This work was supported by the Ministry of Science and Technology of Taiwan under Grant Number 101-2221-E-002-064-MY3.

1. Fabrication of Microfluidic Device
<ol>
	<li>Use a graphic layout software (e.g., AutoCAD) to draw the outline of a T-microchannel. For the T-microchannel, the two feed channels are 90 &#181;m wide and 2,500 &#181;m long, and the confluence channel is 180 &#181;m wide and 3,000 &#181;m long. Connect the end of each channel to an individual circle with a diameter of 1,100 &#181;m.</li>
	<li>Mark &#8216;clear&#8217; and &#8216;dark&#8217; for the exposure and covered areas, respectively. For a negative photoresist (e.g., SU-8), the shape of the T-microchannel is &#8216;clear&#8217; and the surrounding is &#8216;dark.&#8217;</li>
	<li>Use a laser pattern generator with a wavelength of 442 nm and a minimum feature size of 2 &#956;m to transfer the pattern of the T-microchannel onto a chrome-on-glass photomask.</li>
	<li>Use the photomask, a substrate (e.g., single-side polished silicon wafer) and a permanent epoxy photoresist (e.g., SU-8) to make a mold through a standard lithography process. The photoresist layer is 55.2 &#956;m thick. In general, the photoresist thickness should be thinner than the depth of correlation of the objective lens17-19.</li>
	<li>Use the mold and a transparent material such as polydimethylsiloxane (PDMS) to fabricate the T-microchannel20.</li>
	<li>For fluidic connection, use a stainless steel tube of 2 mm in outer diameter to punch through-holes aligned to the circular patterns on the PDMS.</li>
	<li>Treat the surfaces of the PDMS and a glass slide with oxygen plasma at 60 W for 30 sec. Attach the PDMS to the glass slide. The oxidized surfaces of the two materials create a strong bond. Place the bonded PDMS structure on a hot plate for 5 min at 120 &#176;C.</li>
	<li>Insert Teflon tubes into the punched holes for fluidic connection.</li>
</ol>
2. Experimental Setup
<ol>
	<li>Construct the microscale schlieren system from a Hoffman modulation contrast microscope by removing the slit plate in the front focal plane of the condenser and replacing the modulator with a knife-edge in the rear focal plane of the 5X objective3. The depth of correlation, which depends on the numerical aperture of the objective17-19, should be sufficient to cover the entire depth of the microfluidic device. The surface of the knife-edge is blackened by anodic aluminum oxide to reduce its reflectivity.</li>
	<li>Mount the high-speed camera to the trinocular tube of the microscope via a C-mount adapter. Have the camera face the optical path of the microscope through a beam splitter. Connect the camera to a desktop computer via an Ethernet cable. Set the gamma correction to 1 for the camera so that its grayscale readout is proportional to the input luminance.</li>
	<li>Turn on the light source. In order to avoid excess heat, use LED (light emitting diode) lighting.</li>
	<li>Use an image processing software (e.g., function imread in MATLAB) to obtain the grayscale values of an acquired image. Remove the knife-edge, adjust the illumination, the aperture and the exposure time so that the average grayscale readout of the image is about 10% less than the maximal value. This denotes the background intensity for a 0% cutoff and we use a value of 230 for an 8-bit image.</li>
	<li>Insert the knife-edge to block the incident light completely. Record the average grayscale readout of the image. This denotes the background intensity for a 100% cutoff and the value is about 15 for an 8-bit image.</li>
	<li>Adjust the position of the knife-edge such that the average grayscale readout of the acquired image lies in the middle of the values for 0% and 100% cutoff. Now the degree of the cutoff is set to 50%.</li>
	<li>Prepare two transparent fluids with known refractive indices21 that are completely miscible with each other as the constituents. To evaluate the dependence of refractive index on concentration of the mixture, check the literature21 or use the Gladstone-Dale equation22. If the curve is nonlinear over the entire range, pick other fluid components. Then, choose a designated composition below which the refractive index of the solution varies linearly with the concentration. For example, use dilute aqueous ethanol with a mass fraction of 0.05 and water as the working fluids.</li>
	<li>Put the T-microchannel on the specimen stage. Arrange the T-microchannel so with the confluent channel parallel to the knife-edge (Figure 1).</li>
	<li>Prepare two identical syringes: syringe A is filled with the working fluid that serves as the reference fluid (water), and syringe B is filled with the other working fluid (dilute aqueous ethanol). The size of the syringe depends on the desired flow rate Q and the specification of the syringe pump: Q = &#960;d2V/4, where d is the inner diameter of the syringe and V is the speed of the plunger. Flow pulsation usually can be prevented by choosing a small syringe to increase V23.</li>
	<li>Collect the outlet fluid from the T-microchannel in a beaker. Ensure the Teflon outlet tube is fixed to the beaker&#39;s wall and its end is below the liquid level in the beaker in order to avoid vibration that would be caused by droplet breakoff.</li>
</ol>
3. Calibration
<ol>
	<li>Acquire the images of the two fluids mixing and the reference images.
	<ol>
		<li>At a given Reynolds number Re, set the flow rate of the syringe pumps, Q. Q is calculated from Q = &#181;(W+D)Re/4&#961;, where &#181; and &#961; are the viscosity and density of the working fluid, and W and D are the width and depth of the confluence channel of the T-microchannel, respectively.</li>
		<li>Load one pump with syringe A and the other pump with syringe B. Connect the two inlets of the T-microchannel to syringe A and syringe B via Teflon tubing. Start the syringe pumps to deliver the working fluids into the T-microchannel at identical volume flow rates.</li>
		<li>Wait until steady flow establishes. The steady flow condition is defined by the emergence of a stationary schlieren pattern.</li>
		<li>Use the camera controlled software to record twenty frames of fluidic mixing at a frame rate of 30 fps.</li>
		<li>Stop the pump which is loaded with syringe B. Only pump the reference fluid (water) through one inlet into the confluence channel of the T-microchannel at constant rate.</li>
		<li>Wait until steady flow condition is reached and no schlieren pattern is observed.</li>
		<li>Use the camera controlled software to take the reference image, when no optical inhomogeneity is present in the T-microchannel. Record twenty frames at a frame rate of 30 fps.</li>
		<li>Repeat 3.1.1 to 3.1.7 at different Reynolds number: Re = 1, 5, 10, 20 and 50 so that no complex flow structure emerges in the confluence region of the T-microchannel24.</li>
	</ol>
	</li>
	<li>Use an image processing software to divide the acquired image I(i, j) by the reference image I0(i, j)25, where i and j are the pixel indices.</li>
	<li>Employ a CFD (Computational Fluid Dynamics) package to simulate mixing of the designated fluids in the T-microchannel.
	<ol>
		<li>Construct the three-dimensional model for the geometry of the T-microchannel. Discretize the flow domain into structured grids. To increase the accuracy, employ finer mesh in the confluence and the central region of the T-microchannel.</li>
		<li>Assign the physical properties of fluids and establish the boundary conditions to the flow domain. During the solving process, determine the concentration-dependent diffusion coefficient from the concentration obtained in the last iteration26 in order to update the local concentration.</li>
		<li>Examine the sensitivities of the computed results by performing the grid study27.</li>
	</ol>
	</li>
	<li>For each node (xi, yi) on the xy-plane, employ the CFD post-processing tool to take the average values of the concentration field across the channel depth by the trapezoidal rule: w(xi, yj) = {&#8721;k [w(xi, yj, zk) + w(xi, yj, zk+1)]&#8729;(zk+1 - zk)/2}/D28, where D is the channel depth. Use the central differencing scheme to compute the derivative of concentration with respect to the cross-stream direction: (&#8706;w/&#8706;y)i, j = [w(xi, yj+1) - w(xi, yj-1)]/(yj+1 - yj-1).</li>
	<li>For both positive and negative gradients, extract the ratio of grayscale values I/I0 (obtained in 3.2) and the gradient of mass fraction &#8706;w/&#8706;y (obtained in 3.4) at specified locations such as y = 0 (centerline, streamwise direction) or various given x (cross-stream direction).</li>
	<li>Plot the results and determine the relationship between I/I0 and &#8706;w/&#8706;y, I/I0 = C1&#8706;w/&#8706;y + C2 (C1 and C2 are constants), with linear regression.</li>
</ol>
4. Quantitation
<ol>
	<li>Repeat steps 3.1 to 3.2 for mixing in the target microfluidic device. The depth of the target microfluidic device should be identical or close to that of the T-microchannel. If unsteady phenomenon is expected, acquire a video clip (sequence of images) in step 3.1.4 instead. The frame rate should be high enough to resolve the dynamics of the transient flow clearly, while the exposure time should be identical to the value used in 2.4, 2.5, 3.1.4 and 3.1.7.</li>
	<li>Use the relationship obtained in step 3.6 to convert the ratio of grayscale values to the gradient of mass fraction in the target microfluidic device.</li>
</ol>

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The authors have nothing to disclose.

For fluidic mixing in a microfluidic device, the microscale schlieren technique is able to measure the magnitude of concentration gradient through quantifying change in light intensity. Because the principle of this technique relies on detecting the alternation of light propagation, the working fluids and the microfluidic device have to be transparent to the incident light. In addition, the protocol requires a linear relationship between the refractive index of the solution and its composition so that preliminary assessment of the working fluids is essential. Besides aqueous ethanol solution demonstrated herein, microscale schlieren technique is successfully applied to measure salinity gradient29 and solutocapillary convection30. For accurate measurements, aperture range, illumination level, exposure time, objective lens and microchannel depth used in the calibration procedure have to be identical to those used in the quantitation procedure. Moreover, the depth of correlation of the objective lens has to be sufficiently large to cover the entire depth of the microfluidic device.
The calibration process of mixing in the T-microchannel is the most critical step in the accurate quantitation of microscale schlieren technique. For successful implementation of the proposed method, users need to align the tube connection properly, exploit a small syringe or pneumatics for fluid delivery to avoid flow oscillation23, use a LED light source to reduce excess heat, conduct the calibration procedure at low Reynolds numbers24, and place the microfluidic device in focus to eliminate higher-order optical effects31. The lowest measurable gradient (bright pattern, &#8706;w/&#8706;y &#60; 0) is linked to the dynamic range of the camera, whereas the highest measurable gradient (dark pattern, &#8706;w/&#8706;y &#62; 0) is reached when the knife-edge completely blocks the deflected light. To detect a wide range of concentration gradient, a high ISO value is advantageous as long as underexposure or overexposure does not occur. The detection limit, below which the micro schlieren system is unable to discern, depends on the minimal intensity change that the camera is able to resolve. The minimal intensity change is constrained by the degree of noise and the levels of tonal gradation. Hence, a high-sensitivity camera with great pixel depth is desired for low-signal application.
The significance of microscale schlieren technique is two folds; on one hand, it enables unsteady full-field measurements in real time with a simple optical configuration. On the other hand, it is non-invasive so that no alien substance is introduced to disturb the flow field. Because micro-schlieren technique produces two-dimensional projection of the three-dimensional inhomogeneity in a microfluidic device, complex mixing phenomenon that remains veiled by existing methods can be clearly seen. Future applications of this technique include quantifying concentration gradients during an electrochemical process or determining nutrient gradient to study microbial chemotaxis in a micro flow environment.

Fluid mixing is an important issue that is found in many industrial processes and biological systems. With the emergence of microfluidics, mixing in microscale has brought much attention due to its challenge in diffusion domination among the mass transport mechanisms. Since designing an effective micromixer required quantitative validation, several measuring methods were developed5-7. Nevertheless, the three-dimensional structure, commonly found in efficient micromixers5, demands a more accurate representation of the concentration field that the common measuring techniques fail to deliver. Due to the limit of viewing angle8 or reaction kinetics6, the aforementioned methods may produce misleading results that do not correctly account for the homogeneity of the mixture.
For optically transparent fluids mixing in optically transparent microstructures, microscale schlieren technique3,9-14 provides an attractive alternative to analyze mixing inhomogeneity. In the past, microscale schlieren technique has been mostly used to visualize compressible flow9-13,15 or phase gradient16. Microscale schlieren technique benefits from both a simple optical layout and high sensitivity and enables not only the non-invasive investigation of specific flow feature that causes optical disturbance but is well suited for use in assessing mixing. In this paper, we construct the microscale schlieren system by inserting a knife-edge in the back focal plane of the objective of a microscope, describe a calibration procedure to realize quantitative analysis, and report a validation measurement in a microfluidic oscillator4. To implement measurements, the working fluids are properly selected so that the refractive index of the mixed fluids varies linearly with the composition, and the thickness of the target microfluidic device is identical to the one used in calibration. Besides species concentration, microscale schlieren technique can be extended to measure the gradient of other scalar quantity that is linearly correlated to the index of refraction, such as temperature or salinity.

<table><tbody><tr><td>Permanent Epoxy Negative Photoresist</td><td>MicroChem</td><td>SU-8 2150</td><td></td></tr><tr><td>single side polished silicon wafer</td><td>Light Technology</td><td>S4W1PP5SABUP1</td><td>p-type, &lt;br /&gt;diameter: 100&amp;plusmn;0.5 mm, thickness: 525&amp;plusmn;25 mm, orientation: (1 0 0)</td></tr><tr><td>syringe pump</td><td>kdScientific</td><td>kds210</td><td></td></tr><tr><td>syringe, &lt;em&gt;i.e.&lt;/em&gt; 10 ml</td><td>Terumo</td><td>SS-10L2138</td><td></td></tr><tr><td>Hoffman modulation contrast microscope</td><td>Leica Microsystems</td><td>DM IL LED</td><td></td></tr><tr><td>5X objective lens</td><td>Leica Microsystems</td><td>N PLAN</td><td>NA = 0.12</td></tr><tr><td>knife-edge</td><td></td><td></td><td>custom made part</td></tr><tr><td>camera, &lt;em&gt;i.e.&lt;/em&gt; high speed</td><td>Integrated Design Tools</td><td>NX7-S1</td><td></td></tr><tr><td>C-mount adapter HC 0.63x</td><td>Leica Microsystems</td><td>541537</td><td></td></tr><tr><td>camera operating software</td><td>Integrated Design Tools</td><td>MotionPro X Studio 2.02.01</td><td></td></tr><tr><td>polydimethylsiloxane (PDMS)</td><td>Dow Corning</td><td>Sylgard-184 silicone elastomer kit</td><td></td></tr><tr><td>Teflon tubing, &lt;em&gt;i.e.&lt;/em&gt; O.D. x I.D. 1/16 in. x 0.031 in.</td><td>Supelco</td><td>58700-U</td><td></td></tr><tr><td>micro glass slide</td><td>Matsunami Glass</td><td>S2215</td><td></td></tr><tr><td>hot plate</td><td>Yeong-Shin</td><td>HP-303DN</td><td></td></tr><tr><td>distilled water, &lt;em&gt;i.e.&lt;/em&gt; HPLC grade</td><td>Alps Chemicals</td><td></td><td></td></tr><tr><td>ethyl alcohol, &lt;em&gt;i.e.&lt;/em&gt; reagent grade</td><td>Nihon Shiyaku Reagent</td><td>EA448652</td><td></td></tr><tr><td>image processing software</td><td>Mathworks</td><td>MATLAB R2009a</td><td></td></tr><tr><td>computational fluid dynamics package</td><td>ESI group</td><td>CFD-ACE+ 2008</td><td></td></tr></tbody></table>

analyzing mixing inhomogeneity in a microfluidic device by microscale schlieren technique

In this paper, we introduce the use of microscale schlieren technique to measure mixing inhomogeneity in a microfluidic device. The microscale schlieren system is constructed from a Hoffman modulation contrast microscope, which provides easy access to the rear focal plane of the objective lens, by removing the slit plate and replacing the modulator with a knife-edge. The working principle of microscale schlieren technique relies on detecting light deflection caused by variation of refractive index1-3. The deflected light either escapes or is obstructed by the knife-edge to produce a bright or a dark band, respectively. If the refractive index of the mixture varies linearly with its composition, the local change in light intensity in the image plane is proportional to the concentration gradient normal to the optical axis. The micro-schlieren image gives a two-dimensional projection of the disturbed light produced by three-dimensional inhomogeneity.
To accomplish quantitative analysis, we describe a calibration procedure that mixes two fluids in a T-microchannel. We carry out a numerical simulation to obtain the concentration gradient in the T-microchannel that correlates closely with the corresponding micro-schlieren image. By comparison, a relationship between the grayscale readouts of the micro-schlieren image and the concentration gradients presented in a microfluidic device is established. Using this relationship, we are able to analyze the mixing inhomogeneity from associate micro-schlieren image and demonstrate the capability of microscale schlieren technique with measurements in a microfluidic oscillator4. For optically transparent fluids, microscale schlieren technique is an attractive diagnostic tool to provide instantaneous full-field information that retains the three-dimensional features of the mixing process.

The grayscale ratio I/I0 under different Reynolds number for both positive and negative gradients of mass fraction is shown (Figure 2) with a symmetrical band appearing in the middle of the T-microchannel. At low Reynolds number, the tail of the schlieren band is expanded and blurred due to the dispersion across the mixing interface. As the Reynolds number increases, the diffusion length shortens leading to a narrower band. At different downstream locations, the variations of the intensity change &#916;I/I0 along the cross-stream direction are depicted quantitatively (Figure 3). The results from the calibration process are represented (Figure 4A and 4B). The relationship between I/I0 and &#8706;w/&#8706;y is linear and independent of the Reynolds number. From the regression analysis, I/I0 = -110&#8706;w/&#8706;y + 1.03 for &#8706;w/&#8706;y &#62; 0 and I/I0 = -160&#8706;w/&#8706;y + 0.83 for &#8706;w/&#8706;y &#60; 0, &#8706;w/&#8706;y is in &#181;m-1. The relative uncertainties are &#177;3.8% and &#177;3.2% in Figure 4A and 4B, respectively. The detection limit is reached where the data points level out. It is noted that the deviation in slopes of the positive and negative gradients is not uncommon3. Using these equations, the variation of mass fraction gradient with time in a microfluidic oscillator4 is seen (Figure 5). The mixing interface is deflected in the cavity region and flow instability commences. This video figure clearly reveals the oscillating nature of the flow in the microfluidic oscillator and demonstrates the capability of the microscale schlieren technique to capture the time-resolved full-field concentration gradient in a microfluidic device.
<img alt="Figure 1" src="/files/ftp_upload/52915/52915fig1.jpg" /> 
Figure 1. Schematic of the optical setup. The orientation of the knife-edge produces a dark band with a positive gradient of refractive index. The light deflects toward the direction of increasing refractive index. Because the objective lens inverts the image, blocking the &#8211;y region shields the distorted light and produces a dark band.
<img alt="Figure 2" src="/files/ftp_upload/52915/52915fig2.jpg" /> 
Figure 2. Ratio of grayscale readouts for mixing in the T-microchannel under different flow configuration. Positive and negative gradients result in dark and bright bands, respectively. As the Reynolds number increases, the band becomes more concentrated.
<img alt="Figure 3" src="/files/ftp_upload/52915/52915fig3.jpg" /> 
Figure 3. The variation of the intensity change along the cross-stream direction for both positive and negative gradients. Re = 1 and Re = 5.
<img alt="Figure 4" src="/files/ftp_upload/52915/52915fig4.jpg" /> 
Figure 4. Relationship between the gradient of mass fraction and the grayscale ratio. For both positive and negative gradients, grayscale ratio varies linearly with the mass fraction gradient.
<img alt="Figure 5" src="/files/ftp_upload/52915/52915fig5.jpg" /> <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/52915/52915fig5.mp4" target="_blank">Figure 5 (Video Figure).</a> Evolution of mass fraction gradient in a microfluidic oscillator at Re = 250. The mixing characteristic through flow oscillation is successfully captured by microscale schlieren technique.

Watch this Scientific Journal Video about Analyzing Mixing Inhomogeneity in a Microfluidic Device by Microscale Schlieren Technique at JoVE.com

Analyzing Mixing Inhomogeneity in a Microfluidic Device by Microscale Schlieren Technique

Herein, we describe a procedure that employs microscale schlieren technique to measure mixing inhomogeneity in a microfluidic device. Through calibration, distribution of concentration gradient can be derived from the micro-schlieren image.

In this paper, we introduce the use of microscale schlieren technique to measure mixing inhomogeneity in a microfluidic device. The microscale schlieren ...

analyzing-mixing-inhomogeneity-microfluidic-device-microscale

Universidad San Sebastian

Research

JoVE Journal

Bioengineering

8.9K Views.  National Taiwan University. Herein, we describe a procedure that employs microscale schlieren technique to measure mixing inhomogeneity in a microfluidic device. Through calibration, distribution of concentration gradient can be derived from the micro-schlieren image. 

Video: Analyzing Mixing Inhomogeneity in a Microfluidic Device by Microscale Schlieren Technique

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 ...

Engineering

Confocal Imaging of Confined Quiescent and Flowing Colloid-polymer Mixtures

The behavior of confined colloidal suspensions with attractive interparticle interactions is critical to the rational design of materials for directed assembly1-3, drug delivery4, improved hydrocarbon recovery5-7, and flowable electrodes for energy storage8. Suspensions containing fluorescent colloids and non-adsorbing polymers are appealing model systems, as the ratio of the polymer radius of gyration to the particle radius&#160;and concentration of polymer control the range and strength of the interparticle attraction, respectively. By tuning the polymer properties and the volume fraction of the colloids, colloid fluids, fluids of clusters, gels, crystals, and glasses can be obtained9.&#160;Confocal microscopy, a variant of fluorescence microscopy, allows an optically transparent and fluorescent sample to be imaged with high spatial and temporal resolution in three dimensions. In this technique, a small pinhole or slit blocks the emitted fluorescent light from regions of the sample that are outside the focal volume of the microscope optical system. As a result, only a thin section of the sample in the focal plane is imaged. This technique is particularly well suited to probe the structure and dynamics in dense colloidal suspensions at the single-particle scale: the particles are large enough to be resolved using visible light and diffuse slowly enough to be captured at typical scan speeds of commercial confocal systems10. Improvements in scan speeds and analysis algorithms have also enabled quantitative confocal imaging of flowing suspensions11-16,37.&#160;In this paper, we demonstrate confocal microscopy experiments to probe the confined phase behavior and flow properties of colloid-polymer mixtures. We first prepare colloid-polymer mixtures that are density- and refractive-index matched. Next, we report a standard protocol for imaging quiescent dense colloid-polymer mixtures under varying confinement in thin wedge-shaped cells. Finally, we demonstrate a protocol for imaging colloid-polymer mixtures during microchannel flow.

Confocal microscopy is used to image quiescent and flowing colloid-polymer mixtures, which are studied as model systems for attractive suspensions. Image analysis algorithms are used to calculate structural and dynamic metrics for the colloidal particles that measure changes due to geometric confinement.

The behavior of confined colloidal suspensions with attractive interparticle interactions is critical to the rational design of materials for directed ...

Chemistry

Microfluidic Chips Controlled with Elastomeric Microvalve Arrays

Miniaturized microfluidic systems provide simple and effective solutions for low-cost point-of-care diagnostics and high-throughput biomedical assays. Robust flow control and precise fluidic volumes are two critical requirements for these applications. We have developed microfluidic chips featuring elastomeric polydimethylsiloxane (PDMS) microvalve arrays that: 1) need no extra energy source to close the fluidic path, hence the loaded device is highly portable; and 2) allow for microfabricating deep (up to 1 mm) channels with vertical sidewalls and resulting in very precise features.

The PDMS microvalves-based devices consist of three layers: a fluidic layer containing fluidic paths and microchambers of various sizes, a control layer containing the microchannels necessary to actuate the fluidic path with microvalves, and a middle thin PDMS membrane that is bound to the control layer. Fluidic layer and control layers are made by replica molding of PDMS from SU-8 photoresist masters, and the thin PDMS membrane is made by spinning PDMS at specified heights. The control layer is bonded to the thin PDMS membrane after oxygen activation of both, and then assembled with the fluidic layer. The microvalves are closed at rest and can be opened by applying negative pressure (e.g., house vacuum). Microvalve closure and opening are automated via solenoid valves controlled by computer software.

Here, we demonstrate two microvalve-based microfluidic chips for two different applications. The first chip allows for storing and mixing precise sub-nanoliter volumes of aqueous solutions at various mixing ratios. The second chip allows for computer-controlled perfusion of microfluidic cell cultures.

The devices are easy to fabricate and simple to control. Due to the biocompatibility of PDMS, these microchips could have broad applications in miniaturized diagnostic assays as well as basic cell biology studies.

We demonstrate protocols for manufacturing and automating elastomeric polydimethylsiloxane (PDMS)-based microvalve arrays that need no extra energy to close and feature photolithographically defined precise volumes. A parallel subnanoliter-volume mixer and an integrated microfluidic perfusion system are presented.

Miniaturized microfluidic systems provide simple and effective solutions for low-cost point-of-care diagnostics and high-throughput biomedical assays.  ...

Biology

Microfluidic Mixers for Studying Protein Folding

The process by which a protein folds into its native conformation is highly relevant to biology and human health yet still poorly understood. One reason for this is that folding takes place over a wide range of timescales, from nanoseconds to seconds or longer, depending on the protein1. Conventional stopped-flow mixers have allowed measurement of folding kinetics starting at about 1 ms. We have recently developed a microfluidic mixer that dilutes denaturant ~100-fold in ~8 &mu;s2. Unlike a stopped-flow mixer, this mixer operates in the laminar flow regime in which turbulence does not occur. The absence of turbulence allows precise numeric simulation of all flows within the mixer with excellent agreement to experiment3-4.
Laminar flow is achieved for Reynolds numbers Re &le;100. For aqueous solutions, this requires micron scale geometries. We use a hard substrate, such as silicon or fused silica, to make channels 5-10 &mu;m wide and 10 &mu;m deep (See Figure 1). The smallest dimensions, at the entrance to the mixing region, are on the order of 1 &mu;m in size. The chip is sealed with a thin glass or fused silica coverslip for optical access. Typical total linear flow rates are ~1 m/s, yielding Re~10, but the protein consumption is only ~0.5 nL/s or 1.8 &mu;L/hr. Protein concentration depends on the detection method: For tryptophan fluorescence the typical concentration is 100 &mu;M (for 1 Trp/protein) and for FRET the typical concentration is ~100 nM.
The folding process is initiated by rapid dilution of denaturant from 6 M to 0.06 M guanidine hydrochloride. The protein in high denaturant flows down a central channel and is met on either side at the mixing region by buffer without denaturant moving ~100 times faster (see Figure 2). This geometry causes rapid constriction of the protein flow into a narrow jet ~100 nm wide. Diffusion of the light denaturant molecules is very rapid, while diffusion of the heavy protein molecules is much slower, diffusing less than 1 &mu;m in 1 ms. The difference in diffusion constant of the denaturant and the protein results in rapid dilution of the denaturant from the protein stream, reducing the effective concentration of the denaturant around the protein. The protein jet flows at a constant rate down the observation channel and fluorescence of the protein during folding can be observed using a scanning confocal microscope5.

In this work we explain the fabrication and use of a microfluidic mixer capable of mixing two solutions in ~8 &mu;s. We also demonstrate the use of these mixers with spectroscopic detection using UV fluorescence and fluorescence resonance energy transfer (FRET).

The process by which a protein folds into its native conformation is highly relevant to biology and human health yet still poorly understood. One reason ...

A Microfluidic Device for Studying Multiple Distinct Strains

The study of cell responses to environmental changes poses many experimental challenges: cells need to be imaged under changing conditions, often in a comparative manner. Multiwell plates are routinely used to compare many different strains or cell lines, but allow limited control over the environment dynamics. Microfluidic devices, on the other hand, allow exquisite dynamic control over the surrounding conditions, but it is challenging to image and distinguish more than a few strains in them. Here we describe a method to easily and rapidly manufacture a microfluidic device capable of applying dynamically changing conditions to multiple distinct yeast strains in one channel. The device is designed and manufactured by simple means without the need for soft lithography. It is composed of a Y-shaped flow channel attached to a second layer harboring microwells. The strains are placed in separate microwells, and imaged under the exact same dynamic conditions. We demonstrate the use of the device for measuring protein localization responses to pulses of nutrient changes in different yeast strains.

We present a simple method to produce microfluidic devices capable of applying similar dynamic conditions to multiple distinct strains, without the need for a clean room or soft lithography.

The study of cell responses to environmental changes poses many experimental challenges: cells need to be imaged under changing conditions, often in a ...

Combining Microfluidics and Microrheology to Determine Rheological Properties of Soft Matter during Repeated Phase Transitions

The microstructure of soft matter directly impacts macroscopic rheological properties and can be changed by factors including colloidal rearrangement during previous phase changes and applied shear. To determine the extent of these changes, we have developed a microfluidic device that enables repeated phase transitions induced by exchange of the surrounding fluid and microrheological characterization while limiting shear on the sample. This technique is &#181;2rheology, the combination of microfluidics and microrheology. The microfluidic device is a two-layer design with symmetric inlet streams entering a sample chamber that traps the gel sample in place during fluid exchange. Suction can be applied far away from the sample chamber to pull fluids into the sample chamber. Material rheological properties are characterized using multiple particle tracking microrheology (MPT). In MPT, fluorescent probe particles are embedded into the material and the Brownian motion of the probes is recorded using video microscopy. The movement of the particles is tracked and the mean-squared displacement (MSD) is calculated. The MSD is related to macroscopic rheological properties, using the Generalized Stokes-Einstein Relation. The phase of the material is identified by comparison to the critical relaxation exponent, determined using time-cure superposition. Measurements of a fibrous colloidal gel illustrate the utility of the technique. This gel has a delicate structure that can be irreversibly changed when shear is applied. &#181;2rheology data shows that the material repeatedly equilibrates to the same rheological properties after each phase transition, indicating that phase transitions do not play a role in microstructural changes. To determine the role of shear, samples can be sheared prior to injection into our microfluidic device. &#181;2rheology is a widely applicable technique for the characterization of soft matter enabling the determination of rheological properties of delicate microstructures in a single sample during phase transitions in response to repeated changes in the surrounding environmental conditions.

We demonstrate the fabrication and use of a microfluidic device that enables multiple particle tracking microrheology measurements to study the rheological effects of repeated phase transitions on soft matter.

The microstructure of soft matter directly impacts macroscopic rheological properties and can be changed by factors including colloidal rearrangement ...

One-Step Approach to Fabricating Polydimethylsiloxane Microfluidic Channels of Different Geometric Sections by Sequential Wet Etching Processes

Polydimethylsiloxane (PDMS) materials are substantially exploited to fabricate microfluidic devices by using soft lithography replica molding techniques. Customized channel layout designs are necessary for specific functions and integrated performance of microfluidic devices in numerous biomedical and chemical applications (e.g., cell culture, biosensing, chemical synthesis, and liquid handling). Owing to the nature of molding approaches using silicon wafers with photoresist layers patterned by photolithography as master molds, the microfluidic channels commonly have regular cross sections of rectangular shapes with identical heights. Typically, channels with multiple heights or different geometric sections are designed to possess particular functions and to perform in various microfluidic applications (e.g., hydrophoresis is used for sorting particles and in continuous flows for separating blood cells6,7,8,9). Therefore, a great deal of effort has been made in constructing channels with various sections through multiple-step approaches like photolithography using several photoresist layers and assembly of different PDMS thin sheets. Nevertheless, such multiple-step approaches usually involve tedious procedures and extensive instrumentation. Furthermore, the fabricated devices may not perform consistently and the resulted experimental data may be unpredictable. Here, a one-step approach is developed for the straightforward fabrication of microfluidic channels with different geometric cross sections through PDMS sequential wet etching processes, that introduces etchant into channels of planned single-layer layouts embedded in PDMS materials. Compared to the existing methods for manufacturing PDMS microfluidic channels with different geometries, the developed one-step approach can significantly simplify the process to fabricate channels with non-rectangular sections or various heights. Consequently, the technique is a way of constructing complex microfluidic channels, which provides a fabrication solution for the advancement of innovative microfluidic systems.

Several methods are available for the fabrication of channels of non-rectangular sections embedded in polydimethylsiloxane microfluidic devices. Most of them involve multistep manufacturing and extensive alignment. In this paper, a one-step approach is reported for fabricating microfluidic channels of different geometric cross sections by polydimethylsiloxane sequential wet etching.

Polydimethylsiloxane (PDMS) materials are substantially exploited to fabricate microfluidic devices by using soft lithography replica molding techniques. ...

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. ...

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 ...

Polydimethylsiloxane-polycarbonate Microfluidic Devices for Cell Migration Studies Under Perpendicular Chemical and Oxygen Gradients

This paper reports a microfluidic device made of polydimethylsiloxane (PDMS) with an embedded polycarbonate (PC) thin film to study cell migration under combinations of chemical and oxygen gradients. Both chemical and oxygen gradients can greatly affect cell migration in vivo; however, due to technical limitations, very little research has been performed to investigate their effects in vitro. The device developed in this research takes advantage of a series of serpentine-shaped channels to generate the desired chemical gradients and exploits a spatially confined chemical reaction method for oxygen gradient generation. The directions of the chemical and oxygen gradients are perpendicular to each other to enable straightforward migration result interpretation. In order to efficiently generate the oxygen gradients with minimal chemical consumption, the embedded PC thin film is utilized as a gas diffusion barrier. The developed microfluidic device can be actuated by syringe pumps and placed into a conventional cell incubator during cell migration experiments to allow for setup simplification and optimized cell culture conditions. In cell experiments, we used the device to study migrations of adenocarcinomic human alveolar basal epithelial cells, A549, under combinations of chemokine (stromal cell-derived factor, SDF-1&#945;) and oxygen gradients. The experimental results show that the device can stably generate perpendicular chemokine and oxygen gradients and is compatible with cells. The migration study results indicate that oxygen gradients may play an essential role in guiding cell migration, and cellular behavior under combinations of gradients cannot be predicted from those under single gradients. The device provides a powerful and practical tool for researchers to study interactions between chemical and oxygen gradients in cell culture, which can promote better cell migration studies in more in vivo-like microenvironments.

The control of chemical and oxygen gradients is essential for cell cultures. This paper reports a polydimethylsiloxane-polycarbonate (PDMS-PC) microfluidic device capable of reliably generating combinations of chemical and oxygen gradients for cell migration studies, which can be practically utilized in biological labs without sophisticated instrumentation.

This paper reports a microfluidic device made of polydimethylsiloxane (PDMS) with an embedded polycarbonate (PC) thin film to study cell migration under ...