This research was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT and Future Planning (NRF-2014R1A1A2057527 and NRF-2016R1D1A1B03934418).

1. Preparation of through-hole array aluminum plate and magnet array
<ol>
	<li>Prepare two 50 mm x 50 mm (or larger) aluminum plates. The thickness of each plate was 300 &#181;m that is half of the bead diameter.</li>
	<li>Form a 30 x 30 through-hole array on one of the aluminum plates by using a CNC rotary engraver with a &#934;550-&#181;m micro drill bit with 30 mm/s of plunge rate and 8000 RPM of spindle speed. The distance between each hole (center to center) was 1 mm (Figure 1a and Figure 2a, i).</li>
	<li>Form a 30 x 30 array of &#934;750-&#181;m through-holes on the other aluminum plate, using the same procedure as that described in 1.2 (Figure 1a and Figure 2a, ii).</li>
	<li>Attach the two plates each other by using a sticky tape and form &#934;3 mm alignment holes at each of the four corners of both aluminum plates.</li>
	<li>Soak the aluminum plates in 15% sulfuric acid for 12 h to clean their surfaces. Since the thin layer of aluminum oxide on the surface of the aluminum make it corrosion resistant, the hole diameter and thickness of the plate are not changed by this acid treatment.</li>
	<li>Form a 30 &#215; 30 array of 1 x 1 x 1 mm neodymium magnets (with a magnetic strength of 0.363 N). Ensure that each magnet is the opposite polarity to its neighbor. To prevent the breaking or scattering of the magnet array, attach a 30 x 30 mm aluminum plate to the bottom of the magnet array using double-sided tape (Figure 2a,&#160;iii and inset in Figure 2).</li>
</ol>
2. Bead trapping process
<ol>
	<li>Align and stack the two aluminum plates (top plate: 750-&#181;m-hole plate, bottom plate: 550-&#181;m-hole plate) using the prepared alignment holes at the four corners of each plate (Figure 1b).</li>
	<li>Lock the two plates together by inserting M3 bolts into the alignment holes, and then secure the bolts with nuts (Figure 1b).</li>
	<li>Stack the aluminum plate assembly on the prepared magnet array (Figure&#160;1b, 2b, and 2c). Align the array of magnets and the array of through holes in the aluminum plate during the stacking process. Then use a sticky tape to fix the position of the magnet array.</li>
	<li>Place a sufficient number of &#934;600 mm SUJ2 steel beads on the plate assembly and manipulate the beads using an acryl (or non-metallic) plate such that a bead becomes trapped in each hole (Figure 1c, 1d, and 1e) while simultaneously removing the excess beads which have not lodged in the holes.</li>
	<li>Carefully remove the top plate to avoid unwanted scattering and dislocation of the trapped beads (Figure 1f).</li>
</ol>
3. Concave microwell fabrication
<ol>
	<li>Move the concave microwell mold, produced in steps 2.1 to 2.5, above, to a Petri dish.</li>
	<li>Mix polydimethylsiloxane (PDMS) monomer and curing agent according to the manufacturer&#39;s instructions19 with a PDMS monomer:curing agent ratio of 10:1.</li>
	<li>De-gas the PDMS mixture by using a desiccator and vacuum pump to remove any bubbles trapped in the PDMS mixture.</li>
	<li>Pour the PDMS mixture into the concave microwell mold and de-gas again using the same procedure as that described in 3.3 (Figure 1f).</li>
	<li>Bake the PDMS mixture on a hotplate at 80 &#176;C for 2 h to form a bead-embedded PDMS substrate (Figure 1g).</li>
	<li>Remove the cured PDMS substrate from the mold (Figure 1g). In the removing process, spray methanol using washing bottle to detach PDMS substrate from the mold.</li>
	<li>Using a &#934;15 mm x 2 mm magnet, remove the trapped steel beads from the PDMS substrate (Figure 1h). For this process, any magnet that is sufficiently strong to extract the beads from the PDMS substrate can be used.</li>
</ol>
4. Spheroid culture
<ol>
	<li>Cut the concave microwell-patterned PDMS substrate by using a &#934;14 mm biopsy punch to be fitted in 24-well plate in this study.</li>
	<li>Sterilize the resulting &#934;14 mm PDMS substrate in an autoclave sterilizer at 121 &#176;C and 15 psi.</li>
	<li>Place the sterilized PDMS substrate into a 24 well plate.</li>
	<li>Coat the entire PDMS substrate with 4% (w/v) pluronic F-127 solution overnight to prevent cell attachment to the microwell surface. During the coating process, remove any air bubbles entrapped in the concave microwells by pipetting or by using an ultrasonic cleaner.</li>
	<li>Flush the F-127 solution three times by using phosphate-buffered saline (PBS).</li>
	<li>Seed 1 mL of cell-medium (Dulbecco&#39;s Modified Eagle Medium) solution (which contains 2 x 106 cells) on the PDMS substrate. Note that the seeding density can be changed according to the target spheroid size and/or target cell type. Here, adipose-derived stem cells (ASC) were used.</li>
	<li>Aspirate the 1 mL of medium using a 1000 &#181;L pipette to remove any excess cells that were not trapped in the microwells (Figure 3).</li>
	<li>Incubate the cells at 36.5 &#176;C, humidity of &#62;95%, and 5% CO2 condition. In the case of the ASCs used in our study, the cells aggregate to a spheroid in 48 h.</li>
</ol>

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The authors have no conflicts of interest to disclose.

The major challenge facing this fabrication method was the secure fixing of the beads in the through-hole array in the aluminum plate. To solve this challenge, magnetic force in the form of a 30 x 30 magnet array was used to fix the beads securely, as shown in Figures 6 and 7. The magnetic flux density of the magnet array, which has the opposite polarity, is strongest at the center of each magnet surface. Because the strength of the magnetic force depends on the flux density, the beads w...

Cells grown into a spheroid form are more similar to real tissue in the body than a two-dimensional planar culture1.&#160;Given this advantage, the use of spheroids has been adopted to improve the study of cell to cell interaction2,3, immune-activation4, drug screening5, and differentiation6. In addition, spheroids incorporating multiple cell types have recently been applied to organoids (near-physiological three-dimensional (3D) tissue), which are very useful for studying human development and disease7.&#160;Several methods have been used to produce spheroids. The simplest method involves the utilization of a non-adhesive surface, such that the cells aggregate with each other and form spheroids. A Petri dish can be treated with bovine serum albumin, pluronic F-127, or a hydrophobic polymer (e.g. poly 2-hydroxyethl methacrylate) to make its surface non-adhesive8,9. The spinner-flask method is another well-known means of producing large amounts of spheroids10,11. In this method, cells are held in suspension by stirring to prevent them from becoming attached to the substrate. Instead, the floating cells aggregate to form spheroids. Both the non-adhesive surface method and spinner flask method can produce large amounts of spheroids. However, they are subject to limitations including difficulties in controlling the spheroid size, as well as the tracking and monitoring of each spheroid. As a remedy for such problems, another spheroid production method, namely, the hanging drop method can be employed12. This involves depositing cell suspension drops onto the underside of the lid of a culture dish. These drops are usually 15 to 30 &#181;L in size and contain approximately 300 to 3000 cells13. When the lid is inverted, the drops are held in place by surface tension. The microgravity environment in each drop concentrates the cells, which then form single spheroids at the free liquid-air interface. The benefits of the hanging drop method are that it offers a well-controlled size distribution, while it is easy to trace and monitor each spheroid, relative to the non-adhesive surface and spinner flask methods. However, this method incurs one disadvantage in that the massive production of spheroids and the production process itself is excessively labor intensive.
A microwell array is a flat plate with many micro-size wells, each having a diameter ranging from 100 to 1000 &#181;m. The spheroid production principle when using microwells is similar to that of the non-adhesive surface method. Benefits include the fact that microwells provide spaces between the microwells for separating the cells or spheroids, such that it is easy to control the spheroid size, while also making it easy to monitor each single spheroid. With a large number of microwells, high-throughput spheroid production is also possible. Another advantage of microwells is the option to form wells of different shapes (hexahedral, cylindrical, trigonal prismatic) depending on the users&#39; unique experimental purposes. Generally, however, a three-dimensional (3D) concave (or hemispherical) shape is regarded as being the most suitable for producing uniform-sized single spheroids. Therefore, the usefulness of concave microwells has been reported for many cell biology studies such as those examining the cardiomyocyte differentiation of embryonic stem cells14, the insulin secretion of islet cell clusters15, the enzymatic activity of hepatocytes16, and the drug resistance of tumor spheroids17.
Unfortunately, the fabrication of microwells often requires specialized micropatterning facilities; conventional photolithography-based methods require exposure and developing facilities while reactive ion-etching-based methods need plasma and ion-beam equipment. Such equipment is costly which, together with the complicated fabrication process, presents a high barrier to entry for biologists who do not have access to microtechnology. To overcome these problems, other cost-effective methods such as ice lithography18 (using frozen water droplets) and the flexible membrane method14 (using a membrane, through-hole substrate, and a vacuum) have been suggested. However, these methods also incur serious drawbacks such as it being difficult to control the pattern sizes, the attainment of high aspect ratios, and the production of larger-area microwells.
To overcome the above issues, we are proposing a novel concave microwell fabrication method utilizing a through-hole substrate, steel beads, and a magnet array. Using this method, hundreds of concave spherical microwells can be fabricated by taking advantage of the mechanism of magnetic-force-assisted self-locking metallic beads (Figure 1). The fabrication process involves the use of very few expensive and complicated facilities and does not demand many advanced skills. As such, even unskilled persons can easily undertake this fabrication method. To demonstrate the proposed method, human-adipose-derived stem cells were cultured in the concave microwells to produce spheroids.

<table border="0" cellpadding="0" cellspacing="0" width="612">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">CNC rotary engraver</td>
			<td style="width:111px;">Roland DGA</td>
			<td style="width:119px;">EGX-350</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Micro drill bit</td>
			<td style="width:111px;">HAM Pr&#228;zision</td>
			<td style="width:119px;">30-1301 TA</td>
			<td>&#934; 0.55 and 0.75 mm</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:216px;">Sulfuric acid 98%</td>
			<td style="width:111px;">Daejung</td>
			<td style="width:119px;">7683-4100</td>
			<td style="width:167px;">For cleaning aluminum plate. 
			Dilute with distilled water with 15% solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Neodymium magnet</td>
			<td style="width:111px;">Supermagnete</td>
			<td>W-01-N</td>
			<td>1 x 1 x 1 mm</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Bearing ball</td>
			<td style="width:111px;">Agami Modeling</td>
			<td style="width:119px;">SUJ2</td>
			<td>&#934; 600 &#956;m steel bead</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Polydimethylsiloxane (PDMS)</td>
			<td style="width:111px;">Dowcorning</td>
			<td style="width:119px;">Sylgard 184</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Pluronic F-127</td>
			<td style="width:111px;">Sigma Aldrich</td>
			<td style="width:119px;">p2443</td>
			<td>Dilute with phosphate buffered saline to 4% (w/v) solution</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Dulbecco&#39;s modified eaggle&#39;s medium (DMEM)</td>
			<td style="width:111px;">ATCC</td>
			<td style="width:119px;">30-2002</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Dulbecco&#39;s phosphate buffered saline (D-PBS)</td>
			<td style="width:111px;">ATCC</td>
			<td style="width:119px;">30-2200</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Fetal bovine serum</td>
			<td style="width:111px;">ATCC</td>
			<td style="width:119px;">30-2020</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Adipose-derived mesenchymal stem cells</td>
			<td style="width:111px;">ATCC</td>
			<td style="width:119px;">ATCC PCS-500-011</td>
			<td></td>
		</tr>
	</tbody>
</table>

a paired bead and magnet array for molding microwells with variable concave geometries

A spheroid culture is a useful tool for understanding cellular behavior in that it provides an in vivo-like three-dimensional environment. Various spheroid production methods such as non-adhesive surfaces, spinner flasks, hanging drops, and microwells have been used in studies of cell-to-cell interaction, immune-activation, drug screening, stem cell differentiation, and organoid generation. Among these methods, microwells with a three-dimensional concave geometry have gained the attention of scientists and engineers, given their advantages of uniform-sized spheroid generation and the ease with which the responses of individual spheroids can be monitored. Even though cost-effective methods such as the use of flexible membranes and ice lithography have been proposed, these techniques incur serious drawbacks such as difficulty in controlling the pattern sizes, achievement of high aspect ratios, and production of larger areas of microwells. To overcome these problems, we propose a robust method for fabricating concave microwells without the need for complex high-cost facilities. This method utilizes a 30 x 30 through-hole array, several hundred micrometer-order steel beads, and magnetic force to fabricate 900 microwells in a 3 cm x 3 cm polydimethylsiloxane (PDMS) substrate. To demonstrate the applicability of our method to cell biological applications, we cultured adipose stem cells for 3 days and successfully produced spheroids using our microwell platform. In addition, we performed a magnetostatic simulation to investigate the mechanism, whereby magnetic force was used to trap the steel beads in the through-holes. We believe that the proposed microwell fabrication method could be applied to many spheroid-based cellular studies such as drug screening, tissue regeneration, stem cell differentiation, and cancer metastasis.

A convex mold and microwell pattern were successfully fabricated by following steps 2.1 to 3.7. (Figure 4). The commercial steel beads were trapped in the 30 x 30 through-hole array. The beads were held tightly without any gaps between the beads and the corresponding through-holes (Figure 4a). The shape of fabricated concave microwell is concave hemispherical, with a diameter of 600 &#181;m, which is the same as that of the steel...

Watch this Scientific Journal Video about A Paired Bead and Magnet Array for Molding Microwells with Variable Concave Geometries at JoVE.com

A Paired Bead and Magnet Array for Molding Microwells with Variable Concave Geometries

This manuscript introduces a robust method of fabricating concave microwells without the need for complex high-cost facilities. Using magnetic force, steel beads, and a through-hole array, several hundred microwells were formed in a 3 cm x 3 cm polydimethylsiloxane (PDMS) substrate.

A spheroid culture is a useful tool for understanding cellular behavior in that it provides an in vivo-like three-dimensional environment. Various ...

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Unit 1

Magnetic Forces and Fields

SCIENTISTS IN ACTION

8.6K Views.  Chung-Ang University. This manuscript introduces a robust method of fabricating concave microwells without the need for complex high-cost facilities. Using magnetic force, steel beads, and a through-hole array, several hundred microwells were formed in a 3 cm x 3 cm polydimethylsiloxane (PDMS) substrate.

Video: A Paired Bead and Magnet Array for Molding Microwells with Variable Concave Geometries

Investigating Receptor-ligand Systems of the Cellulosome with AFM-based Single-molecule Force Spectroscopy

Cellulosomes are discrete multienzyme complexes used by a subset of anaerobic bacteria and fungi to digest lignocellulosic substrates. Assembly of the enzymes onto the noncatalytic scaffold protein is directed by interactions among a family of related receptor-ligand pairs comprising interacting cohesin and dockerin modules. The extremely strong binding between cohesin and dockerin modules results in dissociation constants in the low picomolar to nanomolar range, which may hamper accurate off-rate measurements with conventional bulk methods. Single-molecule force spectroscopy (SMFS) with the atomic force microscope measures the response of individual biomolecules to force, and in contrast to other single-molecule manipulation methods (i.e.&#160;optical tweezers), is optimal for studying high-affinity receptor-ligand interactions because of its ability to probe the high-force regime (&#62;120 pN). Here we present our complete protocol for studying cellulosomal protein assemblies at the single-molecule level. Using a protein topology derived from the native cellulosome, we worked with enzyme-dockerin and carbohydrate binding module-cohesin (CBM-cohesin) fusion proteins, each with an accessible free thiol group at an engineered cysteine residue. We present our site-specific surface immobilization protocol, along with our measurement and data analysis procedure for obtaining detailed binding parameters for the high-affinity complex. We demonstrate how to quantify single subdomain unfolding forces, complex rupture forces, kinetic off-rates, and potential widths of the binding well. The successful application of these methods in characterizing the cohesin-dockerin interaction responsible for assembly of multidomain cellulolytic complexes is further described.

Cellulosomes are multienzyme complexes designed for digesting cellulose. AFM-based SMFS was used to study the mechanical properties and folding configuration of cellulosome-associated protein assemblies. We present a complete workflow for protein immobilization, data acquisition, and data analysis to study the interactions of individual receptor-ligand complexes involved in cellulosome assembly.

Cellulosomes are discrete multienzyme complexes used by a subset of anaerobic bacteria and fungi to digest lignocellulosic substrates. Assembly of the ...

Microfluidic Fabrication of Polymeric and Biohybrid Fibers with Predesigned Size and Shape

A &#8220;sheath&#8221; fluid passing through a microfluidic channel at low Reynolds number can be directed around another &#8220;core&#8221; stream and used to dictate the shape as well as the diameter of a core stream.&#160;Grooves in the top and bottom of a microfluidic channel were designed to direct the sheath fluid and shape the core fluid.&#160;By matching the viscosity and hydrophilicity of the sheath and core fluids, the interfacial effects are minimized and complex fluid shapes can be formed.&#160;Controlling the relative flow rates of the sheath and core fluids determines the cross-sectional area of the core fluid.&#160;Fibers have been produced with sizes ranging from 300 nm to ~1 mm, and fiber cross-sections can be round, flat, square, or complex as in the case with double anchor fibers. Polymerization of the core fluid downstream from the shaping region solidifies the fibers.&#160;Photoinitiated click chemistries are well suited for rapid polymerization of the core fluid by irradiation with ultraviolet light.&#160;Fibers with a wide variety of shapes have been produced from a list of polymers including liquid crystals, poly(methylmethacrylate), thiol-ene and thiol-yne resins, polyethylene glycol, and hydrogel derivatives. Minimal shear during the shaping process and mild polymerization conditions also makes the fabrication process well suited for encapsulation of cells and other biological components.

Two adjacent fluids passing through a grooved microfluidic channel can be directed to form a sheath around a prepolymer core; thereby&#160;determining both shape and cross-section. Photoinitiated polymerization, such as thiol click chemistry, is well suited for rapidly solidifying the core fluid into a microfiber with predetermined size and shape.

A &#8220;sheath&#8221; fluid passing through a microfluidic channel at low Reynolds number can be directed around another &#8220;core&#8221; stream and ...

Rapid and Low-cost Prototyping of Medical Devices Using 3D Printed Molds for Liquid Injection Molding

Biologically inert elastomers such as silicone are favorable materials for medical device fabrication, but forming and curing these elastomers using traditional liquid injection molding processes can be an expensive process due to tooling and equipment costs. As a result, it has traditionally been impractical to use liquid injection molding for low-cost, rapid prototyping applications. We have devised a method for rapid and low-cost production of liquid elastomer injection molded devices that utilizes fused deposition modeling 3D printers for mold design and a modified desiccator as an injection system. Low costs and rapid turnaround time in this technique lower the barrier to iteratively designing and prototyping complex elastomer devices. Furthermore, CAD models developed in this process can be later adapted for metal mold tooling design, enabling an easy transition to a traditional injection molding process. We have used this technique to manufacture intravaginal probes involving complex geometries, as well as overmolding over metal parts, using tools commonly available within an academic research laboratory. However, this technique can be easily adapted to create liquid injection molded devices for many other applications.

We have devised a method for low-cost and rapid prototyping of liquid elastomer rubber injection molded devices by using fused deposition modeling 3D printers for mold design and a modified desiccator as a liquid injection system.

Biologically inert elastomers such as silicone are favorable materials for medical device fabrication, but forming and curing these elastomers using ...

Bead Aggregation Assays for the Characterization of Putative Cell Adhesion Molecules

Cell-cell adhesion is fundamental to multicellular life and is mediated by a diverse array of cell surface proteins. However, the adhesive interactions for many of these proteins are poorly understood. Here we present a simple, rapid method for characterizing the adhesive properties of putative homophilic cell adhesion molecules. Cultured HEK293 cells are transfected with DNA plasmid encoding a secreted, epitope-tagged ectodomain of a cell surface protein. Using functionalized beads specific for the epitope tag, the soluble, secreted fusion protein is captured from the culture medium. The coated beads can then be used directly in bead aggregation assays or in fluorescent bead sorting assays to test for homophilic adhesion. If desired, mutagenesis can then be used to elucidate the specific amino acids or domains required for adhesion. This assay requires only small amounts of expressed protein, does not require the production of stable cell lines, and can be accomplished in 4 days.

Here we present a simple, rapid method for characterizing the intrinsic adhesive properties of putative cell adhesion molecules. The secreted, epitope-tagged ectodomain of a cell adhesion molecule is captured from the culture medium on small, uniform functionalized beads. These beads can then be used immediately in simple bead aggregation assays.

Cell-cell adhesion is fundamental to multicellular life and is mediated by a diverse array of cell surface proteins. However, the adhesive interactions ...

Polymeric Microneedle Array Fabrication by Photolithography

This manuscript describes the fabrication of polymeric microneedle (MN) arrays by photolithography. It involves a simple mold-free process by using a photomask consisting of embedded micro-lenses. Embedded micro-lenses were found to influence MN geometry (sharpness). Robust MN arrays with tip diameters ranging between 41.5 &#181;m &#177; 8.4 &#181;m and 71.6 &#181;m &#177; 13.7 &#181;m, with two different lengths (1,336 &#181;m &#177; 193 &#181;m and 957 &#181;m &#177; 171 &#181;m) were fabricated. These MN arrays may provide potential applications in delivery of low molecular and macromolecular therapeutic agents through skin.

Here, we present a protocol describing a mold-free fabrication process of the polymeric microneedles by photolithography.

This manuscript describes the fabrication of polymeric microneedle (MN) arrays by photolithography. It involves a simple mold-free process by using a ...

Multi-step Variable Height Photolithography for Valved Multilayer Microfluidic Devices

Microfluidic systems have enabled powerful new approaches to high-throughput biochemical and biological analysis. However, there remains a barrier to entry for non-specialists who would benefit greatly from the ability to develop their own microfluidic devices to address research questions. Particularly lacking has been the open dissemination of protocols related to photolithography, a key step in the development of a replica mold for the manufacture of polydimethylsiloxane (PDMS) devices. While the fabrication of single height silicon masters has been explored extensively in literature, fabrication steps for more complicated photolithography features necessary for many interesting device functionalities (such as feature rounding to make valve structures, multi-height single-mold patterning, or high aspect ratio definition) are often not explicitly outlined. 
Here, we provide a complete protocol for making multilayer microfluidic devices with valves and complex multi-height geometries, tunable for any application. These fabrication procedures are presented in the context of a microfluidic hydrogel bead synthesizer and demonstrate the production of droplets containing polyethylene glycol (PEG diacrylate) and a photoinitiator that can be polymerized into solid beads. This protocol and accompanying discussion provide a foundation of design principles and fabrication methods that enables development of a wide variety of microfluidic devices. The details included here should allow non-specialists to design and fabricate novel devices, thereby bringing a host of recently developed technologies to their most exciting applications in biological laboratories.

Multilayer microfluidic devices often involve the fabrication of master molds with complex geometries for functionality. This article presents a complete protocol for multi-step photolithography with valves and variable height features tunable to any application. As a demonstration, we fabricate a microfluidic droplet generator capable of producing hydrogel beads.

Microfluidic systems have enabled powerful new approaches to high-throughput biochemical and biological analysis. However, there remains a barrier to ...

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 ...

Assembly and Tracking of Microbial Community Development within a Microwell Array Platform

The development of microbial communities depends on a combination of complex deterministic and stochastic factors that can dramatically alter the spatial distribution and activities of community members. We have developed a microwell array platform that can be used to rapidly assemble and track thousands of bacterial communities in parallel. This protocol highlights the utility of the platform and describes its use for optically monitoring the development of simple, two-member communities within an ensemble of arrays within the platform. This demonstration uses two mutants of Pseudomonas aeruginosa, part of a series of mutants developed to study Type VI secretion pathogenicity. Chromosomal inserts of either mCherry or GFP genes facilitate the constitutive expression of fluorescent proteins with distinct emission wavelengths that can be used to monitor community member abundance and location within each microwell. This protocol describes a detailed method for assembling mixtures of bacteria into the wells of the array and using time-lapse fluorescence imaging and quantitative image analysis to measure the relative growth of each member population over time. The seeding and assembly of the microwell platform, the imaging procedures necessary for the quantitative analysis of microbial communities within the array, and the methods that can be used to reveal interactions between microbial species area all discussed.

The development of microbial communities depends on a combination of factors, including environmental architecture, member abundance, traits, and interactions. This protocol describes a synthetic, microfabricated environment for the simultaneous tracking of thousands of communities contained in femtoliter wells, where key factors such as niche size and confinement can be approximated.

The development of microbial communities depends on a combination of complex deterministic and stochastic factors that can dramatically alter the spatial ...

Visual Detection of Multiple Nucleic Acids in a Capillary Array

Multi-target, short time, and resource-affordable methodologies for the detection of multiple nucleic acids in a single, easy to operate test are urgently needed in disease diagnosis, microbial monitoring, genetically modified organism (GMO) detection, and forensic analysis. We have previously described the platform called CALM (Capillary Array-based Loop-mediated isothermal amplification for Multiplex visual detection of nucleic acids). Herein, we describe improved fabrication and performance processes for this platform. Here, we apply a small, ready-to-use cassette assembled by capillary array for multiplex visual detection of nucleic acids. The capillary array is pre-treated into a hydrophobic and hydrophilic pattern before fixing loop-mediated isothermal amplification (LAMP) primer sets in capillaries. After assembly of the loading adaptor, LAMP reaction mixture is loaded and isolated into each capillary, due to capillary force by a single pipetting step. The LAMP reactions are performed in parallel in the capillaries. The results are visually read out by illumination with a hand-held UV flashlight. Using this platform, we demonstrate monitoring of 8 frequently appearing elements and genes in GMO samples with high specificity and sensitivity. In summary, the platform described herein is intended to facilitate the detection of multiple nucleic acids. We believe it will be widely applicable in fields where high-throughput nucleic acid analysis is required.

This protocol describes the fabrication of a small, ready-to-use cassette that can be applied for visual detection of multiple nucleic acids in a single, test that is easy to operate. In this approach, a capillary array was used for multiplex and highly efficient detection of GMO targets.

Multi-target, short time, and resource-affordable methodologies for the detection of multiple nucleic acids in a single, easy to operate test are urgently ...

Fabrication of a Multiplexed Artificial Cellular MicroEnvironment Array

Cellular microenvironments consist of a variety of cues, such as growth factors, extracellular matrices, and intercellular interactions. These cues are well orchestrated and are crucial in regulating cell functions in a living system. Although a number of researchers have attempted to investigate the correlation between environmental factors and desired cellular functions, much remains unknown. This is largely due to the lack of a proper methodology to mimic such environmental cues in vitro, and simultaneously test different environmental cues on cells. Here, we report an integrated platform of microfluidic channels and a nanofiber array, followed by high-content single-cell analysis, to examine stem cell phenotypes altered by distinct environmental factors. To demonstrate the application of this platform, this study focuses on the phenotypes of self-renewing human pluripotent stem cells (hPSCs). Here, we present the preparation procedures for a nanofiber array and the microfluidic structure in the fabrication of a Multiplexed Artificial Cellular MicroEnvironment (MACME) array. Moreover, overall steps of the single-cell profiling, cell staining with multiple fluorescent markers, multiple fluorescence imaging, and statistical analyses, are described.

This article describes the detailed methodology to prepare a Multiplexed Artificial Cellular MicroEnvironment (MACME) array for high-throughput manipulation of physical and chemical cues mimicking in vivo cellular microenvironments and to identify the optimal cellular environment for human pluripotent stem cells (hPSCs) with single-cell profiling.

Cellular microenvironments consist of a variety of cues, such as growth factors, extracellular matrices, and intercellular interactions. These cues are ...