel branch 32, as illustrated in
In contrast, the non-obstructed outlet channel branch 32 receives a higher flow compared to the process without a cell contained in the parent droplet, as an additional flow results from the obstruction of the other outlet channel branch. So, the necking will still be compensated, preventing the step breakup process in this outlet channel branch. Finally, the cell will be encapsulated in a sample droplet created by the central breakup process on one side and step breakups on both steps 33. It is caught between two significantly larger empty child droplets from the same parent droplet. The size of the sample droplet is essentially determined by the geometry, specifically the outlet channel branch length N and its height h.
Due to similar reasons, not all kind of microfluidic devices with a step at the end of the outlet channel branches 32 can produce the step breakup regime. Unlike in the first embodiment, where the droplet fate is either one of the two possible breakup regimes, in the second embodiment, there are different breakup regimes for each droplet consisting of one or more than one breakup regime. This is because these competing breakup regimes happen geometrically far apart, and achieving one will not necessarily stop the other. In fact, there is never a stand-alone step breakup, it is always accompanied with one last breakup close to the junction: be it a central breakup or a lateral breakup (which exactly depends on the same physics as described with the first embodiment.
On the contrary, it is possible to have only the central breakup in the scheme-It just requires the flow from the junction pushes enough the thread such that the step breakup process is suppressed and cannot achieve before the final central breakup. In fact, the ideal scheme as explained above is one where the central breakup actually occurs, but the step breakup development is also close to being finalized.
To enable a step breakup, the T junction needs to connect to the step-like widened channel portion at both ends of the outlet channel branches 32 creating step 37. In addition, the aspect ratio h/wo should be large enough.
It has been examined varying flow conditions on differing geometries. The length N of the outlet channel branch 32 equals 50 and 75 μm. Geometries of θ equal to 90°, 105°, and 120° degrees.
As shown in
By these patterns the best operating scheme can be found by changing the flow conditions.
It has been found that in terms of geometry, small wi/wo and small ho/wo favor central breakup short outlet channel branch length N, large angle θ and large aspect ratio ho/wo favor step breakup. It is preferred to select the width ratio (wi/wo) and aspect ratio (ho/wo) according to the same geometrical condition as in the first embodiment, and explore the influence of the outlet channel branch length N and the angle θ, as can be seen in
On the first geometry N50A90 it has been found the desirable operating scheme that appears at the moderate capillary number Ca range for shorter parent droplets, as can be seen in
Cell experiments demonstrate the described working principle with the successful generation of smaller droplets when and only when there is a sample in the parent droplets. Compared to the first embodiment, the cell-triggered sample droplets have sizes that increased considerably from 21 μm (first embodiment) to 25 μm (N50A90), and 35 um (N75A120) in averaged diameter.
In summary, to design a T junction that can be used in the second embodiment, a “step” structure has to be formed and the geometrical parameters N and 0 (while obeying the same geometrical rule of
have to be tuned such that the desired operating scheme (close to transitions of breakup schemes) of droplet breakup is possible on that geometry.
In
With the straight channel, there is only a step breakup regime. On geometries that fulfill the geometric condition defined by the z number, step breakups always have the tendency to occur. The occurrence of step breakup events depends on the flow condition which is defined by the capillary number and the droplet length L. When flow rate, which affects the capillary number, is high, the droplet will already pass the passage point P before it has the time to actually finish the breakup procedure, and will become stable again (stop the tendency to break) when it enters the step-like widened channel portion 36 thereby leaving the critical part of the geometry. No breakup is effected as shown in the two subsequent operation states of
With the described embodiments, it is possible to provide deformable samples in sample droplets separated from other sampleless droplets which are empty, i.e. containing no deformable samples. The droplets are output at the outlet of the outlet channel into a receiving inlet. However, in practice, there is still a need to separate the sample droplets from the sampleless droplets. In difference to the sorting module mentioned before, a separation module may be provided which is described in the following in conjunction with
The separation chamber 55 is formed by walls receiving inlet 54 is formed by parts of the walls 57, 58 while upper and bottom walls (not shown) may be in parallel to each other to close the separation chamber. The asymmetrical expanding/widening nature at the region of the receiving inlet 54 serves to give each droplet C, S a characteristic velocity component in a lateral direction L across to the axial direction A. The characteristic velocity depends on the stiffness of the droplet and particularly on the stiffness of a deformed sample within the sample droplets S. The velocity of each droplet in axial direction A is independent of its stiffness or deformability.
In other words, sample droplets are pushed less into lateral direction than sampleless droplets thereby forming different trajectories when leaving the receiving inlet.
This effect can be explained as follows: Due to the widening nature in the region of the receiving inlet 54, the sampleless droplets remain longer in contact with the first wall 57 of the separation chamber 55 than the sample droplets S. The longer contact of the sample droplets acts against the droplet relaxation from its deformed state which they had inside the outlet channel 52. The time of droplet relaxation influences the lateral velocity of the droplets.
The widening nature of the separation chamber 55 in the region of the receiving inlet 54 therefore creates a lateral force component effected on the droplet which depends on the stiffness of the droplet and the thereby caused relaxation behavior. A stiffer droplet has more resistance to this extensional deformation (relaxation) so that it travels a longer time following the extension of the first wall 57 than the less stiff droplets (such as the sampleless droplets).
When deformation occurs as long as the droplet is still in the outlet channel, the rear cap of the droplet is deformed when the droplet comes free of the confinement of the outlet channel decides about the duration of the attachment to the outlet opening. Here, sampleless droplets have concave rear caps with interfaces attached to the first and second walls, while in sample droplets the sample occupies the rear cap region, which helps the rear cap withstand the concaving deformation as this region is now more rigid than the fluid of the droplet. This also prevents the attachment of the droplet to the second wall, and further influences the following droplet relaxation process. Eventually, provided with a constant flow rate, the sampleless droplets C are more deformed and inclined towards the second wall, whereas the sample droplets S are more resistant to the relaxing deformation and are free in the original axial direction. This results in different trajectories of the droplets starting from the receiving inlet.
In addition, due to the asymmetric cross-section of the expansion chamber 55 the streamlines of the fluid stream downstream the receiving inlet 54 are divergent what results in a spreading force on the droplets travelling on different trajectories. This leads to an amplification of the spit paths of the sample-filled sample droplets S and empty sampleless droplets C in the separation chamber 55.
Thereby, a deformation and relaxation behavior of the sample and sampleless droplets S, C can be effectively used to create different trajectories starting from the receiving inlet 54 of the separation module 50. Consequently, the presence of a deformable sample in the droplet substantially affecting its stiffness, different trajectories of sample droplets and sampleless droplets can be achieved.
The separation module 50 is mainly formed by the separation chamber 55 which directly connects with the output opening 53 of the outlet channel 52. Here, two walls of the separation chamber 55 provide a widening space, wherein the walls connect with the output of the outlet channel in different inclination angles. In the shown
In other words, the inclination angles of the first and second walls 57, 58 may be different, while the inclination angle of the first wall is smaller than the inclination angle of the second wall, thereby providing streamlines at the axial elongation of the outlet channel which have a stream component perpendicular to the axial direction forcing the droplets away from the axial elongation of the outlet channel 52. Due to the different lateral velocities of sample droplets and sampleless droplets this results in substantially different trajectories within the separation chamber 55.
The separating element 56 is located downstream in the separation chamber 55 and may provide a wall or similar structures which guide the sampleless droplets C and the sample droplets S travelling on different trajectories into different channels 59, thereby completing the separation of the droplets C, S.
| Number | Date | Country | Kind |
|---|---|---|---|
| 21208067.5 | Nov 2021 | EP | regional |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2022/081677 | 11/11/2022 | WO |
