Daniel
Frenzel
and
Christoph A.
Merten
*
European Molecular Biology Laboratory (EMBL), Genome Biology Unit, Heidelberg, Germany. E-mail: merten@embl.de
First published on 17th February 2017
Fluorescence-activated droplet sorting (FADS) has become a widely used technique for high-throughput screening applications. However, existing methods are very sensitive to fluctuating flow rates at the sorting junction, which can be caused by the pulsing effects of mechanical pumps, droplet aggregates or the accumulation of precipitates during lengthy biological screening applications. Furthermore, existing sorting devices allow only 2-way sorting. We present here a dielectrophoretic sorting system in which the droplets are sorted along multiple electrode pairs that run parallel to the channels. This enables highly reliable sorting (no errors were detected for more than 2000 sorting events) even when inverting the relative flow rates at a 2-way sorting junction from 80:
20 to 20
:
80. Furthermore, our toolbox is scalable: we demonstrate on the example of a triple-colour sorting experiment with a total of four decoupled electrodes that multi-way sorting is feasible.
So far, this basic sorting principle has been used for more than ten years. Nonetheless, it has two inevitable limitations: first of all, the approach is very sensitive to minor variations in the fluid flow and the resistance of individual channel sections. The frequently observed pulsing of syringe pumps (having threads which on the microscale cannot be rotated at a 100% constant speed) or the formation of droplet aggregates in widening channels can push non-selected droplets into the collection channel. Similarly, deposits in the channels (dust particles or protein precipitates) can change the fluidic resistance, so that undesired droplets are collected. In consequence, it is difficult to run microfluidic droplet sorting experiments stably over many hours. However, this is required for many biological screening applications in which hundreds of thousands or even millions of droplets have to be sorted based on the endpoint signal of biochemical or cell-based fluorescence assays. Using current approaches which are sensitive to fluctuations in the relative flow rates at the sorting junction this inevitably results in the selection of false positives. For example, on screening more than 300000 hybridoma cells over a period of ∼4 h, we previously reported a false positive rate of more than 5%.4 Similarly other groups have demonstrated 3–4 hour sorting experiments focussing on metabolic11 or genetic6 assays, which could strongly profit from a systematic reduction of the false positive rate.
Another limitation of existing sorting devices is the fact that they hardly allow sorting into more than two channels. This is a significant disadvantage, since multi-way sorting has tremendous potential for screening applications. For example, it could be used to sort samples according to the strength of a phenotype (e.g. very strong = collection channel 1, strong = collection channel 2, intermediate = collection channel 3, etc.) rather than just in a digital on/off fashion. Only in this way can truly quantitative and mechanistic data be obtained from downstream genetic or biochemical analyses of the sorted samples. Alternatively multichannel sorting can be used in multiplexed assays (e.g. sorting droplets according to more than one phenotype). However, existing sorting devices do not allow these operations to be performed on microfluidic droplets.
We have overcome these limitations using dielectrophoretic sorting devices that have an electrode pair not only on the side of the collection channel, but also on the side of the waste channel. Furthermore, in our approach, exactly one electrode pair is activated at any point in time: whenever a droplet is showing a desired readout signal, the electrode pair next to the collection channel is switched on, while for all other times, only the electrode pair next to the waste channel is switched on. This enables reliable sorting without the need to adjust the relative flow rates at the sorting junction (and also without being sensitive to flow rate fluctuations) and furthermore allows multi-way sorting by placing electrode pairs next to each collection channel. This enables an unprecedented level of control by moving droplets on “electric rails”.
To demonstrate the feasibility of this approach, we sorted 100 μm droplets containing 100 μM FITC. To analyse the sorting efficiency, we used an algorithm triggering the electrodes only for every second, third or fifth droplet. This allowed accurate monitoring of the correct sorting of individual droplets by video analysis (Table 1), revealing 100% efficiency. This high level of robustness was further confirmed by sorting every second droplet over a period of five hours, during which videos were taken every hour. On analyzing a total of 4659 sorting events, not a single mistake was observed (Table S1†). After optimizing all flow rates, we further achieved a maximum throughput of ∼240 Hz (Movie S1†), which is approximately 5-fold higher than what we achieved previously for droplets of the same size, using a conventional sorting setup.4 More importantly, we could show that the sorting efficiency is largely independent of the relative flow rates downstream of the sorting junction. By connecting a syringe aspirating fluid from the waste outlet (while leaving the collection outlet unaffected), we aspirated different fractions of the overall flow and monitored the sorting efficiency (using the algorithm for sorting every second droplet) by high-speed imaging (Movies S2–S7†). Without changing any other sorting parameters (e.g. pulse amplitude and/or duration), the sorting efficiency remained completely unaffected at ∼100% (see details in Table 2 and Fig. 1D), while aspirating from 7% to 86% (at 13 Hz) and 40% to 70% (at ∼90 Hz) in terms of the relative flow rates at the waste channel. In contrast, sorting with just one electrode pair (mimicking a conventional setup) worked well when constantly aspirating 80% of the relative flow from the waste outlet, but failed when changing the relative flow rates by more than 5% (Table 2, Fig. 1D and Movies S8–S10†). Similarly, our setup showed much higher robustness when perturbing the system in a real world context; while performing sorting experiments with one and two electrode pairs (at flow rates which usually allow 100% efficiency in both systems; 80:
20 for one and 50
:
50 for two electrode pairs, see Fig. 1D), we gently flicked the tubing connected to the collection outlet. This procedure mimics a perturbation that occurs frequently in long-term biological screens, such as moving the tubing between different collection tubes (e.g. to avoid a single mistake that can ruin an entire screen of valuable samples or when changing sorting gates). For each experiment, the tubing was gently flicked six times within six seconds and the entire process was recorded using high-speed imaging (ESI† Movies S11 and S12). Furthermore, each experiment was repeated three times to obtain high statistical confidence (Table 3). While for the single electrode pair setup the perturbation decreased the sorting efficiency on average to 92.2%, the two electrode setup allowed 99.4% efficiency (analyzing more than 2600 sorting events in both cases), even under such difficult conditions. These values correspond to a 13-fold reduced number of false positives.
Sorting ratio | Droplets | Rate [Hz] | Efficiency [%] |
---|---|---|---|
1![]() ![]() |
761 | 153 | 100 |
1![]() ![]() |
802 | 161 | 100 |
1![]() ![]() |
778 | 157 | 100 |
Number of powered electrode pairs | Relative flow rates (waste/collection channel) | Efficiencya [%] |
---|---|---|
a On average, more than 380 sorting events were analyzed for each data point shown in Fig. 1D. | ||
One | 50![]() ![]() |
0 |
60![]() ![]() |
0 | |
65![]() ![]() |
0 | |
75![]() ![]() |
3.4 | |
78![]() ![]() |
41.5 | |
80![]() ![]() |
99.4 | |
85![]() ![]() |
100 | |
90![]() ![]() |
1.1 | |
Two | 11![]() ![]() |
0 |
20![]() ![]() |
17.2 | |
25![]() ![]() |
53.3 | |
30![]() ![]() |
90.7 | |
40![]() ![]() |
100 | |
50![]() ![]() |
100 | |
60![]() ![]() |
100 | |
70![]() ![]() |
100 | |
72![]() ![]() |
85.5 | |
75![]() ![]() |
0.6 | |
80![]() ![]() |
0 |
Experiment | Efficiency when using one electrode pair [%] | Efficiency when using two electrode pairs [%] |
---|---|---|
#1 | 92.6 | 99.8 |
#2 | 85.7 | 100 |
#3 | 97.5 | 99.4 |
#4 | 92.9 | 98.4 |
To complete the characterization of the 2-way sorting device, we finally had a look at the effect of the constant DEP force on the droplet velocity (Table S2 and Movie S13†) and the ability of our device to sort droplets of different sizes without changing the geometry (Fig. S6 and Movies S7, S12 and S14†). In brief, the constant electric field decreased the droplet velocity very little (by a maximum of 9.4%, even when applying 1688 V rather than 1500 V as done for the sorts) and this is valid over a wide range of different total flow rates. Furthermore, the device enabled reliable sorting of droplets with sizes ranging from approximately 60–120 μm in diameter (corresponding to an 8-fold difference in volume), without changing the geometry. Larger or smaller droplets can probably be sorted as well, but could not be generated using a droplet maker of constant size.
Next, we focused on multi-way sorting. This was implemented by designing a sorting module (Fig. 2A–C) in which four channels sequentially branch off from the waste channel. Each of these collection channels has its own electrode pair running parallel to the channel wall (Fig. 2C). In order to simplify the wiring, we spaced out the connection points for the electrodes to match low cost 2.54 mm pin connectors (Fig. 2D).
In a first experiment, we focussed on high-speed analysis of the sorting process, using red light to illuminate the chip (so that interference with the photomultiplier tubes of the green and blue channels can be ruled out). For this purpose, we produced droplets containing cascade blue (CB), fluorescein isothiocyanate (FITC), and a combination of both (using the droplet maker shown in ESI† Fig. S1; see the Materials and methods section for details on the dye concentrations) and performed dual-colour sorting using a multi-channel optical setup (Fig. S2†). To be able to monitor the sorting efficiency, the droplets were additionally stained with different concentrations of the chromogenic dye naphthol blue black, allowing the different droplet populations to be distinguished by the eye and even outside the laser spot (Fig. 2E and Movie S15†). After determining the optimal pulse durations and – delays (the further downstream a particular collection channel is located, the longer the pulse delay; Tables 2 and 3), we sorted a reinjected emulsion containing the different droplet populations for a total of 2 hours and measured a sorting efficiency of >98% (averaged over all sorting channels in two independent experiments) at a sorting rate of ∼2 Hz.
We then repeated the experiment and determined the long-term sorting efficiency based on subsequent fluorescence microscopy analysis of the sorted fractions. This readout is not dependent on high-speed movies, for which reason we could dim the red light used for illumination, thus allowing the detection of a third red droplet species. We hence generated droplets containing cascade blue (CB), Alexa 488, or Alexa 594, reinjected the mixed emulsion into the sorting device and sorted unattended over 13 hours. During this time, an overall throughput of 3 Hz at ∼90% efficiency (average value based on microscopic analysis of all three sorted fractions; Fig. 2F and Table 3) was achieved. This long-term efficiency clearly demonstrates the suitability of our system, not only for short proof-of-principle experiments, but also for real screening campaigns.
As a final application, we further addressed multi-way sorting according to the signal intensity. Instead of using different fluorophores, we encapsulated different concentrations of Alexa 488 (12.5 μM, 25 μM and 50 μM) and sorted the droplets according to their fluorescence intensity in the green channel. After sorting for a total of eight hours, aliquots from the different collection tubes were taken and analyzed by fluorescence microscopy. Similar to the previous experiments, an overall throughput of 3 Hz at ∼90% efficiency (average value based on microscopic analysis of all three sorted fractions; Fig. 2G and Table 5) was achieved. This clearly demonstrates the possibility of sorting samples not just for different phenotypes, but as well as for the quantitative strength of a particular phenotype.
In addition, we present a toolkit which enables the experimenter to setup a scalable platform for highly multiplexed sorting applications at a slightly lower efficiency of 94% on average (based on almost 2000 analyzed sorting events shown in Tables 4 and 5). By using a total of four decoupled electrode pairs (three for specific sorting channels and one for the waste), we were able to sort three different droplet types, either containing unique dyes (three individual phenotypes), mixtures thereof (two phenotypes including double positives) or even a single dye at different concentrations (different strengths of a particular phenotype). In theory, the number of collection channels sequentially branching off from the waste channel is only limited by the desired throughput: the more channels used, the longer the pulse delay for the most downstream channels and hence the lower the overall throughput. However, for many biological screening applications, the number of different collection reservoirs is far more important than the throughput. For example, a setup as presented here could be used to sort individual droplets into the wells of a microwell plate for further downstream characterization (e.g. single-cell sequencing, biochemical or cell-based assays). Furthermore, sorting channels that sequentially branch off from the waste channel could also be replaced by a star-like geometry, which does not require long pulse delays and hence allows higher throughput. We believe that this would also allow the efficiency of the multi-way sorting device to be further increased, whose error rate is mostly limited by the exact timing of the sorting delay for each collection channel. This should not be the case for a symmetric, star-like geometry operating without sorting delays (similar to the ∼100% efficient 2-way sorting), or for which they could at least be equal and short for all collection channels. While electrode pairs running next to the channels are difficult to implement for such a geometry (because of space constraints), one could place electrode pairs above or below the sorting channels.12,13 This would even enable precise control of the trajectory of droplets along different paths within a single channel; however, much more complex manufacturing methods are required for such a device. In contrast to this, the setup shown here can be easily reproduced by standard soft lithography techniques and we hence look forward to see rapid spreading and further use of the technology.
Content | Channel | Delay [ms] | Rate [Hz] | Droplets | Efficiency [%] |
---|---|---|---|---|---|
FITC/CB | #1 | 260 | 2 | 36 | 97.2 |
FITC | #2 | 370 | 55 | 100.0 | |
CB | #3 | 480 | 67 | 98.5 | |
Sorting with inversed channel order | |||||
CB | #1 | 260 | 2 | 50 | 100.0 |
FITC | #2 | 370 | 36 | 100.0 | |
FITC/CB | #3 | 480 | 15 | 93.3 |
Content | Channel | Delay [ms] | Rate [Hz] | Droplets | Efficiency [%] |
---|---|---|---|---|---|
Sorting for different colours | |||||
Alexa 594 | #1 | 150 | 3 | 274 | 84.5 |
Alexa 488 | #2 | 240 | 504 | 97.4 | |
CB | #3 | 320 | 210 | 92.9 | |
Sorting for different intensities | |||||
12.5 μM Alexa 488 | #1 | 150 | 3 | 333 | 94.6 |
25 μM Alexa 488 | #2 | 240 | 205 | 92.7 | |
50 μM Alexa 488 | #3 | 320 | 154 | 93.5 |
Footnote |
† Electronic supplementary information (ESI) available. See DOI: 10.1039/c6lc01544a |
This journal is © The Royal Society of Chemistry 2017 |