Name: pneumatically driven microfluidic platform for micro-particle concentration
Uploaded: 2022-02-01T14:15:00.000+00:00
Duration: 8 min 43 s
Description: Watch this Scientific Journal Video about Pneumatically Driven Microfluidic Platform for Micro-Particle Concentration at JoVE.com

This work was supported by the National Research Foundation of Korea(NRF) grant funded by the Korea government(Ministry of Science and ICT). (No. NRF-2021R1A2C1011380).

1. Designing the microfluidic platform for particle concentration
<ol>
	<li>Design the pneumatic microfluidic platform consisting of one pneumatic valve for fluid flow in the 3D flow network and three pneumatic valves for sieve (Vs), fluid (Vf), and particle (Vp) valve operation (Figure 1). 
	NOTE: Vs blocks concentrate particles from the liquid, and Vf and Vp allow fluid and particle release after concentration. Three pneumatic ports provide compressed air from the fluid/pneumatic supply layer (normally open) and the pneumatic valve light outlet to actuate the valve. The microfluidic channel network is designed with a CAD program18,19.</li>
	<li>Design the channel to be a pneumatic supply layer and a 3D channel network layer (Figure 2). 
	NOTE: The fluid network is interconnected with the curved channels in the anterior part and the rectangular chamber in the posterior region. Vs block the inlet, and particles accumulate in the collection area of the curved fluid channel. Particle-free fluids (particle-free liquids) are exited through the Qf outlet and the concentrated particles through the Qp outlet (Figure 3).</li>
	<li>According to the above conditions, prepare four types of SU-8 molds. 
	NOTE: The four molds include a mold that allows the valve to be controlled via pneumatics, two molds that create fluid channels, and a clean mold without shape (Figure 4 and Table 1). The four types of molds mentioned are fabricated using standard photolithography processes. This mold making consists of a SU-8 mold on a silicon wafer as per previously published reports18,19. Figure 5 depicts the device chip.</li>
</ol>
2. Fabrication of the microfluidic platform for particle concentration
NOTE: Figure 6 illustrates the fabrication of a microfluidic platform that concentrates particles.
<ol>
	<li>Replicate the PDMS layer using a prepared pneumatic valve channel SU-8 mold (step 1.3) for pneumatically controlling the valve.
	<ol>
		<li>Pour 10 mL of liquid PDMS and 1 mL of curing agent (see Table of Materials) into a prepared pneumatic valve channel mold (step 1.3) and heat-activate at 90 &#176;C for 30 min.</li>
		<li>After the PDMS structures are cured, separate the SU-8 mold of step 2.1.1.</li>
		<li>Punch three 1.5 mm pneumatic ports (Vs, Vf, and Vp) into the pneumatic valve channel manufactured according to step 2.1.2 using a 1.5 mm puncture (see Table of Materials).</li>
		<li>Pour 10 mL of liquid PDMS and 1 mL of curing agent into a prepared clean SU-8 mold prepared in step 1.3 and spin-coat at 1,500 rpm for 15 s using a spin coater (see Table of Materials). Then heat-activate at 90 &#176;C for 30 min.</li>
		<li>After the PDMS structures are cured, separate the SU-8 mold of step 2.1.4. 
		NOTE: The valve diaphragm layer controls fluid flow according to the pneumatic pressure.</li>
		<li>Treat atmospheric plasma (see Table of Materials) to the PDMS structures prepared in steps 2.1.3 and 2.1.5 for 20 s.</li>
		<li>Align directly plasma-treated PDMS structures from step 2.1.6 according to the channel structure by checking with a microscope.</li>
		<li>Bond the aligned PDMS structures prepared in step 2.1.7 by heating at 90 &#176;C for 30 min.</li>
		<li>Punch a 1.5 mm diameter hole in the fluid channel inlet (Qfp) and fluid channel outlets (Qf and Qp) within the pneumatic channel part to which the thin diaphragm layer is bonded, using a 1.5 mm puncture.</li>
	</ol>
	</li>
	<li>Replicate both sides of the PDMS layer using two SU-8 molds to make a microfluidic channel. Use a curved and rectangular microfluidic channel mold on the front and a microfluidic interconnection channel mold on the rear.
	<ol>
		<li>Pour 10 mL of liquid PDMS and 1 mL of curing agent into the curved and rectangular microfluidic channel mold and spin-coat at 1,200 rpm for 15 s. Then create molds for the curved fluid chamber and fluid channels by thermal activation at 90 &#176;C for 30 min (Figure 6A).</li>
		<li>Separate the PDMS layer on which the microfluidic channel is formed, then make a heat-activated mold covering the sealed vent wall by bonding to the glass wafer by treating atmospheric plasma for 20 s (Figure 6B).</li>
		<li>Pour 3 mL of liquid PDMS into the interconnection channel of the SU-8 mold (Figure 6C).</li>
		<li>Arrange the structure fabricated in step 2.2.2 with the interconnection channel mold in liquid PDMS on the microfluidic interconnect channel mold, and dry the superimposed structure at 130 &#176;C for 30 min (Figure 6D). 
		NOTE: While curing the rear structure, the PDMS mold fabricated in step 2.2.2 is inflated by the thermal pressure of the air layer, and the deformed PDMS layer is thermally activated (Figure 6E)16.</li>
		<li>After curing, remove the front SU-8 mold from the microfluidic channel network layer and carefully peel off the rear PDMS mold (Figure 6F). 
		NOTE: The 3D fluidic network layer allows the creation of an anterior curved fluid chamber and microfluidic channels.</li>
		<li>Pour 10 mL of liquid PDMS and 1 mL of curing agent into a clean SU-8 mold. Then heat-activate at 90 &#176;C for 30 min.</li>
		<li>After the PDMS structures are cured, separate the SU-8 mold. 
		NOTE: This step creates the additional sealing layer.</li>
		<li>Treat the atmospheric plasma to PDMS structures prepared in steps 2.2.3 and 2.2.7 for 20 s.</li>
		<li>Align directly plasma-treated PDMS structures according to the channel structure by checking with a microscope.</li>
		<li>Bond the aligned PDMS structures by heating at 90 &#176;C for 30 min.</li>
	</ol>
	</li>
	<li>Align the PDMS structures prepared in steps 2.1 and 2.2 according to the channel structure and bond them by treating atmospheric plasma for 20 s.</li>
</ol>
3. Setting up the device
NOTE: Figure 7 shows fabricating a microfluidic platform that concentrates particles.
<ol>
	<li>Manually fill the microfluidic channel with bubble-free demineralized water using a 10 mL syringe.</li>
	<li>To control the P_Qfp and the three pneumatic valves (P_Vs, P_Vf, and P_Vp) that control the microbead flow, insert a precision pressure controller with four or more output channels (see Table of Materials) for the working fluid (Qfp) into the microfluidic platform. 
	NOTE: A precision pressure controller with four output channels can be replaced with multiple precision pressure controllers. In this experiment, the operating pressure of P_Qfp was 10 kPa, P_Vs was 15 kPa, and P_Vf and P_Vp were both 18 kPa (Figure 8 and Table 2). Figure 8 shows the working fluid flow rate over time as particles are concentrated by the microfluidic platform with P_Vs of 15 kPa, and Table 2 shows the actuation results according to the pneumatic valves.</li>
	<li>Prepare carboxyl polystyrene test particles of various sizes in distilled water (see Table of Materials). 
	NOTE: The particle sizes used in this experiment were 24.9, 8.49, and 4.16 &#181;m; particles of various sizes can be used depending on the pressure of P_Vs.</li>
	<li>To control the flow rate of the working fluid, fill a glass bottle half full with water (working fluid) and connect the glass bottle cap to the controller output channel and microvalve. 
	NOTE: Connect one tube to the microvalve to receive compressed air from the controller and the other tube to inject water.</li>
	<li>Observe platform operation through an inverted microscope for all platform operations and measure the operating flow rate over time at the outlet by a liquid flow meter (see Table of Materials).</li>
</ol>
4. Operation of the device
<ol>
	<li>Inject the particle/fluid mixture under pressure at the inlet (Qfp) with Vp (Figure 9A). 
	NOTE: The flow of particles and clean fluid from the outlet through the interconnected channels are controlled via Vp and Vf, respectively (Table 2).</li>
	<li>Apply pressure to Vs at 15 kPa and Vp at 18 kPa to actuate the valve. 
	NOTE: At this time, the diaphragm is deformed, the particles of the fluid Qfp are blocked in the contact space between the curved fluid channel and the curved fluid cantilever, and the unwanted Qfp fluid is released through the open Qf (Figure 9B,C).</li>
	<li>When the particles are concentrated, apply pressure only to Vf. 
	NOTE: At this time, when pressure is applied only to Vf, the clogged particles are released through Qp (Figure 9D).</li>
</ol>

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The authors have no conflicts of interest to disclose.

This platform provides a simple way to purify and concentrate particles of various sizes. Particles are accumulated and released through pneumatic valve control, and no clogging is observed because there is no passive structure. Using this device, the concentration of particles of three sizes is presented. However, the operating pressure, the time required for concentration, and the rate may vary depending on the device dimensions, particle size magnification, and the pressure at Vs18,<...

Due to the importance of biological analysis, microfluidic and biomedical microelectromechanical systems (BioMEMS) technologies1,2 are used to develop and study devices for the purification and collection of micromaterials2,3,4.&#160;Particle capture is categorized as active or passive. Active traps have been used for external dielectric5, magnetophoretic6, auditory7, visual8, or thermal9 forces acting on independent particles, enabling precise control of their movements. However, an interaction between the particle and external force is required; thus, the throughput is low. In microfluidic systems, controlling the flow rate is very important because the external forces are transmitted to the target particles.
In general, passive microfluidic devices have micropillars in microchannels10,11. Particles are filtered through interaction with a flowing fluid, and these devices are easy to design and inexpensive to manufacture. However, they cause particle clogging in micro-pillars, so more complex devices have been developed to prevent particle clogging12. Microfluidic devices with complex structures are generally suitable for managing a limited number of particles13,14,15,16,17,18.
This article describes a method to fabricate and operate a pneumatically driven microfluidic platform for large particle concentrations that overcomes the shortcomings18 as mentioned above. This platform can block and concentrate particles by deformation and actuation of the thin diaphragm layer of the sieve valve (Vs) plate that accumulates in curved microfluidic channels. Particles accumulate in curved microfluidic channels, and the concentrated particles can separate by discharging the working fluid via the actuation of two PDMS seals on/off valves18. This method makes it possible to process a limited number of particles or concentrate a large number of small particles. Operating conditions such as the magnitude of flow rate and compressed air pressure can prevent unwanted cell damage and increase cell trapping efficiency.

<table><tbody><tr><td>1.5 mm puncture</td><td>Self procduction</td><td>Self procduction</td><td>This puncture was made by requesting a mold maker based on the Miltex&amp;reg; Biopsy Punch with Plunger (15110-15) product.</td></tr><tr><td>4 inch Silicon Wafer/SU-8 mold</td><td>4science</td><td>29-03573-01</td><td>4 inch (100) Ptype silicon wafer/SU-8 mold</td></tr><tr><td>Carboxyl Polystyrene Crosslinked Particle(24.9 &amp;mu;m)</td><td>Spherotech</td><td>CPX-200-10</td><td>Concentrated bead sample1</td></tr><tr><td>Flow meter</td><td>Sensirion</td><td>SLI-1000</td><td>Flow measurement</td></tr><tr><td>High-speed camera</td><td>Photron</td><td>FASTCAM Mini</td><td>Observation of concentration</td></tr><tr><td>Hot plate</td><td>As one</td><td>HI-1000</td><td>heating plate for curing of liquid PDMS</td></tr><tr><td>KOVAX-SYRINGE 10 mL/Syringe</td><td>Koreavaccine</td><td>22G-10ML</td><td>Fill the microfluidic channel with bubble-free demineralized water.</td></tr><tr><td>Laboratory Conona treater/Atmospheric plasma</td><td>Electro-Technic</td><td>BD-20AC</td><td>Chip bonding/atmospheric plasma</td></tr><tr><td>Liquid polydimethylsiloxane, PDMS</td><td>Dow Corning Inc.</td><td>Sylgard 184</td><td>Components of chip</td></tr><tr><td>Microscope</td><td>Olympus</td><td>IX-81</td><td>Observation of concentration</td></tr><tr><td>PEEK Tubes</td><td>SAINT-GOBAIN PPL CORP.</td><td>AAD04103</td><td>Inject or collect particles</td></tr><tr><td>Polystyrene Particle(4.16 &amp;mu;m)</td><td>Spherotech</td><td>PP-40-10</td><td>Concentrated bead sample3</td></tr><tr><td>Polystyrene Particle(8.49 &amp;mu;m)</td><td>Spherotech</td><td>PP-100-10</td><td>Concentrated bead sample2</td></tr><tr><td>Pressure controller/&amp;mu;flucon</td><td>AMED</td><td>&amp;mu;flucon</td><td>Control of air pressure</td></tr><tr><td>Spin coater</td><td>iNexus</td><td>ACE-200</td><td>spread the liquid PDMS on SU-8 mold</td></tr></tbody></table>

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.

Figure 8 shows the flow rate of the fluid rates for a four-stage platform operation, as mentioned in Table 2. The first stage is the loading state (a state). The platform was supplied with fluid with all valves open, and the working fluid (Qf) and particles (Qp) are almost identical as the microfluidic channel network exhibits structural symmetry. In the second stage (b state), compressed air was transported to Vs to block the particles, and as the Vs diaphragm deformed, the...

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Pneumatically Driven Microfluidic Platform for Micro-Particle Concentration

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. ...

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2.3K Views.  Inje University. The present protocol describes a pneumatic microfluidic platform that can be used for efficient microparticle concentration.

Video: Pneumatically Driven Microfluidic Platform for Micro-Particle Concentration

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 ...

Bioengineering

Fluorescence detection methods for microfluidic droplet platforms

The development of microfluidic platforms for performing chemistry and biology has in large part been driven by a range of potential benefits that accompany system miniaturisation. Advantages include the ability to efficiently process nano- to femoto- liter volumes of sample, facile integration of functional components, an intrinsic predisposition towards large-scale multiplexing, enhanced analytical throughput, improved control and reduced instrumental footprints.1
In recent years much interest has focussed on the development of droplet-based (or segmented flow) microfluidic systems and their potential as platforms in high-throughput experimentation.2-4 Here water-in-oil emulsions are made to spontaneously form in microfluidic channels as a result of capillary instabilities between the two immiscible phases. Importantly, microdroplets of precisely defined volumes and compositions can be generated at frequencies of several kHz. Furthermore, by encapsulating reagents of interest within isolated compartments separated by a continuous immiscible phase, both sample cross-talk and dispersion (diffusion- and Taylor-based) can be eliminated, which leads to minimal cross-contamination and the ability to time analytical processes with great accuracy. Additionally, since there is no contact between the contents of the droplets and the channel walls (which are wetted by the continuous phase) absorption and loss of reagents on the channel walls is prevented.
Once droplets of this kind have been generated and processed, it is necessary to extract the required analytical information. In this respect the detection method of choice should be rapid, provide high-sensitivity and low limits of detection, be applicable to a range of molecular species, be non-destructive and be able to be integrated with microfluidic devices in a facile manner. To address this need we have developed a suite of experimental tools and protocols that enable the extraction of large amounts of photophysical information from small-volume environments, and are applicable to the analysis of a wide range of physical, chemical and biological parameters. Herein two examples of these methods are presented and applied to the detection of single cells and the mapping of mixing processes inside picoliter-volume droplets. We report the entire experimental process including microfluidic chip fabrication, the optical setup and the process of droplet generation and detection.

Droplet-based microfluidic platforms are promising candidates for high throughput experimentation since they are able to generate picoliter, self-compartmentalized vessels inexpensively at kHz rates. Through integration with fast, sensitive and high resolution fluorescence spectroscopic methods, the large amounts of information generated within these systems can be efficiently extracted, harnessed and utilized.

The development of microfluidic platforms for performing chemistry and biology has in large part been driven by a range of potential benefits that ...

Capillary-based Centrifugal Microfluidic Device for Size-controllable Formation of Monodisperse Microdroplets

Here, we demonstrate a simple method for the rapid production of size-controllable, monodisperse, W/O microdroplets using a capillary-based centrifugal microfluidic device. W/O microdroplets have recently been used in powerful methods that enable miniaturized chemical experiments. Therefore, developing a versatile method to yield monodisperse W/O microdroplets is needed. We have developed a method for generating monodisperse W/O microdroplets based on a capillary-based centrifugal axisymmetric co-flowing microfluidic device. We succeeded in controlling the size of microdroplets by adjusting the capillary orifice. Our method requires equipment that is easier-to-use than with other microfluidic techniques, requires only a small volume (0.1-1 &#181;l) of sample solution for encapsulation, and enables the production of hundreds of thousands number of W/O microdroplets per second. We expect this method will assist biological studies that require precious biological samples by conserving the volume of the samples for rapid quantitative analysis biochemical and biological studies.

Here, we demonstrate a simple production method for size-controllable, monodisperse, water-in-oil (W/O) microdroplets using a capillary-based centrifugal microfluidic device. This method requires only a small sample volume and enables high-yield production. We expect this method will be useful for rapid biochemical and cellular analyses.

Here, we demonstrate a simple method for the rapid production of size-controllable, monodisperse, W/O microdroplets using a capillary-based centrifugal ...

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 ...

High Speed Droplet-based Delivery System for Passive Pumping in Microfluidic Devices

A novel microfluidic system has been developed that uses the phenomenon of passive pumping along with a user controlled droplet based fluid delivery system. Passive pumping is the phenomenon by which surface tension induced pressure differences drive fluid movement in closed channels. The automated fluid delivery system consists of a set of voltage controlled valves with micro-nozzles connected to a fluid reservoir and a control system. These voltage controlled valves offer a volumetrically precise way to deliver fluid droplets to the inlet of a microfluidic device in a high frequency manner. Based on the  dimensions demonstrated in the current study example, the system is capable of flowing 4 milliliters per minute (through a 2.2mm by 260um cross-sectional channel). Based on these same channel dimensions, fluid exchange of a point inside the channel can be achieved in as little as eight milliseconds. It is observed that there is interplay between momentum of the system (imparted by a combination of the droplets created by the valves and the fluid velocity in the channel), and the surface tension of the liquid. Where momentum provides velocity to the fluid flow (or vice-versa), equilibration of surface tension at the inlet provides a sudden stop to any flow. This sudden stop allows the user to control the flow characteristics of the channel and opens the door for a variety of biological applications, ranging anywhere from reagent delivery to drug-cell studies. It is also observed that when nozzles are aimed at the inlet at shallow angles, the droplet momentum can cause additional interesting fluid phenomena, such as mixing of multiple droplets in the inlet.

A novel microfluidic system has been developed using the phenomenon of passive pumping and a user controlled fluid delivery system. This microfluidic system has the potential to be used in a wide variety of biological applications given its low cost, ease of use, volumetric precision, high speed, repeatability and automation.

A novel microfluidic system has been developed that uses the phenomenon of passive pumping along with a user controlled droplet based fluid delivery ...

Biology

A Microfluidic Platform to Study Bioclogging in Porous Media

Bacterial biofilms are found in several environmental and industrial porous media, including soils and filtration membranes. Biofilms grow under certain flow conditions and can clog pores, thereby redirecting the local fluid flow. The ability of biofilms to clog pores, the so-called bioclogging, can have a tremendous effect on the local permeability of the porous medium, creating a pressure buildup in the system, and impacting the mass flow through it. To understand the interplay between biofilm growth and fluid flow under different physical conditions (e.g., at different flow velocities and pore sizes), in the present study, a microfluidic platform is developed to visualize biofilm development using a microscope under externally-imposed, controlled physical conditions. The biofilm-induced pressure buildup in the porous medium can be measured simultaneously using pressure sensors and, later, correlated with the surface coverage of the biofilm. The presented platform provides a baseline for a systematic approach to investigate bioclogging caused by biofilms in porous media under flow conditions and can be adapted to studying environmental isolates or multispecies biofilms.

The present protocol describes a microfluidic platform to study biofilm development in quasi-2D porous media by combining high-resolution microscopy imaging with simultaneous pressure difference measurements. The platform quantifies the influence of pore size and fluid flow rates in porous media on bioclogging.

Bacterial biofilms are found in several environmental and industrial porous media, including soils and filtration membranes. Biofilms grow under certain ...

Environment

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 ...

Aqueous Droplets Used as Enzymatic Microreactors and Their Electromagnetic Actuation

For the successful implementation of microfluidic reaction systems, such as PCR and electrophoresis, the movement of small liquid volumes is essential. In conventional lab-on-a-chip-platforms, solvents and samples are passed through defined microfluidic channels with complex flow control installations. The droplet actuation platform presented here is a promising alternative. With it, it is possible to move a liquid drop (microreactor) on a planar surface of a reaction platform (lab-in-a-drop). The actuation of microreactors on the hydrophobic surface of the platform is based on the use of magnetic forces acting on the outer shell of the liquid drops which is made of a thin layer of superhydrophobic magnetite particles. The hydrophobic surface of the platform is needed to avoid any contact between the liquid core and the surface to allow a smooth movement of the microreactor. On the platform, one or more microreactors with volumes of 10 &#181;L can be positioned and moved simultaneously. The platform itself consists of a 3 x 3 matrix of electrical double coils which accommodate either neodymium or iron cores. The magnetic field gradients are automatically controlled. By variation of the magnetic field gradients, the microreactors' magnetic hydrophobic shell can be manipulated automatically to move the microreactor or open the shell reversibly. Reactions of substrates and corresponding enzymes can be initiated by merging the microreactors or bringing them into contact with surface immobilized catalysts.

Lab-in-a-drop reaction systems allow the versatile implementation of complex reactions in a microfluidic scale. An automated actuation platform consisting of a 3 x 3 matrix of electromagnetic coils was developed and successfully used to merge two 10 &#181;L microreactors and thereby initiate an enzymatic reaction in the resulting liquid marbles.

For the successful implementation of microfluidic reaction systems, such as PCR and electrophoresis, the movement of small liquid volumes is essential. In ...

Microfluidic Platform with Multiplexed Electronic Detection for Spatial Tracking of Particles

Microfluidic processing of biological samples typically involves differential manipulations of suspended particles under various force fields in order to spatially fractionate the sample based on a biological property of interest. For the resultant spatial distribution to be used as the assay readout, microfluidic devices are often subjected to microscopic analysis requiring complex instrumentation with higher cost and reduced portability. To address this limitation, we have developed an integrated electronic sensing technology for multiplexed detection of particles at different locations on a microfluidic chip. Our technology, called Microfluidic CODES, combines Resistive Pulse Sensing with Code Division Multiple Access to compress 2D spatial information into a 1D electrical signal. In this paper, we present a practical demonstration of the Microfluidic CODES technology to detect and size cultured cancer cells distributed over multiple microfluidic channels. As validated by the high-speed microscopy, our technology can accurately analyze dense cell populations all electronically without the need for an external instrument. As such, the Microfluidic CODES can potentially enable low-cost integrated lab-on-a-chip devices that are well suited for the point-of-care testing of biological samples.

We demonstrate a microfluidic platform with an integrated surface electrode network that combines resistive pulse sensing (RPS) with code division multiple access (CDMA), to multiplex detection and sizing of particles in multiple microfluidic channels.

Microfluidic processing of biological samples typically involves differential manipulations of suspended particles under various force fields in order to ...