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

1. Microfluidic acoustophoresis chip design
NOTE: Figure 1 shows a schematic of the separation and collection of target microbeads from microchannels by acoustophoresis. The microfluidic acoustophoresis chip is designed with a CAD program.
<ol>
	<li>Design a microfluidic acoustophoresis chip that uses a mixture of aptamer-modified beads and streptavidin-coated polystyrene (PS) beads corresponding to the size of bacteria to study the separation performance of the device.</li>
	<li>Design a microfluidic acoustophoresis chip that collects PS beads at the target outlet and discards the rest through the outlet after injecting a PS sample mixture into the sample inlet (see Figure 2 and Table 1). 
	NOTE: For this purpose, the acoustofluidic chip is designed consisting of two inlets for injecting samples and buffer, a main channel with an attached piezoelectric transducer (PZT) to allow the microbeads to be aligned centrally, and two outlets through which specimens are collected and waste are discharged (Figure 3A)</li>
	<li>Design a microfluidic acoustophoresis chip in which microbeads, buffer, bacteria, and aptamers pass through the main channel, with the microbeads aligned centrally via in-chip acoustophoresis induced by the PZT (Figure 3B,C)</li>
</ol>
2. Microfluidic acoustophoresis chip fabrication
NOTE: Assemble four layers in the following order: a borosilicate glass-silicon layer, a silicon layer, a borosilicate glass layer, and a PZT layer, as shown in Figure 3A,B.
<ol>
	<li>Prepare borosilicate glass (the uppermost layer) with 2-mm-diameter holes by sandblast17 at the inlet and outlet for connecting polyetheretherketone (PEEK) tubes. The borosilicate glass measures 20 &#215; 80 &#215; 0.5 mm3.</li>
	<li>Prepare a 200-&#181;m-thick silicon channel layer with a microchannel with a cross-sectional area of 0.2 &#215; 0.2 mm2 formed using a photoresist and a silicon pattern obtained via deep reactive ion etching (RIE)18. Drill 1 mm diameter holes for the sample and buffer inlet channels and the collection and waste outlet channels during reactive ion etching. 
	NOTE: Here, ion-etching uses the RIE process to form microchannels. A photoresist was applied in the shape of a channel on a silicon wafer for the silicon channel layer. The PR-coated silicon wafer was etched with plasma generated by applying 13.56 Mhz to fluorine18.</li>
	<li>Prepare a chip with the layers above and below the silicon layer (20 &#215; 80 &#215; 0.5 mm3) prepared in step 2.2 (20 &#215; 80 &#215; 0.5 mm3) bonded to the borosilicate glass in step 2.1 and a third borosilicate glass layer using anodization at 1,000 V and 400 &#176;C19.</li>
	<li>Attach a PZT (20 &#215; 40 mm2) to the borosilicate glass layer along the microfluidic channel using a cyanoacrylate adhesive (10 &#181;L or less). 
	&#8203;NOTE: Apply the adhesive as a very thin layer in the channel using a cotton swab to minimize any change in height. Figure 3C is a picture of the device.</li>
</ol>
3. Bacterial strains and culture
NOTE: Refer to Table 2 to select and incubate GN and Gram-positive (GP) bacteria for experiments. For the culture method, refer to steps 3.1-3.4. All bacteria should be incubated under aerobic conditions until an absorbance of 0.4 at 600 nm (OD600) is obtained.
<ol>
	<li>For GN bacteria such as Escherichia coli DH5&#945;, Escherichia coli KCTC2571, Sphingomonas insulae, and Pseudomonas pictorum&#160;and GP bacteria such as&#160;Staphylococcus epidermidis&#160;and&#160;Staphylococcus pasteuri, incubate in Luria-Bertani medium at 37 &#176;C and 220 rpm for 16 h.</li>
	<li>For Enterobacter (GN) and Bacillus megaterium (GP; KCTC 1021), incubate in nutrient broth medium at 37 &#176;C and 220 rpm for 16 h.</li>
	<li>For Enterococcus thailandicus (GP), incubate in de Man, Rogosa, and Sharpe (MRS) medium at 37 &#176;C and 220 rpm for 16 h.</li>
	<li>For Listeria grayi (GP), incubate in brain heart infusion medium at 37 &#176;C and 220 rpm for 16 h.</li>
	<li>Centrifuge (9056 x g) the cultured bacteria for 1 min at room temperature (RT), then wash twice with 1x phosphate-buffered saline (PBS) buffer.</li>
	<li>Prepare the selected GN and GP bacteria for analysis by resuspending in PBS buffer.</li>
</ol>
4. Microbeads and immobilization of aptamer onto microbeads
<ol>
	<li>Resuspend the streptavidin-coated microbeads (10 &#181;m) mixture (Table of Materials) before use (mix via vortexing for 20 s).</li>
	<li>Prepare aptamer by denaturing at 95 &#176;C for 3 min and then refolding at 0 &#176;C for 2 min.</li>
	<li>Transfer 250 &#181;L of the resuspended streptavidin-coated microbeads mixture to a 1.5 mL tube and wash with Tris-HCl buffer (50 mM Tris, pH 7.4, 1 mM MgCl2, 5 mM KCL, 100 mM NaCl) at RT. Then add 100 &#181;L of biotinylated DNA aptamer to the tube.</li>
	<li>Incubate the mixture at RT for 30 min while rotating (25 rpm).</li>
	<li>After centrifugation (9056 x g), wash the tube twice with 200 &#181;L of Tris-HCl buffer at RT.</li>
	<li>Add 10 &#181;L of BSA (100 mg/mL) to the washed sample tube and incubate for 30 min at RT with rotation (25 rpm).</li>
	<li>Finally, wash the aptamer-modified microbeads twice by centrifugation (9056 x g) in Tris-HCl buffer at RT.</li>
</ol>
5. Acoustophoresis setup and operation
<ol>
	<li>Connect PEEK tubes to the two inlets for injecting two samples and buffer and the two outlets for collecting and discharging waste (Figure 4).</li>
	<li>Manually fill the microfluidic acoustophoresis channel with bubble-free demineralized water using a 10 mL syringe.</li>
	<li>Prepare a precision pressure controller with two or more output channels to control the fluid flow. Then, half-fill the vials with sample and buffer with two holes in their caps, respectively, and connect to the chip inlet. 
	NOTE: A precision pressure controller with two or more output channels can be replaced with multiple precision pressure controllers. At the buffer inlet, a buffer capable of generating a laminar flow that prevents the sample from moving to the center during sample injection is injected through a flow controller.</li>
	<li>After preparing the device, inject the sample and buffer by applying a pressure of 2 kPa to the sample inlet and 4 kPa to the buffer inlet using the precision pressure control device. 
	NOTE: At this time, for smooth laminar flow, the injection pressure of the buffer should be higher than the injection pressure of the sample. The flow is controlled by a flow controller fixed to the aspirator connected to the inlet channel.</li>
	<li>Focus on a bead to move it into the center of the microfluidic channel using the PZT while checking through the microscope. 
	NOTE: The larger the bead, the greater the effect on the waveform, so it is easier to align with the node point. Function generator with amplifier applies power to PZT to generate sine wave in the microchannel. Since the upper and lower portions of the microchannel are made of glass, the generated sine wave is reflected and creates a node point7.</li>
	<li>Generate a resonance frequency of 3.66 MHz using a single-channel function generator and amplify a typical signal by 16 dB (about nine-fold) using a power amplifier (Figure 4). 
	NOTE: The resonance frequency of the actuator must match the size of the channel; because the channel is square, the PZT operates at an accurate frequency to create a single node.</li>
	<li>Observe the separation and enrichment processes on the acoustofluidic chip with a fluorescence microscope and a high-speed camera operating at 1,200 fps.</li>
	<li>Quantify and analyze the presence or absence of GN bacteria and GP bacteria by checking the images taken with the fluorescence microscope camera of the bacteria-bound beads and bacteria discharged through the collection and waste outlet samples. 
	NOTE: Microbeads with buffer, bacteria, and aptamers pass through the main channel, and the microbeads are aligned centrally via in-chip acoustophoresis induced by the PZT. Finally, microbeads that have bound GN bacteria are collected at the collection outlet, and the uncollected bacteria are discharged through the waste outlet.</li>
</ol>

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The authors have no conflicts of interest to disclose.

We developed a sonic levitation microfluidic device for capturing and transferring GN bacteria from culture samples at high speed based on a continuous running method according to their size and type, and aptamer-modified microbeads. The long, square microchannel enables a simpler design and greater cost-efficiency for 2D acoustophoresis than previously reported20,21,22,23,<sup class...

Microfluidic platforms are being developed to isolate bacteria from medical and environmental samples, in addition to methods based on dielectric transfer, magnetophoresis, bead extraction, filtering, centrifugal microfluidics and inertial effects, and surface acoustic waves1,2.&#160;The detection of pathogenic bacteria is continued using polymerase chain reaction (PCR), but it is usually laborious, complex, and time-consuming3,4. Microfluidic acoustophoresis systems are an alternative to address this through reasonable throughput and non-contact cell isolation5,6,7. Acoustophoresis is a technology that separates or concentrates beads using the phenomenon of material movement through a sound wave. When sound waves enter the microchannel, they are sorted according to the size, density, etc., of the beads, and cells can be separated according to the biochemical and electrical properties of the suspension medium7,8. Accordingly, many acoustophoretic studies have been actively pursued9,10,11, and recently, 3D numerical simulations of acoustophoretic motion induced by boundary-driven acoustic streaming in standing surface acoustic wave microfluidics have been introduced12.
Studies in various fields are examining how to replace antibodies2,3. Aptamer is a target material having high selectivity and specificity, and many studies are being conducted2,9,10,13. Aptamers have advantages of small size, excellent biological stability, low cost, and high reproducibility compared to antibodies and are being studied in diagnostic and therapeutic applications2,3,14.
Here, this article describes a microfluidic acoustophoresis technology protocol that can be used for the rapid, efficient separation of Gram-negative (GN) bacteria from a medium using aptamer-modified microbeads. This system generates a two-dimensional (2D) acoustic standing wave through single piezoelectric actuation by simultaneously stimulating two orthogonal resonances within a long rectangular microchannel to align and focus aptamer-attached microbeads at the node and anti-node points for separation efficiency2,11,15,16.&#160;There is a bifurcated channel at both the inlet and outlet, enabling simultaneous separation, purification, and concentration.
This protocol can be helpful in the field of early diagnosis of bacterial infectious diseases, as well as a rapid, selective, and sensitive response to pathogenic bacterial infections through real-time water monitoring.

<table><tbody><tr><td>1 &amp;micro;m polystyrene microbeads</td><td>Bang Laboratories</td><td>PS04001</td><td>Cell size beads</td></tr><tr><td>10 &amp;micro;m Streptavidin-coated microbeads</td><td>Bang Laboratories</td><td>CP01007</td><td>Aptamer affinity beads</td></tr><tr><td>4-inch Silicon Wafer/SU-8 mold</td><td>4science</td><td>29-03573-01</td><td>Components of chip</td></tr><tr><td>Aptamer</td><td>Integrated DNA Technologies</td><td>GN3-6&#039;</td><td>RNA for bacteria conjugation</td></tr><tr><td>Borosilicate glass</td><td>Schott</td><td>BOROFLOAT 33</td><td>Components of chip</td></tr><tr><td>Centrifuge</td><td>Daihan</td><td>CF-10</td><td>Wasing particles</td></tr><tr><td>Cyanoacrylate glue</td><td>3M</td><td>AD100</td><td>Attach PZT to microchip</td></tr><tr><td>Escherichia coli DH5&amp;alpha;</td><td>KCTC</td><td>KCTC2571</td><td>Target bacteria</td></tr><tr><td>Functional generator</td><td>GW Instek</td><td>AFG-2225</td><td>Generate frequency</td></tr><tr><td>High-speed camera</td><td>Photron</td><td>FASTCAM Mini</td><td>Observation of separation</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 acoustophoresis channel with bubble-free demineralized water.</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>LB Broth Miller</td><td>BD Difco</td><td>244620</td><td>Cell culture (Luria-Bertani medium)</td></tr><tr><td>Microscope</td><td>Olympus Corp.</td><td>IX-81</td><td>Observation of separation</td></tr><tr><td>PBS buffer</td><td>Capricorn scientific</td><td>PBS-1A</td><td>Wasing bacteria</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>Piezoelectric transducer</td><td>Fuji Ceramics</td><td>C-213</td><td>Generate specific wave in channel</td></tr><tr><td>Power amplifier</td><td>Amplifier Research</td><td>75A250A</td><td>Amplify frequency</td></tr><tr><td>Pressure controller/&amp;mu;flucon</td><td>AMED</td><td>AMED-&amp;mu;flucon</td><td>Control of air pressure/flow controller</td></tr><tr><td>Tris-HCl buffer</td><td>invitrogen</td><td>15567027</td><td>Wasing particles</td></tr><tr><td>Tube rotator</td><td>SeouLin Bioscience</td><td>SLRM-3</td><td>Modifiying aptamer and bead</td></tr></tbody></table>

microfluidic acoustophoresis for flowthrough separation of gram-negative bacteria using aptamer affinity beads

This article describes the fabrication and operation of microfluidic acoustophoretic chips using a microfluidic acoustophoresis technique and aptamer-modified microbeads that can be used for the fast, efficient isolation of Gram-negative bacteria from a medium. This method enhances the separation efficiency using a mix of long, square microchannels. In this system, the sample and buffer are injected into the inlet port through a flow controller. For bead centering and sample separation, AC power is applied to the piezoelectric transducer via a function generator with a power amplifier to generate acoustic radiation force in the microchannel. There is a bifurcated channel at both the inlet and outlet, enabling simultaneous separation, purification, and concentration. The device has a recovery rate of &gt;98% and purity of 97.8% up to a 10x dose concentration. This study has demonstrated a recovery rate and purity higher than the existing methods for separating bacteria, suggesting that the device can separate bacteria efficiently.

Figure 5 shows the image of bead flow as a function of PZT voltage (OFF, 0.1 V, 0.5 V, 5 V). In the case of the acoustophoretic chip introduced in this study, it was confirmed that as the voltage of the PZT increased, the central concentration of the 10 &#181;m-sized beads increased. Most of the 10 &#181;m-sized beads were concentrated in the center at 5 V of the PZT voltage. Through this result, a resonant frequency of 3.66 MHz was generated in a single channel function generator, and a gen...

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Microfluidic Acoustophoresis for Flowthrough Separation of Gram-Negative Bacteria using Aptamer Affinity Beads

This paper describesthe fabrication and operation of microfluidic acoustophoretic chips using the microfluidic acoustophoresis technique and aptamer-modified microbeads that can be used for fast, efficient isolation of Gram-negative bacteria from a medium.

This article describes the fabrication and operation of microfluidic acoustophoretic chips using a microfluidic acoustophoresis technique and ...

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2.4K Views.  Inje University. Dit protocol beschrijft onze microfluïdische acoustophoresis-technologie die kan worden gebruikt voor de snelle, efficiënte scheiding van Gram-negatieve bacteriën uit een medium met behulp van aptamer-gemodificeerde microparels. Microfluïdische acoustophoresesystemen maken de Gram-negatieve bacteriescheiding mogelijk met een redelijke doorvoer en contactloze celisolatie. Microfluïdische acoustophorese kan worden geoptimaliseerd om bacteriën te isoleren uit medische en omgevingsmonsters....

Video: Microfluidic Acoustophoresis for Flowthrough Separation of Gram-Negative Bacteria using Aptamer Affinity Beads

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 ...

Bioengineering

Fabrication, Operation and Flow Visualization in Surface-acoustic-wave-driven Acoustic-counterflow Microfluidics

Surface acoustic waves (SAWs) can be used to drive liquids in portable microfluidic chips via the acoustic counterflow phenomenon. In this video we present the fabrication protocol for a multilayered SAW acoustic counterflow device. The device is fabricated starting from a lithium niobate (LN) substrate onto which two interdigital transducers (IDTs) and appropriate markers are patterned. A polydimethylsiloxane (PDMS) channel cast on an SU8 master mold is finally bonded on the patterned substrate. Following the fabrication procedure, we show the techniques that allow the characterization and operation of the acoustic counterflow device in order to pump fluids through the PDMS channel grid. We finally present the procedure to visualize liquid flow in the channels. The protocol is used to show on-chip fluid pumping under different flow regimes such as laminar flow and more complicated dynamics characterized by vortices and particle accumulation domains.

In this video we first describe fabrication and operation procedures of a surface acoustic wave (SAW) acoustic counterflow device. We then demonstrate an experimental setup that allows for both qualitative flow visualization and quantitative analysis of complex flows within the SAW pumping device.

Surface acoustic waves (SAWs) can be used to drive liquids in portable microfluidic chips via the acoustic counterflow phenomenon. In this video we ...

Fabrication and Operation of Acoustofluidic Devices Supporting Bulk Acoustic Standing Waves for Sheathless Focusing of Particles

Acoustophoresis refers to the displacement of suspended objects in response to directional forces from sound energy. Given that the suspended objects must be smaller than the incident wavelength of sound and the width of the fluidic channels are typically tens to hundreds of micrometers across, acoustofluidic devices typically use ultrasonic waves generated from a piezoelectric transducer pulsating at high frequencies (in the megahertz range). At characteristic frequencies that depend on the geometry of the device, it is possible to induce the formation of standing waves that can focus particles along desired fluidic streamlines within a bulk flow. Here, we describe a method for the fabrication of acoustophoretic devices from common materials and clean room equipment. We show representative results for the focusing of particles with positive or negative acoustic contrast factors, which move towards the pressure nodes or antinodes of the standing waves, respectively. These devices offer enormous practical utility for precisely positioning large numbers of microscopic entities (e.g., cells) in stationary or flowing fluids for applications ranging from cytometry to assembly.

Acoustofluidic devices use ultrasonic waves within microfluidic channels to manipulate, concentrate and isolate suspended micro and nanoscopic entities. This protocol describes the fabrication and operation of such a device supporting bulk acoustic standing waves to focus particles in a central streamline without the aid of sheath fluids.

Acoustophoresis refers to the displacement of suspended objects in response to directional forces from sound energy. Given that the suspended objects must ...

Combining Fluidic Devices with Microscopy and Flow Cytometry to Study Microbial Transport in Porous Media Across Spatial Scales

Understanding the transport, dispersion and deposition of microorganisms in porous media is a complex scientific task comprising topics as diverse as hydrodynamics, ecology and environmental engineering. Modeling bacterial transport in porous environments at different spatial scales is critical to better predict the consequences of bacterial transport, yet current models often fail to up-scale from laboratory to field conditions. Here, we introduce experimental tools to study bacterial transport in porous media at two spatial scales. The aim of these tools is to obtain macroscopic observables (such as breakthrough curves or deposition profiles) of bacteria injected into transparent porous matrices. At the small scale (10-1000 &#181;m), microfluidic devices are combined with optical video-microscopy and image processing to obtain breakthrough curves and, at the same time, to track individual bacterial cells at the pore scale. At larger scale, flow cytometry is combined with a self-made robotic dispenser to obtain breakthrough curves. We illustrate the utility of these tools to better understand how bacteria are transported in complex porous media such as the hyporheic zone of streams. As these tools provide simultaneous measurements across scales, they pave the way for mechanism-based models, critically important for upscaling. Application of these tools may not only contribute to the development of novel bioremediation applications but also shed new light on the ecological strategies of microorganisms colonizing porous substrates.

Breakthrough curves (BTCs) are efficient tools to study the transport of bacteria in porous media. Here we introduce tools based on fluidic devices in combination with microscopy and flow cytometric counting to obtain BTCs.

Understanding the transport, dispersion and deposition of microorganisms in porous media is a complex scientific task comprising topics as diverse as ...

Environment

Microparticle Manipulation by Standing Surface Acoustic Waves with Dual-frequency Excitations

We demonstrate a method for increasing the tuning ability of a standing surface acoustic wave (SSAW) for microparticles manipulation in a lab-on-a-chip (LOC) system. The simultaneous excitation of the fundamental frequency and its third harmonic, which is termed as dual-frequency excitation, to a pair of interdigital transducers (IDTs) could generate a new type of standing acoustic waves in a microfluidic channel. Varying the power and the phase in the dual-frequency excitation signals results in a reconfigurable field of the acoustic radiation force applied to the microparticles across the microchannel (e.g., the number and location of the pressure nodes and the microparticle concentrations at the corresponding pressure nodes). This article demonstrates that the motion time of the microparticle to only one pressure node can be reduced ~2-fold at the power ratio of the fundamental frequency greater than ~90%. In contrast, there are three pressure nodes in the microchannel if less than this threshold. Furthermore, adjusting the initial phase between the fundamental frequency and the third harmonic results in different motion rates of the three SSAW pressure nodes, as well as in the percentage of microparticles at each pressure node in the microchannel. There is a good agreement between the experimental observation and the numerical predictions. This novel excitation method can easily and non-invasively integrate into the LOC system, with a wide tenability and only a few changes to the experimental set-up.

A protocol for manipulating the microparticles in a microfluidic channel with a dual-frequency excitation is presented.

We demonstrate a method for increasing the tuning ability of a standing surface acoustic wave (SSAW) for microparticles manipulation in a lab-on-a-chip ...

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. ...

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.

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 ...

Window on a Microworld: Simple Microfluidic Systems for Studying Microbial Transport in Porous Media

Microbial growth and transport in porous media have important implications for the quality of groundwater and surface water, the recycling of nutrients in the environment, as well as directly for the transmission of pathogens to drinking water supplies. Natural porous media is composed of an intricate physical topology, varied surface chemistries, dynamic gradients of nutrients and electron acceptors, and a patchy distribution of microbes. These features vary substantially over a length scale of microns, making the results of macro-scale investigations of microbial transport difficult to interpret, and the validation of mechanistic models challenging. Here we demonstrate how simple microfluidic devices can be used to visualize microbial interactions with micro-structured habitats, to identify key processes influencing the observed phenomena, and to systematically validate predictive models. Simple, easy-to-use flow cells were constructed out of the transparent, biocompatible and oxygen-permeable material poly(dimethyl siloxane). Standard methods of photolithography were used to make micro-structured masters, and replica molding was used to cast micro-structured flow cells from the masters. The physical design of the flow cell chamber is adaptable to the experimental requirements: microchannels can vary from simple linear connections to complex topologies with feature sizes as small as 2 &mu;m. Our modular EcoChip flow cell array features dozens of identical chambers and flow control by a gravity-driven flow module. We demonstrate that through use of EcoChip devices, physical structures and pressure heads can be held constant or varied systematically while the influence of surface chemistry, fluid properties, or the characteristics of the microbial population is investigated. Through transport experiments using a non-pathogenic, green fluorescent protein-expressing Vibrio bacterial strain, we illustrate the importance of habitat structure, flow conditions, and inoculums size on fundamental transport phenomena, and with real-time particle-scale observations, demonstrate that microfluidics offer a compelling view of a hidden world.

Microfluidic devices can be used to visualize complex natural processes in real time and at the appropriate physical scales. We have developed a simple microfluidic device that mimics key features of natural porous media for studying growth and transport of bacteria in the subsurface.

Microbial growth and transport in porous media have important implications for the quality of groundwater and surface water, the recycling of nutrients in ...

Biology

A Microfluidic Device for Quantifying Bacterial Chemotaxis in Stable Concentration Gradients

Chemotaxis allows bacteria to approach sources of attractant chemicals or to avoid sources of repellent chemicals. Bacteria constantly monitor the concentration of specific chemoeffectors by comparing the current concentration to the concentration detected a few seconds earlier. This comparison determines the net direction of movement. Although multiple, competing gradients often coexist in nature, conventional approaches for investigating bacterial chemotaxis are suboptimal for quantifying migration in response to concentration gradients of attractants and repellents. Here, we describe the development of a microfluidic chemotaxis model for presenting precise and stable concentration gradients of chemoeffectors to bacteria and quantitatively investigating their response to the applied gradient. The device is versatile in that concentration gradients of any desired absolute concentration and gradient strength can be easily generated by diffusive mixing. The device is demonstrated using the response of Escherichia coli RP437 to gradients of amino acids and nickel ions.

This protocol describes the development of a microfluidic device for investigating bacterial chemotaxis in stable concentration gradients of chemoeffectors.

Chemotaxis allows bacteria to approach sources of attractant chemicals or to avoid sources of repellent chemicals. Bacteria constantly monitor the ...

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.

Microfluidic Acoustophoresis for Flowthrough Separation of Gram-Negative Bacteria using Aptamer Affinity Beads

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