Kind gift of THP-1 cells from Dr. Mark Chong and assistance in microfabrication from Dr. Yuejun Kang and Dr. Nishanth V. Menon (School of Chemical and Biomedical Engineering, Nanyang Technological University) were greatly acknowledged. This project was funded by NTU-Northwestern Institute of Nanomedicine (Nanyang Technological University). H.W.H. was supported by Lee Kong Chian School of Medicine (LKCMedicine) postdoctoral fellowship.

1. Nanoparticles (NPs) Labeling of Mesenchymal Stem Cells and Monocytes
<ol>
	<li>Culture mesenchymal stem cells (MSCs) in Dulbecco&#39;s modified Eagle&#39;s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and antibiotics to &#8805;80% confluency prior to labeling. Similarly, culture THP-1 cells (ATCC) in Roswell Park Memorial Institute (RPMI) 1640 medium supplemented with 10% FBS to a density of ~106 cells/ml.</li>
	<li>Load silica NPs (~500 &#181;m) with calcein dye solution (200 &#181;M) using overnight stirring. Fabricate PLGA-calcein AM (CAM) using a protocol described previously.22
	<ol>
		<li>Dissolve 250 &#181;g CAM and 100 mg PLGA (50:50) in chloroform at 4 &#176;C.</li>
		<li>Generate single emulsion NPs using a high speed homogenizer (13,600 x g, 60 sec) at room temperature. Evaporate the chloroform in a chemical hood (&#8805;3 hr) before collection using centrifugation (3,400 x g for 5 min), washing (double distilled water), freeze-drying and storage in -20 &#176;C.</li>
	</ol>
	</li>
	<li>Incubate CAM-PLGA NPs (1 mg) or calcein silica NPs (150 &#181;g) in 0.01% poly-L-lysine (PLL) solution at room temperature for 15 - 20 min.</li>
	<li>Centrifuge at 3,400 x g for 5 min to remove excess PLL supernatant before NPs resuspension in 1 ml of respective culture medium.</li>
	<li>Incubate the NPs with cells (MSCs or THP-1, ~1 - 2 x 106 cells in total) for approximately 24 hr (0.1 mg&#183;ml&#8722;1 labeling concentration).</li>
	<li>Dissociate and harvest the labeled adherent MSCs using 2 ml of 0.25% trypsin (5 min, 37 &#176;C) and quench with 6 ml of DMEM. Spin down the cells at (1,000 x g, 4 min) and resuspend to a concentration of 105 - 106 cells/ml for microfluidic processing. Use labeled THP-1 cells (105 - 106 cells/ml) directly for microfluidics purification.</li>
</ol>
2. Microfluidic Device Preparation
<ol>
	<li>Device Fabrication
	<ol>
		<li>Fabricate the microfluidic spiral device (500 &#181;m (w) &#215; 115 &#181;m (h)) with polydimethylsiloxane (PDMS) from a commercial kit using standard soft lithography steps.23</li>
		<li>Mix 30 g of base prepolymer and 3 g curing agent thoroughly in a weighing boat. De-gas the mixture in a desiccator for 60 min to remove any air bubbles.</li>
		<li>Pour the PDMS mixture onto the silicon wafer master mold patterned with the spiral channel design carefully to a height of ~5 - 10 mm.</li>
		<li>De-gas the mixture in a desiccator vacuum for 60 min again to remove any air bubbles. Repeat the process until all bubbles are eliminated.</li>
		<li>Cure the PDMS mixture in an 80 &#176;C oven for 2 hr until the PDMS is set. Ensure the wafer is not tilted during curing to have a constant device height.</li>
		<li>Cut out the PDMS spiral device using a scalpel and carefully peel the PDMS slab from the master mold.</li>
		<li>Trim the edges of the device with a scalpel to ensure smooth surface for bonding.</li>
		<li>Punch two holes (1.5 mm) for the inlets and two holes (1.5 mm) for the outlets on the PDMS device using a 1.5 mm biopsy puncher.</li>
		<li>Wash the device with isopropanol (IPA) to remove any debris and dry the device in an 80 &#176;C oven for 5 min.</li>
		<li>Clean the bottom surface of the PDMS device (with channel features) using masking tape.</li>
		<li>Clean one side of a glass slide (2&#34; by 3&#34;) using masking tape.</li>
		<li>Carefully place and expose the cleaned surfaces of the PDMS device and glass slide in the chamber of a plasma cleaner and subject them to vacuum for 60 sec. Next, switch on the plasma power to maximum and lower the chamber pressure until the chamber turns pink in color.
		<ol>
			<li>Expose the surfaces to air plasma for 60 sec. The plasma creates reactive species on the exposed surfaces of the PDMS and glass which enables tight bonding when brought into physical contact. Turn off the plasma power and release the pressure from the plasma cleaner to retrieve the device and glass slide.</li>
		</ol>
		</li>
		<li>Bond the PDMS device and glass slide together by pressing the plasma-exposed surfaces tightly and ensuring that no bubbles are trapped between the two surfaces.</li>
		<li>Heat the bonded device using a hotplate set at 80 &#176;C for 2 hr to strengthen the bonding.</li>
	</ol>
	</li>
	<li>Device Operation
	<ol>
		<li>Cut two pieces of tubing (1.52 mm OD) of ~15 - 20 cm for the inlet syringes and attach a syringe tip (gauge 23) on one end of each tubing.</li>
		<li>Cut two pieces of tubing (1.52 mm OD) of ~5 - 10 cm for the outlets and attach to the outlet holes of the PDMS device.</li>
		<li>Prior to sample running, manually prime the device with a syringe containing 70% ethanol until it flows out of the outlet tubing. Allow the ethanol sit for 30 sec to 1 min to sterilize the device.</li>
		<li>Load 30 ml of filtered (0.2 &#181;m pore) sheath buffer (Phosphate-Buffered Saline (PBS) with 0.1% Bovine Serum Albumin (BSA)) into the 60 ml syringe and secure the syringe on a syringe pump. Set the pump to the correct settings (Syringe size: 60 ml, Volume: 60,000 &#181;l). 
		Note: The addition of BSA to PBS is to minimize cell-cell binding and non-specific binding between cells and the PDMS device.</li>
		<li>Load 3 ml of labeled cells into the 3 ml syringe and secure the syringe on a separate syringe pump. Set the pump to the correct settings (Syringe size: 3 ml, Volume: 3,000 &#181;l).</li>
		<li>Check that no air bubbles are trapped in the syringes to ensure a stable flow. Remove any air bubbles by gently ejecting few drops of liquid out of the nozzle.</li>
		<li>Connect the inlet syringe tips and tubing to the syringes, and insert them into the respective inlets of the device (sheath and sample inlet). Ensure that there are no bubbles along the tubing.</li>
		<li>Mount the devices onto an inverted phase-contrast microscope for real time imaging during cell sorting process.</li>
		<li>Secure a small waste beaker and two 15 ml tubes close to the device on the microscope stage using adhesives.</li>
		<li>Place the outlet tubings into the waste beaker.</li>
		<li>Set the flow ratio for sample to sheath buffer to 1:10 and start both syringe pumps to initiate the sorting process (Example: 120 &#181;l/min for sample syringe and 1,200 &#181;l/min for sheath syringe; channel flow velocity ~0.38 m/sec).</li>
		<li>Run the device for 1.5 min for the flow rate to stabilize. This can be confirmed by presence of inertially focused cells near the channel inner wall under bright-field with phase contrast using a high-speed camera (~5,000 - 10,000 frames per second (fps), exposure time: 10-50 &#181;sec).</li>
		<li>Position the outlet tubings into different tubes to collect the eluents from the cell outlet and waste outlet.</li>
	</ol>
	</li>
</ol>

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The authors have nothing to disclose.

The DFF cell purification technology described herein enables rapid and continuous separation of labeled cells in a high throughput manner. This separation approach is ideal for large sample volume or high cell concentration sample processing, and is better than conventional membrane-based filtration which is prone to clogging after extended use. Similarly, affinity-based magnetic separation requires additional cell labelling steps which are laborious and expensive. The purified cells are shown to retain their labeled agents and cell morphology well (Figure 3) with minimal flow/shear-induced effects (shear stress &#964;, ~200-250 dyne/cm2) due to short residence time within the channel (&#60; 1 sec) 20,24. In addition, the user can choose the eluted solution of the sorted labeled cells by simply substituting the sheath buffer (saline) with the desired media or buffer solution.
It is critical that there is no dirt or debris in the microchannel prior use which can adversely affect cell focusing behavior. This can be achieved by cleaning the inlet holes thoroughly with IPA, as well as using masking tape to clean the PDMS surface before plasma bonding. Since the microdevice operates under high flow conditions (~1 - 1.5 ml/min), it is also important to heat the bonded PDMS devices on a hotplate to ensure strong bonding between the PDMS and the glass slide. During device operation, if the cells are not focusing well, it means that the flow rates are not sufficient. One can troubleshoot by checking the device inlet region and the syringe tips to ensure that there are no leakages.
Removal of unbound NPs from labeled cells is not trivial since common laboratory separation techniques (e.g., centrifugation) cannot separate particles/cells of different sizes (e.g., unbound particles and labeled cells). While one can argue that repeated washing and changing of culture media can remove free NPs from adherent cells to a certain extent, the process is laborious and ineffective for suspension cell cultures. The DFF microfluidic device utilizes size-based sorting and is highly versatile as it can be used for both suspension and adherent cells with a large size range, as well as removal of NPs of different materials. If the target cells are of different sizes, the DFF device can be optimized by changing the flow conditions or channel geometry (e.g., channel height and ratio of inner and outer channel widths) to achieve efficient separation. It has been previously demonstrated that DFF is superior compared to conventional centrifugation in NPs removal with minimal cell loss.22 Furthermore, cell recovery performance remained high (&#62;90%) when the sample concentration was increased to 107 cells/ml. This is a key improvement over existing inertial microfluidic cell separation methods which are generally limited to 105 cells/ml capacity due to cell-cell interaction and cell overcrowding at equilibrium positions.25
While the DFF technology achieves separation based on size difference, it cannot be used for other cell engineering processes such as separation of labeled and unlabeled cells due to negligible change in cell size. Cell surface markers for affinity-based sorting and other common separation modalities (magnetic susceptibility and electrical conductivity) could be considered for such potential applications. DFF technology is also not suitable for multiplexed cell sorting based on fluorescence. A better option would be to use fluorescence activated cell sorting (FACS) for single-step sorting of labelled cells with different fluorescence which operates at high throughput, but requires expensive and bulky equipment.
Finally, this protocol is highly useful for the purification of engineered cells when NPs containing transformative agents (growth factors, drugs, corticosteroids, etc.) are used for bioimaging and/or cell therapy. This is because remnant free NPs can exert off-target transformative effects on neighboring cells or immune cells that respond to these foreign particles when injected in vivo. It may also unintentionally generate signals that can be misleading during bioimaging. Removal of these free NPs prior to usage thus reduce risk to non-target cells, minimize compilations in immune system, and reduce contamination issues between steps during sequential labeling/engineering of cells. Its successful implementation greatly facilitates cell labeling and enables the interference-free usage of particle based cell engineering in therapy.

Engineering cells by agent-loaded micro/nanoparticles (NPs) is a simple, genomic integration-free, and versatile method to enhance bioimaging capability and augment/supplement its native therapeutic properties in regenerative medicine.1-3 Cellular modifications are achieved by labeling the plasma membrane or cytoplasm with an excess concentration of agent-loaded NPs to saturate the binding sites. However, a major drawback of this method is the significant quantities of unbound particles remaining in solution after cell labeling processes, which can potentially confound precise identification of particle-engineered cells or complicate therapeutic outcomes.4,5 In addition, exposure to NPs containing transformative agents (growth factors, corticosteroids, etc.) at excessively high concentration can cause cytotoxicity and misdirected exposure may induce unintended consequences on non-target cells. Even particulate carriers comprising of &#34;biocompatible&#34; materials [e.g., poly(lactic co-glycolic acid), PLGA] can incite potent immune cell responses under certain conditions as well.6 This is especially risky in individuals with impaired immunity (e.g., rheumatoid arthritis) which potentially delays systemic nanoparticle clearance.7 Thus, efficient removal of free particles prior to the introduction of particle-engineered cells is of great importance to minimize toxicity profile and reduce misdirected exposure to agent-loaded particles in vivo.
Conventional gradient centrifugation is often used to separate engineered cells from free particles but is laborious and operated in batch mode. Moreover, shear stresses experienced by cells during high-speed centrifugation and the constituents of the density gradient medium may compromise cell integrity and/or influence cell behavior.8 Microfluidics is an attractive alternative with several separation technologies including deterministic lateral displacement (DLD)9, dieletrophoresis10,11 and acoustophoresis12 developed for small particles separation and buffer exchange applications. However, these methods suffer from low throughput (1-10 &#181;l&#183;min&#8722;1) and are prone to clogging issues. Active separations such as dielectrophoresis-based methods also require differences in intrinsic dielectrophoretic cell phenotypes or additional cell labeling steps to achieve separation. A more promising approach involves inertial microfluidics &#8212; the lateral migration of particles or cells across streamlines to focus at distinct positions due to dominant lift forces (FL) at high Reynolds number (Re).13 Due to its high flow conditions and superior size resolution, it has often been exploited for size-based cell separation14,15 and buffer exchange applications.16-18 However, buffer exchange performance remains poor with &#8764;10&#8722;30% contaminant solution as the separated cells usually remain close to the boundary between original and new buffer solutions.16-18 More importantly, the size distribution of target cells has to be similar to achieve precise inertial focusing and separation from the original buffer solution which poses an issue especially in the processing of heterogeneous-sized cell types such as mesenchymal stem cells (MSCs).19
We have previously developed a new inertial microfluidics cell sorting technique termed Dean Flow Fractionation (DFF) for isolating circulating tumor cells (CTCs)20 and bacteria21 from whole blood using a 2-inlet, 2-outlet spiral microchannel device. In this video protocol, we will describe the process of labeling THP-1 (human acute monocytic leukemia cell line) suspension monocytic cells (~15 &#181;m) and MSCs (10-30 &#181;m) with calcein-loaded NPs, followed by fabrication and operation of the DFF spiral microdevice for efficient recovery of labeled cells and removal of unbound NPs.22 This single step purification strategy enables continuous recovery of labeled suspension and adherent cells suspended in fresh buffer solution without centrifugation. Moreover, it can process up to 10 million cells&#183;ml&#8722;1, a cell density amenable for regenerative medicine applications.

<table><tbody><tr><td>&lt;strong&gt;Cell lines &amp;amp; Media&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Mesenchymal Stem Cells (MSCs)</td><td>Lonza</td><td>PT-2501</td><td></td></tr><tr><td>Dulbecco&amp;rsquo;s modified Eagle&amp;rsquo;s medium (DMEM)</td><td>Lonza</td><td>12-614F</td><td></td></tr><tr><td>Fetal Bovine Serum (FBS)</td><td>Gibco</td><td>10270-106</td><td></td></tr><tr><td>THP-1 monocyte cells (THP-1)</td><td>ATCC</td><td>TIB-202</td><td></td></tr><tr><td>Roswell Park Memorial Institute (RPMI) 1640 media</td><td>Lonza</td><td>12-702F</td><td></td></tr><tr><td>&lt;strong&gt;Reagents &amp;amp; Materials&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>0.01% poly-L-lysine (PLL)</td><td>Sigma-Aldrich</td><td>P8920</td><td></td></tr><tr><td>3 ml Syringe</td><td>BD</td><td>302113</td><td>Syringe 3 ml Luer-Lock</td></tr><tr><td>60 ml Syringe</td><td>BD</td><td>309653</td><td>Syringe 60 ml Luer-Lock</td></tr><tr><td>Bovine Serum Albumin (BSA)</td><td>Biowest</td><td>P6154-100GR</td><td></td></tr><tr><td>Calcein, AM (CAM)</td><td>Life Technologies</td><td>C1430</td><td></td></tr><tr><td>Calcein</td><td>Sigma-Aldrich</td><td>C0875</td><td></td></tr><tr><td>Isopropanol</td><td>Fisher Chemical</td><td>#P/7507/17</td><td>HPLC Grade 2.5 L</td></tr><tr><td>Phosphate-Buffered Saline (PBS)</td><td>Lonza</td><td>17-516Q/12</td><td></td></tr><tr><td>Plain Microscope Slides</td><td>Fisher Scientific</td><td>FIS#12-550D</td><td>75 x 25 x 1 mm&lt;sup&gt;3&lt;/sup&gt;</td></tr><tr><td>Polydimethylsiloxane (PDMS)</td><td>Dow Corning</td><td>SYLGARD&amp;reg; 184</td><td></td></tr><tr><td>Scotch tape</td><td>3M</td><td>21200702044</td><td>18 mm x 25 m</td></tr><tr><td>Silica NPs (&amp;sim;200 &amp;mu;m)</td><td>Sigma-Aldrich</td><td>748161</td><td>Pore size 4 nm</td></tr><tr><td>Syringe Tip</td><td>JEC Technology</td><td>7018302</td><td>23 G 0.013 x 0.25</td></tr><tr><td>Trypsin-EDTA (0.25%)</td><td>Life Technologies</td><td>25200-056</td><td></td></tr><tr><td>Tygon Tubing</td><td>Spectra-Teknik</td><td>06419-01</td><td>0.02 x 0.06&quot; 100</td></tr><tr><td>Poly (D,L-lactide-co-glycolide) (PLGA; 50:50)</td><td>Sigma-Aldrich</td><td>P2191</td><td></td></tr><tr><td>&lt;strong&gt;Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Biopsy punch</td><td>Harris Uni-Core</td><td>69036-15</td><td>1.50 mm</td></tr><tr><td>Dessicator</td><td>Scienceware</td><td>111/4 IN OD</td><td></td></tr><tr><td>High-speed Camera</td><td>Phantom&amp;nbsp;</td><td>V9.1</td><td></td></tr><tr><td>Inverted phase-contrast microscope&amp;nbsp;</td><td>Nikon</td><td>Eclipse Ti</td><td></td></tr><tr><td>Plasma cleaner</td><td>Harrick Plasma</td><td>PDC-002</td><td></td></tr><tr><td>Syringe Pump</td><td>Chemyx</td><td>CX Fusion 200</td><td></td></tr></tbody></table>

microfluidic buffer exchange for interference-free micro/nanoparticle cell engineering

Engineering cells with active-ingredient-loaded micro/nanoparticles (NPs) is becoming an increasingly popular method to enhance native therapeutic properties, enable bio imaging and control cell phenotype. A critical yet inadequately addressed issue is the significant number of particles that remain unbound after cell labeling which cannot be readily removed by conventional centrifugation. This leads to an increase in bio imaging background noise and can impart transformative effects onto neighboring non-target cells. In this protocol, we present an inertial microfluidics-based buffer exchange strategy termed as Dean Flow Fractionation (DFF) to efficiently separate labeled cells from free NPs in a high throughput manner. The developed spiral microdevice facilitates continuous collection (&#62;90% cell recovery) of purified cells (THP-1 and MSCs) suspended in new buffer solution, while achieving &#62;95% depletion of unbound fluorescent dye or dye-loaded NPs (silica or PLGA). This single-step, size-based cell purification strategy enables high cell processing throughput (106 cells/min) and is highly useful for large-volume cell purification of micro/nanoparticle engineered cells to achieve interference-free clinical application.

After labeling the cells with bio imaging agent-loaded NPs overnight, the labeled cells (containing free particles) are harvested and purified by DFF spiral microdevice to remove free NPs in a single step process (Figure 1A). The 2-inlet, 2-outlet spiral microchannel is designed by engineering software and microfabricated using SU-8 photoresist. The patterned silicon wafer is then used as a template for PDMS replica molding using soft lithography techniques (Figure 1B). To perform cell sorting, the cell sample is pumped into the outer wall inlet of the spiral microdevice while the inner wall inlet runs fresh buffer solution at a higher flow rate (1:10) to pinch the cell sample flow. Separation is achieved by inertial focusing of larger labeled cells at the inner wall and Dean-induced recirculation of small unbound NPs towards the outer wall (Figure 2A). The device was first applied to sort labeled suspension monocytic cells (THP-1) from fluorescent silica NPs loaded with calcein (~500 nm in size). High separation efficiencies were obtained as confirmed by high speed imaging (Figure 2A) and flow cytometry analysis (Figure 2B). Images of eluents collected from device cell outlet also indicated efficient removal of silica and PLGA NPs (~1 - 5 &#181;m) from THP-1 cells. In addition, fluorescence imaging showed that internalized NPs in the labeled THP-1 cells were well retained during DFF purification (insets) (Figure 3). Because of MSC size heterogeneity, the spiral microchannel geometry was subsequently modified by changing the channel height and bifurcation design to enable efficient collection of MSCs (Figure 4).
<img alt="Figure 1" src="/files/ftp_upload/54327/54327fig1.jpg" /> 
Figure 1. (A) Overall workflow for rapid single-step micro/nanoparticle (NPs) removal from suspension and adherent cells post-labeling using Dean Flow Fractionation (DFF) microfluidic technology. (B) The DFF spiral device is designed by engineering software and patterned on silicon wafer using microfabrication. PDMS devices are subsequently produced by soft lithography methods. Experimental setup consists of 2 syringe pumps to infuse labeled cells and fresh saline buffer at different flow rates into the device for continuous purification and collection of labeled cells. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54327/54327fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/54327/54327fig2.jpg" /> 
Figure 2. Nanoparticles removal from labeled THP-1 cells via DFF. (A) Illustration of DFF separation principle. Larger labeled cells experience strong inertial lift forces (FL, red arrows) and Dean drag forces (FD, yellow arrows) and focus near the microchannel inner wall. Smaller NPs and buffer solution are solely affected by Dean drag and recirculate to the outer wall to achieve separation. Representative high speed and fluorescence images indicating equilibrium positions of THP-1 cells (red arrows) and silica NPs loaded with calcein dye at the outlet region. (B) Separation performance based on flow cytometry analysis with gating determined by size (FSC-A) and granularity (SSC-A) characteristics. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54327/54327fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/54327/54327fig3.jpg" /> 
Figure 3. Representative bright field images of purified labeled THP-1 cells (red arrows) and NPs removal efficiency using DFF. Insets show the respective phase contract (above) and fluorescence microscopy images of individual cells. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54327/54327fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/54327/54327fig4.jpg" /> 
Figure 4. Nanoparticles removal from mesenchymal stem cells (MSCs) via DFF. (A) Representative high speed image illustrating efficient separation of MSCs (red arrows) and PLGA NPs (blue arrows) into different outlets. Yellow dashed lines indicate the approximate position of the microchannel walls. (B) Separation performance of MSCs from silica and PLGA NPs. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54327/54327fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Microfluidic Buffer Exchange for Interference-free Micro/Nanoparticle Cell Engineering at JoVE.com

Microfluidic Buffer Exchange for Interference-free Micro/Nanoparticle Cell Engineering

This protocol describes the use of an inertial microfluidics-based buffer exchange strategy to purify micro/nanoparticle engineered cells with efficient depletion of unbound particles.

Engineering cells with active-ingredient-loaded micro/nanoparticles (NPs) is becoming an increasingly popular method to enhance native therapeutic ...

microfluidic-buffer-exchange-for-interference-free-micronanoparticle

Universidad San Sebastian

Research

JoVE Journal

Bioengineering

9.0K Views.  Nanyang Technological University. This protocol describes the use of an inertial microfluidics-based buffer exchange strategy to purify micro/nanoparticle engineered cells with efficient depletion of unbound particles.

Video: Microfluidic Buffer Exchange for Interference-free Micro/Nanoparticle Cell Engineering

A Microfluidic-based Hydrodynamic Trap for Single Particles

The ability to confine and manipulate single particles in free solution is a key enabling technology for fundamental and applied science. Methods for particle trapping based on optical, magnetic, electrokinetic, and acoustic techniques have led to major advancements in physics and biology ranging from the molecular to cellular level. In this article, we introduce a new microfluidic-based technique for particle trapping and manipulation based solely on hydrodynamic fluid flow. Using this method, we demonstrate trapping of micro- and nano-scale particles in aqueous solutions for long time scales. The hydrodynamic trap consists of an integrated microfluidic device with a cross-slot channel geometry where two opposing laminar streams converge, thereby generating a planar extensional flow with a fluid stagnation point (zero-velocity point). In this device, particles are confined at the trap center by active control of the flow field to maintain particle position at the fluid stagnation point. In this manner, particles are effectively trapped in free solution using a feedback control algorithm implemented with a custom-built LabVIEW code. The control algorithm consists of image acquisition for a particle in the microfluidic device, followed by particle tracking, determination of particle centroid position, and active adjustment of fluid flow by regulating the pressure applied to an on-chip pneumatic valve using a pressure regulator. In this way, the on-chip dynamic metering valve functions to regulate the relative flow rates in the outlet channels, thereby enabling fine-scale control of stagnation point position and particle trapping. The microfluidic-based hydrodynamic trap exhibits several advantages as a method for particle trapping. Hydrodynamic trapping is possible for any arbitrary particle without specific requirements on the physical or chemical properties of the trapped object. In addition, hydrodynamic trapping enables confinement of a "single" target object in concentrated or crowded particle suspensions, which is difficult using alternative force field-based trapping methods. The hydrodynamic trap is user-friendly, straightforward to implement and may be added to existing microfluidic devices to facilitate trapping and long-time analysis of particles. Overall, the hydrodynamic trap is a new platform for confinement, micromanipulation, and observation of particles without surface immobilization and eliminates the need for potentially perturbative optical, magnetic, and electric fields in the free-solution trapping of small particles.

In this article, we present a microfluidic-based method for particle confinement based on hydrodynamic flow. We demonstrate stable particle trapping at a fluid stagnation point using a feedback control mechanism, thereby enabling confinement and micromanipulation of arbitrary particles in an integrated microdevice.

The ability to confine and manipulate single particles in free solution is a key enabling technology for fundamental and applied science.  Methods for ...

Separating Beads and Cells in Multi-channel Microfluidic Devices Using Dielectrophoresis and Laminar Flow

Microfluidic devices have advanced cell studies by providing a dynamic fluidic environment on the scale of the cell for studying, manipulating, sorting and counting cells. However, manipulating the cell within the fluidic domain remains a challenge and requires complicated fabrication protocols for forming valves and electrodes, or demands specialty equipment like optical tweezers. Here, we demonstrate that conventional printed circuit boards (PCB) can be used for the non-contact manipulation of cells by employing dielectrophoresis (DEP) for bead and cell manipulation in laminar flow fields for bioactuation, and for cell and bead separation in multichannel microfluidic devices. First, we present the protocol for assembling the DEP electrodes and microfluidic devices, and preparing the cells for DEP. Then, we characterize the DEP operation with polystyrene beads. Lastly, we show representative results of bead and cell separation in a multichannel microfluidic device. In summary, DEP is an effective method for manipulating particles (beads or cells) within microfluidic devices.

Dielectrophoresis (DEP) is an effective method to manipulate cells. Printed circuit boards (PCB) can provide inexpensive, reusable and effective electrodes for contact-free cell manipulation within microfluidic devices. By combining PDMS-based microfluidic channels with coverslips on PCBs, we demonstrate bead and cell manipulation and separation within multichannel microfluidic devices.

Microfluidic devices have advanced cell studies by providing a dynamic fluidic environment on the scale of the cell for studying, manipulating, sorting ...

Creating Sub-50 Nm Nanofluidic Junctions in PDMS Microfluidic Chip via Self-Assembly Process of Colloidal Particles

Polydimethylsiloxane (PDMS) is the prevailing building material to make microfluidic devices due to its ease of molding and bonding as well as its transparency. Due to the softness of the PDMS material, however, it is challenging to use PDMS for building nanochannels. The channels tend to collapse easily during plasma bonding. In this paper, we present an evaporation-driven self-assembly method of silica colloidal nanoparticles to create nanofluidic junctions with sub-50 nm pores between two microchannels. The pore size as well as the surface charge of the nanofluidic junction is tunable simply by changing the colloidal silica bead size and surface functionalization outside of the assembled microfluidic device in a vial before the self-assembly process. Using the self-assembly of nanoparticles with a bead size of 300 nm, 500 nm, and 900 nm, it was possible to fabricate a porous membrane with a pore size of ~45 nm, ~75 nm and ~135 nm, respectively. Under electrical potential, this nanoporous membrane initiated ion concentration polarization (ICP) acting as a cation-selective membrane to concentrate DNA by ~1,700 times within 15 min. This non-lithographic nanofabrication process opens up a new opportunity to build a tunable nanofluidic junction for the study of nanoscale transport processes of ions and molecules inside a PDMS microfluidic chip.

We propose a simple self-assembly technique of silica colloidal nanoparticles to create a nanofluidic junction between two microchannels in polydimethylsiloxane (PDMS). Using this technique, a nanoporous bead membrane with a pore size down to ~45 nm was built inside a microchannel and applied to electrokinetic preconcentration of DNA samples.

Polydimethylsiloxane (PDMS) is the prevailing building material to make microfluidic devices due to its ease of molding and bonding as well as its ...

Engineering

A Microfluidic Chip for the Versatile Chemical Analysis of Single Cells

We present a microfluidic device that enables the quantitative determination of intracellular biomolecules in multiple single cells in parallel. For this purpose, the cells are passively trapped in the middle of a microchamber. Upon activation of the control layer, the cell is isolated from the surrounding volume in a small chamber. The surrounding volume can then be exchanged without affecting the isolated cell. However, upon short opening and closing of the chamber, the solution in the chamber can be replaced within a few hundred milliseconds. Due to the reversibility of the chambers, the cells can be exposed to different solutions sequentially in a highly controllable fashion, e.g. for incubation, washing, and finally, cell lysis. The tightly sealed microchambers enable the retention of the lysate, minimize and control the dilution after cell lysis. Since lysis and analysis occur at the same location, high sensitivity is retained because no further dilution or loss of the analytes occurs during transport. The microchamber design therefore enables the reliable and reproducible analysis of very small copy numbers of intracellular molecules (attomoles, zeptomoles) released from individual cells. Furthermore, many microchambers can be arranged in an array format, allowing the analysis of many cells at once, given that suitable optical instruments are used for monitoring. We have already used the platform for proof-of-concept studies to analyze intracellular proteins, enzymes, cofactors and second messengers in either relative or absolute quantifiable manner.

In this article we present a microfluidic chip for single cell analysis. It allows the quantification of intracellular proteins, enzymes, cofactors, and second messengers by means of fluorescent assays or immunoassays.&#160;

We present a microfluidic device that enables the quantitative determination of intracellular biomolecules in multiple single cells in parallel. For this ...

A Modular Microfluidic Technology for Systematic Studies of Colloidal Semiconductor Nanocrystals

Colloidal semiconductor nanocrystals, known as quantum dots (QDs), are a rapidly growing class of materials in commercial electronics, such as light emitting diodes (LEDs) and photovoltaics (PVs). Among this material group, inorganic/organic perovskites have demonstrated significant improvement and potential towards high-efficiency, low-cost PV fabrication due to their high charge carrier mobilities and lifetimes. Despite the opportunities for perovskite QDs in large-scale PV and LED applications, the lack of fundamental and comprehensive understanding of their growth pathways has inhibited their adaptation within continuous nanomanufacturing strategies. Traditional flask-based screening approaches are generally expensive, labor-intensive, and imprecise for effectively characterizing the broad parameter space and synthesis variety relevant to colloidal QD reactions. In this work, a fully autonomous microfluidic platform is developed to systematically study the large parameter space associated with the colloidal synthesis of nanocrystals in a continuous flow format. Through the application of a novel translating three-port flow cell and modular reactor extension units, the system may rapidly collect fluorescence and absorption spectra across reactor lengths ranging 3 - 196 cm. The adjustable reactor length not only decouples the residence time from the velocity-dependent mass transfer, it also substantially improves the sampling rates and chemical consumption due to the characterization of 40 unique spectra within a single equilibrated system. Sample rates may reach up to 30,000 unique spectra per day, and the conditions cover 4 orders of magnitude in residence times ranging 100 ms - 17 min. Further applications of this system would substantially improve the rate and precision of the material discovery and screening in future studies. Detailed within this report are the system materials and assembly protocols with a general description of the automated sampling software and offline data processing.

Detailed herein are the operation and assembly protocols of a modular microfluidic screening platform for the systematic characterization of colloidal semiconductor nanocrystal syntheses. Through fully adjustable system arrangements, highly efficient spectra collection may be carried out across 4 orders of magnitude reaction time scales within a mass transfer-controlled sampling space.

Colloidal semiconductor nanocrystals, known as quantum dots (QDs), are a rapidly growing class of materials in commercial electronics, such as light ...

A Microfluidic Platform for Precision Small-volume Sample Processing and Its Use to Size Separate Biological Particles with an Acoustic Microdevice

A major advantage of microfluidic devices is the ability to manipulate small sample volumes, thus reducing reagent waste and preserving precious sample. However, to achieve robust sample manipulation it is necessary to address device integration with the macroscale environment. To realize repeatable, sensitive particle separation with microfluidic devices, this protocol presents a complete automated and integrated microfluidic platform that enables precise processing of 0.15&#8211;1.5 ml samples using microfluidic devices. Important aspects of this system include modular device layout and robust fixtures resulting in reliable and flexible world to chip connections, and fully-automated fluid handling which accomplishes closed-loop sample collection, system cleaning and priming steps to ensure repeatable operation. Different microfluidic devices can be used interchangeably with this architecture. Here we incorporate an acoustofluidic device, detail its characterization, performance optimization, and demonstrate its use for size-separation of biological samples. By using real-time feedback during separation experiments, sample collection is optimized to conserve and concentrate sample. Although requiring the integration of multiple pieces of equipment, advantages of this architecture include the ability to process unknown samples with no additional system optimization, ease of device replacement, and precise, robust sample processing.

This protocol describes a system architecture for performing automated small volume (0.15&#8211;1.5 ml) particle separations using a microfluidic device, and discusses methods to optimize acoustofluidic device performance and operation.

A major advantage of microfluidic devices is the ability to manipulate small sample volumes, thus reducing reagent waste and preserving precious sample. ...

A Microfluidic Platform for High-throughput Single-cell Isolation and Culture

Studying the heterogeneity of single cells is crucial for many biological questions, but is technically difficult. Thus, there is a need for a simple, yet high-throughput, method to perform single-cell culture experiments. Here, we report a microfluidic chip-based strategy for high-efficiency single-cell isolation (~77%) and demonstrate its capability of performing long-term single-cell culture (up to 7 d) and cellular heterogeneity analysis using clonogenic assay. These applications were demonstrated with KT98 mouse neural stem cells, and A549 and MDA-MB-435 human cancer cells. High single-cell isolation efficiency and long-term culture capability are achieved by using different sizes of microwells on the top and bottom of the microfluidic channel. The small microwell array is designed for precisely isolating single-cells, and the large microwell array is used for single-cell clonal culture in the microfluidic chip. This microfluidic platform constitutes an attractive approach for single-cell culture applications, due to its flexibility of adjustable cell culture spaces for different culture strategies, without decreasing isolation efficiency.

Here, we present a protocol for isolating and culturing single cells with a microfluidic platform, which utilizes a new microwell design concept to allow for high-efficiency single cell isolation and long-term clonal culture.

Studying the heterogeneity of single cells is crucial for many biological questions, but is technically difficult. Thus, there is a need for a simple, yet ...

Pneumatically Driven Microfluidic Platform for Micro-Particle Concentration

The present article introduces a method for fabricating and operating a pneumatic valve to control particle concentration using a microfluidic platform. This platform has a three-dimensional (3D) network with curved fluid channels and three pneumatic valves, which create networks, channels, and spaces through duplex replication with polydimethylsiloxane (PDMS). The device operates based on the transient response of a fluid flow rate controlled by a pneumatic valve in the following order: (1) sample loading, (2) sample blocking, (3) sample concentration, and (4) sample release. The particles are blocked by thin diaphragm layer deformation of the sieve valve (Vs) plate and accumulate in the curved microfluidic channel. The working fluid is discharged by the actuation of two on/off valves. As a result of the operation, all particles of various magnifications were successfully intercepted and disengaged. When this technology is applied, the operating pressure, the time required for concentration, and the concentration rate may vary depending on the device dimensions and particle size magnification.

The present protocol describes a pneumatic microfluidic platform that can be used for efficient microparticle concentration.

The present article introduces a method for fabricating and operating a pneumatic valve to control particle concentration using a microfluidic platform. ...

Merging Ion Concentration Polarization between Juxtaposed Ion Exchange Membranes to Block the Propagation of the Polarization Zone

The ion concentration polarization (ICP) phenomenon is one of the most prevailing methods to preconcentrate low-abundance biological samples. The ICP induces a noninvasive region for charged biomolecules (i.e., the ion depletion zone), and targets can be preconcentrated on this region boundary. Despite the high preconcentration performances with ICP, it is difficult to find the operating conditions of non-propagating ion depletion zones. To overcome this narrow operating window, we recently developed a new platform for spatiotemporally fixed preconcentration. Unlike preceding methods that only use ion depletion, this platform also uses the opposite polarity of the ICP (i.e., ion enrichment) to stop the propagation of the ion depletion zone. By confronting the enrichment zone with the depletion zone, the two zones merge together and stop. In this paper, we describe a detailed experimental protocol to build this spatiotemporally defined ICP platform and characterize the preconcentration dynamics of the new platform by comparing them with those of the conventional device. Qualitative ion concentration profiles and current-time responses successfully capture the different dynamics between the merged ICP and the stand-alone ICP. In contrast to the conventional one that can fix the preconcentration location at only ~5 V, the new platform can produce a target-condensed plug at a specific location in the broad ranges of operating conditions: voltage (0.5-100 V), ionic strength (1-100 mM), and pH (3.7-10.3).

The protocol for a novel ion concentration polarization (ICP) platform that can stop the propagation of the ICP zone, regardless of the operating conditions is described. This unique ability of the platform lies in the use of merging ion depletion and enrichment, which are two polarities of the ICP phenomenon.

The ion concentration polarization (ICP) phenomenon is one of the most prevailing methods to preconcentrate low-abundance biological samples. The ICP ...

Computer Numerical Control Micromilling of a Microfluidic Acrylic Device with a Staggered Restriction for Magnetic Nanoparticle-Based Immunoassays

Microfluidic systems have greatly improved immunoassay techniques. However, many microfabrication techniques require specialized, expensive, or complicated equipment, making fabrication costly and incompatible with mass production, which is one of the most important preconditions for point-of-care tests (POCT) to be adopted in low-resource settings. This work describes the fabrication process of an acrylic (polymethylmethacrylate, PMMA) device for nanoparticle-conjugated enzymatic immunoassay testing using the computer numerical control (CNC) micromilling technique. The functioning of the microfluidic device is shown by performing an immunoassay to detect a commercial antibody using lysozyme as a model antigen conjugated to 100 nm magnetic nanoparticles. This device integrates a physical staggered restriction of only 5 &#181;m in height, used to capture magnetic microparticles that make up a magnetic trap by placing an external magnet. In this way, the magnetic force on the immunosupport of conjugated nanoparticles is enough to capture them and resist flow drag. This microfluidic device is particularly suitable for low-cost mass production without the loss of precision for immunoassay performance.

Microfluidics is a powerful tool for the development of diagnostic tests. However, expensive equipment and materials, as well as laborious fabrication and handling techniques, are often required. Here, we detail the fabrication protocol of an acrylic microfluidic device for magnetic micro- and nanoparticle-based immunoassays in a low-cost and simple-to-use setting.