Patent ID: 12239981

DETAILED DESCRIPTION

The disclosure is presented in several embodiments in the following description with reference to the Figures.

This collection system consists of a three-branch sorter, three independently-controlled valves and corresponding collection platforms, as shown inFIG.1. The quantitative collection is realized through alternatively driving subgroups of droplets into three branch channels, thus leaving enough interval between adjacent subgroups going into the same channel to complete each collection. The collection process can be divided into three steps: droplet generation, three-branch sorting, and droplet collection.

A flow-focusing microfluidic channel was used for the generation and gapping of droplets, as shown inFIG.2. To realize stable and accurate sorting in next step, two requirements should be met in this part: sufficiently small droplet size to prevent channel blocking, and adequate gap between adjacent droplets to complete individual sorting. Therefore, a relatively low ratio of inner phase to outer phase flow rate (normally from 1:100 to 1:30 according to different aqueous samples) was used, corresponding to the droplet diameter of around 60 μm. And a sufficiently high gapping flow rate (normally higher than 200 μL/h) is used to guarantee enough gap.

When droplet flowed through the detection area, its arrival can be detected by an optical detector beneath the microfluidic device, which can identify the fluorescence with a wavelength of 488 nm, as shown inFIG.3. Based on the detected signals, an automatic program edited by LabVIEW was used to process the input data at 100 kHz. And the output sorting signal was amplified by a high voltage amplifier followed by being supplied to the electrode to sort the droplets. As shown inFIG.2, the three branch channels were designed with different fluidic resistance: channel 1 is the widest and shortest, while branch channel 3 is the narrowest and longest. Thus, droplets are supposed to choose channel 1 due to the lowest fluidic resistance. Furthermore, the three channels were designed asymmetric to the horizontal line: branch channel 1 and 2 has the same horizontal angle of 15°, while channel 3 has a larger one of 45°, resulting in the effect of inertial force being higher on branch channel 1 and 2, but lower on branch channel 3. Therefore, at normal conditions, droplets tended to flow into channel 1; while applying a lower sorting voltage of 370 Vpp with the frequency of 10 kHz, droplets were driven into branch channel 2; with a higher voltage of 650 Vpp, droplets were driven into branch channel 3, as shown inFIG.4. In one embodiment, the lower sorting voltage is in a range of 350-450 Vpp and the higher voltage is in a range of 600-900 Vpp. Based on this, subgroups of droplets were alternatively driven into three channels for continuous collection, while negative droplets flowing into branch channel 1 and subgroups of positive droplets alternatively driven into branch channel 2 and 3 for sorting combined with collection.

After one subgroup of droplets being sorted into corresponding channel, the connected valve (FIG.5) and rotation platform (FIG.6) were successively operated to complete this collection. A compressed air-driven outer phase flow was utilized to rapidly pump out droplets into the PCR tube through a curved needle. To ensure that droplets have flowed into the outlet while opening valve to start pumping, delay times of 1.5 s, 2 s and 2.5 s were applied to channel 1, 2 and 3 for valve opening, respectively. And in consideration of the fluidic perturbation caused by pumping flow, which may induce sorting instability, a DC voltage of 1.2 V was output to the valve, corresponding to the air pressure of around 8 kPa and pumping flow rate of around 12,000 μL/h. At this relatively high flow rate, the inner phase flow was temporarily stopped to avoid this instability. To guarantee droplets be completely pumped out, the pumping volume and valve open time of were set at 5 μL and 1.5 s, respectively. Finally, after another delay time of 0.5 s for the droplets to drip into the PCR tube, 20 cycles of TTL signal with the frequency of 150 Hz was output to the step motor, driving the collection platform to rotate 36° to another tube in around 0.13 s for the next subgroup collection, as shown in movie 1.

To perform continuous collection of homogeneous droplets, a kind of fluorescent dye was tested as the inner phase to continuously generate and collect positive droplets. The inner phase, outer phase and gapping flow rates were set as 5 μL/h, 500 μL/h and 200 μL/h, corresponding to the droplet diameter of 54 μm and generation frequency of around 14 Hz. Firstly, a symmetric collection was tested, which means that three channels collect the same number of droplets for each subgroup. To verify the interval between adjacent subgroups going into the same channel be longer than each collection time, the collection number for each subgroup should be beyond a minimum value of 25. In one embodiment, the collection number is adjusted as a parameter in LabVIEW. Based on this, the collection number was respectively set as 30, 40, 50 and 100 to test this system. The optical images of collected droplets are shown inFIG.7, and statistical results of droplet number are shown inFIG.8. It can be seen from the figure that this system can accurately collect different numbers of droplets, with only a small quantitative deviation. Furthermore, if the collection numbers in two channels are increased to supply longer interval, the collection number in the other channel can be accordingly reduced, which is called asymmetric collection and can realize a low-number collection in one channel. To verify this, the collection numbers in channel 1 and 3 were both set as 50, and the number in channel 2 was set as a lower value of 20, 15, 10, 5, and 1, respectively. Experimentally, this method can accurately collect a relatively low number of droplets, and even realize the single-droplet collection, as shown inFIG.9.

While reserving branch channel 1 for negative sample and the other two channels for alternative collection, this system can effectively combine droplet sorting with quantitative collection, thus collecting a certain number of desired droplets into each PCR tube, as shown inFIG.10. To verify this, a kind of cancer cell (KYSE 150) with the diameter of 10-50 μm was tested for this system. The flow rates of inner phase, outer phase and gapping flow were set at 10 μL/h, 300 μL/h and 300 μL/h, respectively, corresponding to the droplet size of 66 μm and generation frequency of 18 Hz. Limited by the collection time, there is a threshold for the minimum collection number, n, which is closely related to the average number of microspheres in each droplet, A. Experimentally, while the ratio between n and A is beyond 100, a stable quantitative collection can be achieved. Based on this, the concentration of fluorescent microspheres was diluted to three different levels, corresponding to the A of 0.2, 0.05 and 0.01, and sorting frequency of around 4 Hz, 1 Hz and 0.2 Hz, respectively. To test the system, the collection numbers of 20, 30 and 50 were tested for the A of 0.2; the collection numbers of 5, 10, 15 were tested for the A of 0.05; and single positive droplet collection was tested for the A of 0.01. Fluorescence images of collected droplets are illustrated to show the droplet number and the encapsulation of cancer cells, as shown inFIG.11. In addition,FIG.12illustrates the counted droplet numbers for different collection numbers, which shows a high accuracy at a large working range.

On the basis of on-demand collection of microfluidic droplets, a novel recovery method to extract the encapsulants inside the droplets was also developed. Specifically, it is completed by collecting the droplet emulsions into a culture plate filled with aqueous medium. Under the effect of interfacial tension, the oil phase will spread to a thin layer and the droplets rapidly aggregate at the center. Then as the oil layer gradually evaporates, the droplet phase will directly merge into the aqueous medium when the oil phase completely evaporates (FIG.13). This method realizes non-invasive and lossless droplet extraction in around 20 s, allowing integration with conventional biological assays to directly analyze the target encapsulants, such as cells, microbes and genetic molecules, in aqueous phase.

Unless otherwise indicated in the following examples and elsewhere in the specification and claims, all parts and percentages are by weight, all temperatures are in degrees Centigrade, and pressure is at or near atmospheric pressure.

With respect to any figure or numerical range for a given characteristic, a figure or a parameter from one range may be combined with another figure or a parameter from a different range for the same characteristic to generate a numerical range.

Other than in the operating examples, if any, or where otherwise indicated, all numbers, values and/or expressions referring to parameters, measurements, conditions, etc., used in the specification and claims are to be understood as modified in all instances by the term “about.”

While the invention is explained in relation to certain embodiments, it is to be understood that various modifications thereof will become apparent to those skilled in the art upon reading the specification. Therefore, it is to be understood that the invention disclosed herein is intended to cover such modifications as fall within the scope of the appended claims.