- Open Access
High-throughput magnetic particle washing in nanoliter droplets using serial injection and splitting
© The Author(s) 2018
Received: 16 May 2018
Accepted: 18 June 2018
Published: 21 June 2018
Droplet microfluidics has emerged as a promising technique to perform high-throughput, massively-parallel chemical and molecular biological reactions. Droplet microfluidic operations such as droplet generation, sorting, and fluid addition are well established; however, fluid exchange (i.e. washing) at high-throughput is challenging to implement. Here we present a microfluidic device architecture that utilizes wash buffer injection preceding a splitting junction in proximity to a magnetic field to transfer paramagnetic microparticles across a concentration gradient within a single droplet. The device can operate at high throughput (50 Hz) while preserving input droplet volume at the collection outlet as verified using high speed imaging. Using a two-stage device, combined microparticle retention rates (up to 97.5%) and high wash efficiency (92.9%) is demonstrated using dye absorbance and fluorescence. This method can be performed in a serial array to obtain an arbitrary degree of wash efficiency and integrated into lab-on-a-chip systems for use in multi-step microfluidic bioassays or single-cell genomic applications requiring high-fidelity washing steps within droplets.
Droplet microfluidics involves sample partitioning into nanoliter or smaller volumes and has been extensively used in a variety of fields including biology, medicine, chemistry and physics [1–4]. Most commonly, aqueous fluids are encapsulated into droplets surrounded by a continuous phase of an oil surfactant mixture. Droplet-based sample partitioning into miniscule volumes allows for faster reaction kinetics due to decreased diffusion times compared to bulk scale reactions . The ability to generate droplets at kHz frequency permits massively parallel reaction schemes with precision control of reaction volumes due to the highly monodisperse nature of microfluidic droplet generation. A major goal of microfluidics is to mimic bench-level fluid manipulations within the droplet format in order to take advantage of smaller reaction volumes, massively-parallel scalability, compartmentalization and decreased reagent use.
Magnetic and non-magnetic oligonucleotide barcoded microparticles have recently been used in conjunction with droplet microfluidic or microwell partitioning to profile the surface proteomes and or transcriptomes of thousands of cells in a massively parallel manner [6–13]. Magnetic microparticles are used extensively in molecular biology and genomics as versatile systems for genomic immunoassays, cell capture and sorting, and nucleic acid binding and size selection [14–16]. Their ease-of-use, high efficiency and low cost have made magnetic microparticles a ubiquitous tool and the solid support of choice for washing and capture at the bench-top and in high throughput robotic protocols. In order to translate standard molecular biology reactions using magnetic microparticles into the droplet format and to expand the repertoire of high-throughput single cell genomic applications, magnetic microparticles should be seamlessly integrated into on-chip droplet assays. The majority of on-chip droplet microfluidic assays can be described by primary unit operations which include droplet generation, fluid injection, sorting, splitting and washing. Additive functions such as generation and injection are commonplace, however subtractive functions such as washing are less established, especially in a robust manner at high throughput [1, 17, 18].
Washing magnetic beads in droplets on digital microfluidic (DMF) devices using electrowetting on demand (EWOD) based platforms is straightforward; typically an electromagnet is activated beneath an array of electrode pads which are used to manipulate droplets containing magnetic particles . DMF and EWOD platforms are easily automated, however they are inherently limited in throughput due to constraints of fabricating and addressing large arrays of electrode pads each of which can accommodate at most one droplet. In light of this limitation, continuous flow droplet washing approaches have been developed to increase throughput. Magnetic tweezers have been used to controllably immobilize magnetic particles derived from a droplet within a section of tubing [20, 21]. This approach showed extremely high recovery of magnetic microparticles and excellent wash efficiency, however the throughput was severely limited due to active switching of the magnetic tweezers electromagnet. Additionally, the magnetic tweezers apparatus is large and not amenable to miniaturization and integration into microfluidic systems. Another approach involves the synchronization of two droplet trains followed by electrocoalescence and splitting . One droplet train contained magnetic particles in a background solution to be washed away, the other droplet train contained wash buffer into which the magnetic particles are transferred. The droplet trains are forced through a rail-road network of channels to equilibrate pressure and synchronize the positions and velocities of the droplets. Droplets are then electrocoalesced at a junction proximal to a stationary magnetic field, thereby transferring magnetic microparticles into the wash buffer droplet prior to physical splitting of the electrocoalesced droplet into two daughter droplets. In spite of relatively efficient washing in a single step, multiple failure mechanisms were observed and throughput was low due to the required synchronization of droplet trains in the rail-road network.
Device design and fabrication
Dark field photolithography transparency masks (Advance Reproductions, North Andover, MA) were designed in AutoCAD 2016. Silicon wafers were cleaned with piranha solution for 30 min at room temperature. SU-8 3050 was spin coated to a thickness of 100 μm and exposed through the transparency mask using a Karl Suss MJB3 mask aligner. After development, microstructures were used in soft lithography PDMS molding. After PDMS slabs were cut from the wafer, inlet and outlet ports were punched using a 1 mm biopsy punch. Next, three square regions ~ 3.5 mm2 were cut from the slab to accommodate three square (1/8″ × 1/8″) grade N52 neodymium magnets (B222G-N52, KJ magnetics). Next the PDMS slab was bonded to a glass slide with oxygen plasma for 45 s and heat cured for 10 min in an oven at 75 °C. Next, Aquapel (Pittsburgh glass works Pittsburgh, PA) was perfused through droplet channels and dried at 75 °C oven for 10 min. Next, 50 mM (3-mercaptopropyl)trimethoxysilane (MPTMS) in acetonitrile was perfused through electrode array channels for 30 s and purged. The device was then placed in the oven for 10 min at 75 °C. Next, The device was placed on a hotplate and heated to 215 °C for 10 min. Using rubber tipped tweezers a short strip (~ 5 mm) of 0.030″ diameter 52In/48Sn low melting temperature solder (Indium Corporation, Clinton, NY) was forced into the electrode array channels pre-treated with MPTMS. After all electrode array channels were filled with solder, the device was removed from the hotplate using wafer tweezers and 60 mm copper wire segments obtained from stripping and separating 28 gauge copper wire (AlphaWire, Elizabeth, NJ) were placed into the electrode array ports using the rubber tweezers. Finally, the device was cooled (10 min) and epoxy was used to cover the fragile copper/metal electrode array contacts and let set overnight.
Experimental setup and device operation
Devices were operated using an inverted microscope at 4× and 10× magnification and videos were imaged using a high-speed camera (FASTCAM mini AX200 Photron, Tokyo, Japan) at 5000 FPS. Electrode array contacts were linked and connected to a high-voltage fluorescent light bulb inverter (CXA-L0505-NJL, TDK Corporation) powered at 5 V. Syringe pumps were used to flow fluorinated oil at 325 µl/h and magnetic particles of varying diameter and magnetic type (1 µm superparamagnetic, 1 µm paramagnetic, 5 µm paramagnetic, and 22 µm paramagnetic, Spherotech Inc.) suspended in 1× PBS at 180 µl/h. 22 µm diameter magnetic particles were suspended using a PTFE coated winged magnetic stir raft (773W-5,9, V&P scientific) and stirred using a magnetic stirrer (710D2, V&P scientific). Flow from injection channels was obtained by pressurizing the head space of custom made vials with microtubing connections (New England Small Tube). Pressure was monitored using two independent pressure sensors (Honeywell, Trustability 015PGAA5) connected to regulators (R-800-90W/K, Airtrol Inc.), one for each injection channel in the two-stage device. Operating pressures for the injection channels were 1.3 PSI and 1.1 PSI for the first and second channels respectively. Pressure actuation was controlled through solenoid valve manifold (Festo) in LabView via a DAQ (National Instruments, X Series USB-6343).
For optimization experiments bromophenol blue was used as a visual aid and calibration solution for input buffer removal. Bromophenol blue dye was imaged under a compatible band pass filter FWHM = 10 ± 2 nm (FB590-10, Thor Labs Inc.). Fluorescein salt was also used as a visual aid for washing efficiency. Serial dilutions of bromophenol blue were generated and absorbance spectra measured on NanoDrop 2000 spectrophotometer for each experiment for calibration purposes. For droplet washing efficiency experiments, emulsions were broken using 1H,1H,2H,2H-perfluoro-1-octanol and the absorbance of the aqueous portion was measured. Droplets were visualized in Fuchs-Rosenthal hemacytometers (InCyto). High-speed videos were analyzed using droplet morphometry and velocimetry (DMV) software, generously supplied by Amar Basu. Post processing and additional data analysis was performed in MATLAB (MathWorks).
Results and discussion
Magnetic microparticle recovery and droplet volume preservation
We assessed the microparticle recovery as a function of three paramagnetic microparticle sizes 1, 5, and 22 µm in diameter. For the optimal flow rates determined the observed recovery (fraction of magnetic microparticles recovered in the final collection outlet compared to the combined waste channels) was most efficient (97.5%) for the largest diameter microparticles (22 µm) (Fig. 3b). This is due to the larger magnetic core and resulting higher velocity in the magnetic field. Smaller diameter (~ 1 µm) superparamagnetic particles were also tested, but due to higher net magnetic susceptibilities and resulting higher velocities, the injection channel required modifications to achieve efficient recovery (Additional file 2: Video S1). This represents a limitation of the current device design, which may need to be tailored to the properties of the magnetic particles used in the droplet based assay. Because high speed imaging and tracking of larger diameter particles is less challenging than for smaller particles, the 22 µm diameter magnetic microparticles were used for the remainder of proof-of-principle experiments in this study. In the absence of a magnetic field adjacent to the injection channels, collective recovery for all microparticles was approximately 40% presumably due to the force imparted through injection (Fig. 3b) which biases additional microparticles to the waste outlet channels during splitting. Using high-speed imaging the trajectories of individual 22 µm microparticles were tracked using the DMV software  and superimposed (Fig. 3c). The injection induced Y-axis deflection is typically less than 70 µm and particles return to the top portion of the droplet within about 500 µm distance travelled in the X direction. The distance between the injection channel and the splitting junction is 1 mm for the proof-of-principle devices used in this study indicating that higher throughput is achievable. Using high speed imaging the velocity of the particles was tracked just after injection. Typically maximum velocities did not exceed 2500 µm/s in fair agreement with the expected value (Eq. 4). Slight deviations from the expected velocity may be due to microparticle and magnetic core size dispersity. In addition, the placement of the stationary magnet varies slightly from device to device causing small variations in the magnetic field gradient experienced by the microparticles. In order to verify preservation of droplet volume after splitting, high-speed video of the injection and splitting cycle was acquired. Droplet area was tracked prior-to and during injection and after the bifurcation channel. Droplet area is seen to increase linearly during injection followed by an abrupt decrease at the splitting junction as the two daughter droplets are tracked separately. The area was integrated along the channel height to obtain the volume change for the injection-splitting cycle. Injection and splitting cycle traces were superimposed and compiled into a histogram. The histogram was best fit by two Gaussian distributions indicating identical input droplet and output daughter droplet volumes of 2.2 nl upon injection of 2.1 nl and equal volume splitting (Fig. 3d).
Magnetic microparticle washing efficiency
We have presented a robust method to wash magnetic microparticles within droplet microfluidic systems at high throughput using a serial array of injection channels. Using high speed imaging, absorbance and fluorescence measurements the wash efficiency was determined to be 92.9% while retaining 96% of 22 µm magnetic microparticles. Smaller diameter microparticles could be implemented with minor alteration to the microfluidic device, namely the injection channel geometry and the margination channel length prior to droplet splitting. Magnetic microparticles in this study were washed in droplets at high throughput (~ 50 Hz) representing a tenfold improvement in throughput over alternative approaches with comparable wash efficiency. Further flow rate optimization will allow for even higher throughput washing. The serial nature of this flexible method allows for ‘daisy-chaining’ multiple wash modules for applications requiring strict high-fidelity washing. Importantly, this microfluidic device does not rely on bulky off-chip instrumentation for magnetic microparticle manipulation and is amenable to miniaturization. This device is a step towards robust high-throughput microparticle washing for lab-on-a-chip systems and can be used to complement the droplet microfluidic unit operations. We envision this device can be integrated into microfluidic workflows for the development of complex droplet based bioassays or used in single-cell genomic applications requiring high-throughput washing of captured analytes of interest.
WS conceived of the method, fabricated devices, performed experiments, analyzed the data, and wrote the manuscript. The author read and approved the final manuscript.
We gratefully acknowledge financial support from the New York Genome Center and technical support from Kunal Pandit and critical reading of the Manuscript by Peter Smibert, in addition to support from members of the Technology Innovation Lab at the New York Genome Center. We also thank Professor Amar Basu (Wayne State University) for developing and providing the Droplet Morphometry Velocimetry (DMV) software.
The author declares no competing interests.
Availability of data and materials
The datasets supporting the conclusions of this article are included within the article and its additional files.
This study was supported from funding through the New York Genome Center.
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