We gratefully acknowledge funding from DARPA sponsored Micro/Nano Fluidics Fundamentals Focus (MF3) Center under contract number N66001-10-1-4003, and from DARPA grant N660001-09-C-2082.

1. Physics of ITP
ITP forms a sharp moving boundary between ions of like charge. The technique can be performed with anionic or cationic samples, but we tailor this introduction to anionic ITP and note the same principles apply to cationic ITP. We choose LE and TE buffers such that LE ions have higher magnitude effective electrophoretic mobility. The effective electrophoretic mobility, &mu; = U/E, is the proportionality constant between applied electric field, E, and ion drift velocity, U.13 We establish a diffuse interface between the LE and TE and apply an electric field directed from the high-conductivity LE zone to the low-conductivity TE zone. The system quickly establishes a strong gradient in electric field at the ITP interface, due to the non-uniform conductivity profile. As per its name (from Greek, "isos" means "equal", "takhos" means "speed"), TE and LE ions travel at the same, uniform velocity, as a result of the non-uniform electric field and conservation of current (this is the so-called "ITP condition", see Figure 1).
The ITP interface is self-sharpening: LE ions that diffuse into the TE zone experience a strong restoring flux and return to the leading zone (and vice versa for TE ions in the LE zone). Sample ions focus at this interface if their effective mobility in the TE zone is greater than those of the TE co-ions, and if their effective mobility in the LE zone is less than that of the LE co-ions (see Figure 1). The self-sharpening and focusing properties of ITP contribute to the robustness of this technique and make ITP relatively insensitive to disturbances of the interface (e.g. due to pressure-driven flow or changes in geometry, such as contractions, expansions, and turns).
In peak mode ITP (see Figure 2a and Videos 1-2), sample ion concentrations are at all times significantly lower than LE and TE ion concentrations and therefore contribute negligibly to local conductivity. The distribution of sample ions is determined by the self-sharpening interface between neighboring zones (here the TE and LE) and the value of the sample effective mobility relative to these zones.14 Multiple sample ions focus within the same narrow ITP interface region as largely overlapping peaks. The interface and peak widths, as well as the associated preconcentration factor, scale inversely with the applied current (see experiments in Figure 2b).14
For sufficiently high initial sample concentrations and sufficient accumulation time, sample ions reach a threshold concentration value. For fully-ionized species, this value is determined by the Kohlrausch regulating function (KRF).15 For weak electrolytes, it is determined by the Alberty and Jovin functions.16,17 In plateau mode, as depicted in Figure 3 and Video 3, sample ions separate and purify into zones of locally uniform and constant concentration in an order determined by their effective mobility. Very dilute ions may still focus in peak mode between plateau zones. In ITP, sample ions can be introduced in a finite injection between the TE and LE (see Figure 3) or alternately mixed together with the TE and/or LE (see Figure 2). We refer to mixing in the TE zone as "semi-infinite" injection, which can be used to accumulate analyte ions continuously. Continuous sample accumulation increases sensitivity in both peak and plateau mode assays. However, finite injections are more common in plateau mode. This is likely because high initial analyte concentrations in semi-infinite injection can substantially increase TE conductivity and lower focusing rates. Also, full purification is not possible in semi-infinite injection (since there remains a finite concentration in TE).
2. Device cleaning and preparation
For the assays presented in this protocol we use isotropically wet-etched (roughly D-shaped cross section) glass microfluidic chips with a cross-channel design (see Figure 1). The following cleaning and preparation protocol is optimized for borosilicate and fused silica channels, but can also be used with glass/PDMS chips. Perform this cleaning procedure prior to experiments to ensure run-to-run repeatability and successful application of dynamic coatings needed to suppress electroosmotic flow (EOF). Omission of this protocol may result in strong dispersion of the ITP interface.14
<ol>
	<li>To decontaminate the channel, fill the North, East, and South reservoirs with 10-20 &mu;L of 10% bleach and apply vacuum at the West reservoir for 2 min. If using a standard Caliper chip caddy, vacuum can be effectively applied by simply attaching the wide end of a 200 &mu;L (unfiltered) pipette tip to the chip reservoir and connecting the vacuum line to a 2 mm inner diameter tube.</li>
	<li>Empty the reservoirs and rinse the channel (as in step 1) with 1 M sodium hydroxide for 2 min. This gently etches the channel walls, yielding a clean borosilicate surface to help establish uniform surface properties.</li>
	<li>Empty the reservoirs and clean with de-ionized water (DI), then rinse the channel with LE for ~2 min. During this period surface properties and dynamic coatings equilibrate within the channel.</li>
</ol>
3. Fluorophore focusing in peak mode ITP
<ol>
	<li>Prepare 1 mL LE consisting of 100 mM HCl, 200 mM tris, and 1% PVP.</li>
	<li>Prepare 1 mL TE consisting of 100 mM HEPES and 200 mM tris. Combine 90 &mu;l of TE with 10 &mu;l of 1 &mu;M Alexa Fluor 488 (AF488).</li>
	<li>After rinsing with LE as described in Part 2, empty the West reservoir and clean a few times with DI in order to dilute any LE remaining in the reservoir. Fill this reservoir with 20 &mu;L TE containing AF488.</li>
	<li>Place the positive electrode in the East reservoir and the ground (negative) electrode in the West reservoir and apply 2 &mu;A (constant current). The sample peak will migrate at a constant velocity from the West reservoir to the East reservoir (see Figure 1) and the voltage between these reservoirs will increase as the lower conductivity TE fills the channel.</li>
</ol>
4. Extraction and purification of nucleic acids from cultured E. coli
The ability to selectively focus ionic species makes ITP an ideal technique for biological sample preparation. We purify nucleic acids from untreated cell lysate by selecting a trailing anion with an effective mobility magnitude lower than the target nucleic acid but higher than co-ionic PCR inhibitors (e.g. anionic detergents, proteins, and organic solvents, even if present in high concentration). Cationic PCR inhibitors (e.g. alkali metals and cationic proteins and detergents) migrate in the opposite direction and so are also left behind. ITP extracts and focuses target nucleic acids from the sample reservoir, while leaving slower PCR-inhibiting species behind (see Figure 4).
<ol>
	<li>Obtain a sample of or culture E. coli cells to a density greater than 108 CFU/mL.</li>
	<li>Transfer 1 mL cell culture into a safe-lock microcentrifuge tube and pellet by centrifugation at 4000g for 6 min.</li>
	<li>Re-suspend the pellet in 80 &mu;L RNase-free water and add 10 &mu;L of lysing agent consisting of 10 mM tricine, 10 mM bis-tris, 2 mM EDTA, 0.1 % Triton-X, and 5 mg/mL lysozyme. Mix gently and incubate for 5 mins. at room temperature (lysing protocol adapted from Bercovici et al.11).</li>
	<li>Add 10 &mu;L of 1 M sodium hydroxide to raise the lysate pH to ~12.5. Gently actuate the pipette up and down until the solution becomes clear, at which point lysing is complete.</li>
	<li>Combine 10 &mu;L of lysate with 90 &mu;L of 50 mM tricine and 100 mM bis-tris. This solution can now be used as the TE.</li>
	<li>Prepare 1 mL of LE consisting of 500 mM bis-tris, 250 mM HCl, 1% PVP, and 1X SYBR Green II. Fill the microfluidic chip with LE as described in Part 3.</li>
	<li>In order to extract the purified nucleic acid for off-chip analysis following ITP, replace the contents of the East reservoir with a PCR-compatible LE containing 50mM bis-tris, 25 mM HCl, and 0.1% PVP. Apply 1000 V between the East and West wells to begin the experiment. The current between these reservoirs will decrease.</li>
	<li>At the end of the experiment the sample elutes into the LE reservoir. Coincident with this elution, the current versus time for this system typically reaches a plateau value (since resistance is now dominated by TE ions uniformly distributed within channel). Gently mix the reservoir contents by repeated pipetting and extract a 5 &mu;L volume for analysis by quantitative RT-PCR.</li>
</ol>
5. Separation of amino acids with cationic plateau mode ITP and non-focusing tracer (NFT) for visualization
ITP can be used to separate and focus small ions into adjoining and detectable plateaus between the TE and LE. This allows detection and identification based on physicochemical properties, such as local conductivity, UV absorbance, temperature sensing, or index of refraction. Here we demonstrate a non-focusing tracer (NFT) assay where a fluorescent, co-ionic species is added to the LE. This fluorescent species does not focus, but its concentration adapts to a local electric field and thereby enables visualization of purified plateau zones (see Figure 5).
<ol>
	<li>Prepare 1 mL LE consisting of 100 mM ethanolamine, 200 mM tricine, and 1% PVP, and 100 &mu;M of the cationic fluorophore Rhodamine 6G.</li>
	<li>Prepare 1 mL TE consisting of 20 mM tris, 40 mM tricine. Prepare sample by mixing 90 &mu;L of TE with 10 &mu;L each of 50 mM arginine and 50 mM lysine.</li>
	<li>Dispense 20 &mu;L LE in the West and North reservoirs and sample in the East reservoir. Apply vacuum at South reservoir for 1 min.</li>
	<li>Rinse the East well with DI and replace with TE (TE without sample).</li>
	<li>Apply 500 V between the East and West reservoirs.</li>
</ol>
6. Representative Results
We show isotachopherograms of peak mode experiments in Figure 2b and nucleic acid extraction experiments in Figure 4. In peak mode experiments with a fluorescent reporter (e.g. AF488, SYBR Green II), the overall fluorescence intensity can be integrated and compared against a calibration curve to obtain quantitative concentration information.12 Additionally, in nucleic acid extraction experiments, sample is allowed to elute into the LE reservoir and extracted with a pipette for analysis by quantitative RT-PCR.10,12 We show plateau mode isotachopherograms for amino acid separation in Figure 5. Fluorescence intensity (relative to LE or TE zone intensity) can be used for zone identification, while zone widths enable quantitation.
<img alt="Figure 1." src="/files/ftp_upload/3890/3890fig1.jpg" /> 
Figure 1. Isotachophoresis (ITP) is an electrokinetic technique used in microfluidic applications for sensitive detection and separation of ions. ITP offers separation, selective focusing, and preconcentration capabilities. In addition, it is extremely robust and insensitive to physical disturbances because of its self-sharpening nature. Sample ions selectively focus between a leading (LE) and trailing electrolyte (TE) and travel through the microchannel at a constant speed determined by the speed of the leading ions in the LE zone. Sample ions focus if their effective electrophoretic mobility is bracketed by the effective mobility of the LE and TE ions. The schematic depicts a model ITP experiment where sample focuses continuously between the LE and TE zones.
<img alt="Figure 2." src="/files/ftp_upload/3890/3890fig2.jpg" /> 
Figure 2. Dilute sample ions focus in "peak mode" ITP. a) Two distinct dilute (csample &lt;&lt; cLE) species focus in peak mode at the interface formed between the LE and TE zones. Solely the LE and TE determine the electric field, as sample species do not significantly contribute to current. Sample ions are mixed with TE (in a semi-infinite injection) and focus together in a roughly Gaussian peak at the ITP interface. Focusing criterion (shown as inequalities) applies to strongly ionized species. b) Experiments showing Alexa Fluor 488 (AF488) focused in peak mode for a range of applied currents (see also Video 1). Sample peak width is inversely proportional to current (for negligible advective dispersion due to electroosmotic flow).14 The self-sharpening interface is resistant to dispersion due to pressure-driven flow (see Video 2).
<img alt="Figure 3." src="/files/ftp_upload/3890/3890fig3.jpg" /> 
Figure 3. Sample ions at a sufficiently high concentration focus in "plateau mode" and govern local conductivity. We typically introduce sample ions in a finite injection between the TE and LE. In this modality, sample ions separate and order according to their effective electrophoretic mobility (see Video 3). For strongly ionized species, the TE and plateau zone concentrations are determined by the Kohlrausch regulating function (KRF, an invariant set initially by the LE zone). Dilute ions continue to focus in peak mode between plateau zones bracketing their effective mobility. Focusing criterion (shown as inequalities) applies to strongly ionized species.
<img alt="Figure 4." src="/files/ftp_upload/3890/3890fig4.jpg" /> 
Figure 4. Lysate preparation and nucleic acid extraction with peak mode ITP. Nucleic acid is extracted from E. coli cell culture using lysozyme-assisted alkaline lysing and purified by selective electrophoretic focusing via ITP. TE mixed with lysate (brown) contains target nucleic acid (green), proteins, and potential PCR-inhibiting chemistries. Appropriate selection of trailing and leading ions enables selective focusing of target nucleic acid while leaving PCR inhibitors behind. Total nucleic acid ITP peak often takes on a non-ideal shape, as shown here in the inset image. As a demonstration of this assay, we extracted total nucleic acid from gram-negative bacteria, Escherichia coli (lysed with sodium hydroxide solution alone), purified NA from lysate using ITP, collected the extracted genetic material, and performed qRT-PCR analyses to verify successful purification of 16S rRNA (red) and 16S rDNA (green) from bacterial culture. Negative control threshold cycles for 16S rRNA (blue) and 16S rDNA (yellow) were each above 30 cycles. We performed qRT-PCR using Power SYBR Green RNA-to-CT 1-Step Kit from Applied Biosystems with 150 nM forward (5'-CGGATTGGAGTCTGCAACTCG) and reverse (5'-CACAAAGTGGTAAG CGCCCTC) primers, at the recommended thermal cycling conditions.
<img alt="Figure 5." src="/files/ftp_upload/3890/3890fig5.jpg" /> 
Figure 5. Non-focusing tracer (NFT) assay for separation and detection of unlabeled amino acids. a) Schematic of the NFT assay. A finite injection of sample ions is introduced into the East channel. We mix the LE with a tracer cationic fluorophore with effective mobility lower than the TE ions (&mu;tracer &lt; &mu;TE). The fluorophore is said to act as an "underspeeding tracer". As the fluorophore electromigrates from the LE into zones now occupied by sample plateaus and TE, it experiences a higher electric field and thus its concentration increases. This change in concentration creates steps in the isotachopherogram. b) Separation of two amino acids, arginine and lysine, using the NFT assay with Rhodamine 6G as the underspeeding fluorescent tracer. The slight peak between TE and arginine is common in ITP, is not well understood, and here does not interfere with detecting or quantifying the plateaus.
Supplemental Movie 1. <a href="https://www.jove.com/files/ftp_upload/3890/3890movie1.mpg" target="_blank">Click here to view the supplemental movie</a>.
Supplemental Movie 2. <a href="https://www.jove.com/files/ftp_upload/3890/3890movie2.mpg" target="_blank">Click here to view the supplemental movie</a>.
Supplemental Movie 3. <a href="https://www.jove.com/files/ftp_upload/3890/3890movie3.mpg" target="_blank">Click here to view the supplemental movie</a>.

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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isotachophoresis:+Theory,+instrumentation,+and+applications.">Isotachophoresis: Theory, instrumentation, and applications.</a> Elsevier Science &#38; Technology. , (1976).</li><li>Khurana, T. K., Santiago, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preconcentration,+separation,+and+indirect+detection+of+nonfluorescent+analytes+using+fluorescent+mobility+markers.">Preconcentration, separation, and indirect detection of nonfluorescent analytes using fluorescent mobility markers.</a> Anal. Chem. 80 (1), 279-279 (2008).</li><li>Bercovici, M., Kaigala, G. V., Backhouse, C. J., Santiago, J. 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We have nothing to disclose.

The ITP methods presented here enable rapid, sensitive, and robust detection and handling of ionic molecules. The main challenge in using ITP is proper choice of LE and TE buffers. In anionic ITP, we typically choose chloride as the leading anion because it is a strong acid with very high absolute mobility and thus has very predictable properties. Therefore, buffer choice in anionic ITP is typically reduced to choosing a proper TE and counterion. For selective focusing experiments, TE choice is critical to exclusion of contaminating ions. In applications where contaminants are not present, lower TE effective mobility leads to faster rate of focusing. We recommend using the following, relatively well-behaved anionic TE ions: MES, MOPS, HEPES, tricine. Choice of counterion affects both the system pH and TE effective mobility. Common counterions are tris (pKa 8.1) and bis-tris (pKa 6.4). For assays requiring lower or higher pH, we recommend pyridine (pKa 5.25) and ethanolamine (pKa 9.5), respectively. In Tables 1 and 2, for anionic and cationic ITP, respectively, we summarize several useful buffer examples. We take HCl as the anionic LE ion and sodium as the cationic LE ion, and we assume 100 mM ionic strength of the LE buffer.
 The reader will note that buffer ionic strength in our protocols (and in Tables 1&#x2013;2) is always greater than or equal to 10 mM. While in theory the physics of ITP applies at ionic strength lower than 10 mM, natural contaminants (e.g. carbonic acid from reaction between water and atmospheric carbon dioxide) near the 1 mM level often limit the practical use of low ionic strength buffers. 
 We note that signal transduction mechanism is not limited to the assays presented in this protocol. As discussed briefly in Part 5, in plateau mode purified zones can be detected via changes in local conductivity, UV absorbance, temperature, or index of refraction. In our own group, we have developed a method which utilizes fluorescent carrier ampholytes for sensitive detection and identification of unknown analytes.5 In peak mode experiments, specific probes can be used to label focused analytes. In one example, we use molecular beacons &#x2013; oligonucleotide probes that fluoresce upon hybridization to their target sequence &#x2013; to detect target DNA or RNA molecules with high specificity.11,18
While ITP is relatively robust to dispersion caused by pressure-driven flow and EOF, excessive EOF can lead to stagnation of the ITP interface, where the mean EOF velocity becomes equal to the ITP velocity.14 Such strong EOF occurs especially in conditions of high pH and low ionic strength. For this reason, we recommend the use of buffers with pH 8 or lower and with ionic strength on the order of 100 mM if convenient. In our experience, PVP is the most effective coating for EOF suppression in borosilicate chips. However, because of its sieving properties, PVP may not be appropriate for some applications, such as focusing genomic DNA. In experiments, we observe that addition of PVP can greatly reduce genomic DNA absolute mobility (to the point where it does not focus). In these cases a silanol coating such as Sigmacote in conjuction with a surfactant (e.g. Triton-X 100) can also be effective in reducing EOF.10
<table border="1">
<tbody>
<tr>
 <td>&nbsp;</td>
 <td colspan="4">TE anion (pKa -1)</td>
</tr>
<tr>
 <td>Buffering counterion (pKa +1)</td>
 <td>MES (6.10)</td>
 <td>MOPS (7.20)</td>
 <td>HEPES (7.50)</td>
 <td>tricine (8.15)</td>
</tr>
<tr>
 <td>ethanolamine (9.50)</td>
 <td>21.41</td>
 <td>20.40</td>
 <td>17.38</td>
 <td>22.85</td>
</tr>
<tr>
 <td>tris (8.08)</td>
 <td>21.00</td>
 <td>19.23</td>
 <td>15.84</td>
 <td>17.56</td>
</tr>
<tr>
 <td>bis-tris (6.40)</td>
 <td>18.22</td>
 <td>10.41</td>
 <td>7.30</td>
 <td>5.23</td>
</tr>
<tr>
 <td>pyridine (5.18)</td>
 <td>1.01</td>
 <td>3.72</td>
 <td>2.42</td>
 <td>1.48</td>
</tr>
</tbody>
 </table> 
Table 1. Effective mobility magnitude (&times; 10-9 m2/V/s) of adjusted pure analyte zone in anionic ITP where the LE is 100 mM HCl and 200 mM buffering counterion. 
<table border="1"> 
<tbody>
<tr>
 <td>&nbsp;</td>
 <td colspan="4">TE cation (pKa +1)</td>
</tr>
<tr>
 <td>Buffering counterion (pKa&nbsp;-1)</td>
 <td>ethanolamine (9.50)</td>
 <td>tris (8.08)</td>
 <td>bis-tris (6.40)</td>
 <td>pyridine (5.18)</td>
 </tr>
<tr>
 <td>MES (6.10)</td>
 <td>35.57</td>
 <td>21.96</td>
 <td>16.39</td>
 <td>10.45</td>
</tr>
<tr>
 <td>MOPS (7.20)</td>
 <td>35.47</td>
 <td>20.92</td>
 <td>9.76</td>
 <td>3.93</td>
</tr>
<tr>
 <td>HEPES (7.50)</td>
 <td>35.35</td>
 <td>19.97</td>
 <td>7.77</td>
 <td>2.88</td>
</tr>
<tr>
 <td>Tricine (8.15)</td>
 <td>34.77</td>
 <td>16.72</td>
 <td>4.50</td>
 <td>1.44</td>
</tr>
 </tbody>
</table>
Table 2. Effective mobility magnitude (&times; 10-9 m2/V/s) of adjusted pure analyte zone in cationic ITP where the LE is 100 mM sodium and 200 mM buffering counterion.

<table border="1">
<tbody>
<tr>
 <td colspan="4">REAGENTS</td>
</tr>
<tr>
 <td>Name</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Comments</td>
</tr>
<tr>
 <td>Distilled water</td>
 <td>GIBCO</td>
 <td>10977</td>
 <td>RNase/DNase free</td>
</tr>
<tr>
 <td>Clorox Ultra</td>
 <td>Clorox</td>
 <td>02489CT</td>
 <td></td>
</tr>
<tr>
 <td>sodium hydroxide (NaOH)</td>
 <td>Mallinckrodt</td>
 <td>7708</td>
 <td></td>
</tr>
<tr>
 <td>hydrochloric acid (HCl)</td>
 <td>EMD Chemicals</td>
 <td>HX0603-4</td>
 <td></td>
</tr>
<tr>
 <td>Trizma base (tris)</td>
 <td>Sigma-Aldrich</td>
 <td>T6066</td>
 <td></td>
</tr>
<tr>
 <td>polyvinylpyrrolidone (PVP)</td>
 <td>Polysciences Inc.</td>
 <td>06067</td>
 <td>MW 1,000,000</td>
</tr>
<tr>
 <td>HEPES</td>
 <td>Sigma-Aldrich</td>
 <td>H-4034</td>
 <td></td>
</tr>
<tr>
 <td>Alexa Fluor 488 carboxylic acid</td>
 <td>Invitrogen</td>
 <td>A20000</td>
 <td></td>
</tr>
<tr>
 <td>tricine</td>
 <td>Sigma-Aldrich</td>
 <td>T-9784</td>
 <td></td>
</tr>
<tr>
 <td>bis-tris</td>
 <td>Sigma-Aldrich</td>
 <td>B4429</td>
 <td></td>
</tr>
<tr>
 <td>EDTA</td>
 <td>GIBCO</td>
 <td>AM9260G</td>
 <td></td>
</tr>
<tr>
 <td>Triton-X 100</td>
 <td>Sigma-Aldrich</td>
 <td>X100</td>
 <td></td>
</tr>
<tr>
 <td>lysozyme</td>
 <td>Sigma-Aldrich</td>
 <td>L6876</td>
 <td></td>
</tr>
<tr>
 <td>SYBR Green II</td>
 <td>Invitrogen</td>
 <td>S7564</td>
 <td></td>
</tr>
<tr>
 <td>ethanolamine</td>
 <td>Sigma-Aldrich</td>
 <td>411000</td>
 <td></td>
</tr>
<tr>
 <td>Rhodamine 6G</td>
 <td>Acros Organics (Geel, Belgium) </td>
 <td>CAS 989-38-8</td>
 <td></td>
</tr>
<tr>
 <td>L-Amino Acids</td>
 <td>Sigma-Aldrich</td>
 <td>LAA21</td>
 <td></td>
</tr>
<tr>
 <td>Power SYBR Green RNA-to-CT 1-Step Kit</td>
 <td>Applied Biosystems</td>
 <td>4389986</td>
 <td></td>
</tr>
<tr>
 <td>PCR primers</td>
 <td>Integrated DNA Technologies</td>
 <td></td>
 <td></td>
</tr>
<tr>
 <td colspan="4">EQUIPMENT</td>
</tr>
<tr>
 <td>Name</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Comments</td>
</tr>
<tr>
 <td>Borosilicate microfluidic chip</td>
 <td>Caliper Life Sciences Inc.</td>
 <td>NS12A</td>
 <td>Supplied with or without plastic caddy</td>
</tr>
<tr>
 <td>Vacuum pump</td>
 <td>Gast</td>
 <td>DOA-P104-AA</td>
 <td></td>
</tr>
<tr>
 <td>Sourcemeter</td>
 <td>Keithley </td>
 <td>2410 </td>
 <td>Constant current and constant voltage operation modes</td>
</tr>
<tr>
 <td>Inverted epifluorescent microscope</td>
 <td>Olympus</td>
 <td>IX70</td>
 <td>Use mercury lamp (Olympus) or LED (Thorlabs) for illumination</td>
</tr>
<tr>
 <td>Filter cube</td>
 <td>Omega</td>
 <td>XF115-2</td>
 <td>Excitation/emission: blue/green</td>
</tr>
<tr>
 <td>Safe-lock microcentrifuge tubes</td>
 <td>Eppendorf</td>
 <td>022363204</td>
 <td>1.5&nbsp;mL capacity</td>
</tr>
<tr>
 <td>Centrifuge</td>
 <td>Eppendorf</td>
 <td>5417C</td>
 <td></td>
</tr>
 </tbody>
</table>
Table 3. Specific reagents and equipment.

on-chip isotachophoresis for separation of ions and purification of nucleic acids

Electrokinetic techniques are a staple of microscale applications because of their unique ability to perform a variety of fluidic and electrophoretic processes in simple, compact systems with no moving parts. Isotachophoresis (ITP) is a simple and very robust electrokinetic technique that can achieve million-fold preconcentration1,2 and efficient separation and extraction based on ionic mobility.3 For example, we have demonstrated the application of ITP to separation and sensitive detection of unlabeled ionic molecules (e.g. toxins, DNA, rRNA, miRNA) with little or no sample preparation4-8 and to extraction and purification of nucleic acids from complex matrices including cell culture, urine, and blood.9-12
ITP achieves focusing and separation using an applied electric field and two buffers within a fluidic channel system. For anionic analytes, the leading electrolyte (LE) buffer is chosen such that its anions have higher effective electrophoretic mobility than the anions of the trailing electrolyte (TE) buffer (Effective mobility describes the observable drift velocity of an ion and takes into account the ionization state of the ion, as described in detail by Persat et al.13). After establishing an interface between the TE and LE, an electric field is applied such that LE ions move away from the region occupied by TE ions. Sample ions of intermediate effective mobility race ahead of TE ions but cannot overtake LE ions, and so they focus at the LE-TE interface (hereafter called the "ITP interface"). Further, the TE and LE form regions of respectively low and high conductivity, which establish a steep electric field gradient at the ITP interface. This field gradient preconcentrates sample species as they focus. Proper choice of TE and LE results in focusing and purification of target species from other non-focused species and, eventually, separation and segregation of sample species.
We here review the physical principles underlying ITP and discuss two standard modes of operation: "peak" and "plateau" modes. In peak mode, relatively dilute sample ions focus together within overlapping narrow peaks at the ITP interface. In plateau mode, more abundant sample ions reach a steady-state concentration and segregate into adjoining plateau-like zones ordered by their effective mobility. Peak and plateau modes arise out of the same underlying physics, but represent distinct regimes differentiated by the initial analyte concentration and/or the amount of time allotted for sample accumulation.
We first describe in detail a model peak mode experiment and then demonstrate a peak mode assay for the extraction of nucleic acids from E. coli cell culture. We conclude by presenting a plateau mode assay, where we use a non-focusing tracer (NFT) species to visualize the separation and perform quantitation of amino acids.

Watch this Scientific Journal Video about On-chip Isotachophoresis for Separation of Ions and Purification of Nucleic Acids at JoVE.com

On-chip Isotachophoresis for Separation of Ions and Purification of Nucleic Acids

Isotachophoresis (ITP) is a robust electrokinetic separation and preconcentration technique with applications ranging from toxin detection to sample preparation. We review the physical principles of ITP and the methodology of applying this technique to two specific example applications: separation and detection of small molecules and purification of nucleic acids from cell culture lysate.

Electrokinetic techniques are a staple of microscale applications because of their unique ability to perform a variety of fluidic and electrophoretic ...

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24.2K Views.  Stanford University. Isotachophoresis (ITP) is a robust electrokinetic separation and preconcentration technique with applications ranging from toxin detection to sample preparation. We review the physical principles of ITP and the methodology of applying this technique to two specific example applications: separation and detection of small molecules and purification of nucleic acids from cell culture lysate.

Video: On-chip Isotachophoresis for Separation of Ions and Purification of Nucleic Acids

Fabrication of Electrochemical-DNA Biosensors for the Reagentless Detection of Nucleic Acids, Proteins and Small Molecules

As medicine is currently practiced, doctors send specimens to a central laboratory for testing and thus must wait hours or days to 
receive the results. Many patients would be better served by rapid, bedside tests. To this end our laboratory and others have developed a versatile, reagentless 
biosensor platform that supports the quantitative, reagentless, electrochemical detection of nucleic acids (DNA, RNA), proteins (including antibodies) and small 
molecules analytes directly in unprocessed clinical and environmental samples. In this video, we demonstrate the preparation and use of several biosensors in this 
"E-DNA" class. In particular, we fabricate and demonstrate sensors for the detection of a target DNA sequence in a polymerase chain reaction mixture, an HIV-specific antibody and the drug cocaine. The preparation procedure requires only three hours of hands-on effort followed by an overnight incubation, and their use requires only minutes.

"E-DNA" sensors, reagentless, electrochemical biosensors that perform well even when challenged directly in blood and other complex matrices, have been adapted to the detection of a wide range of nucleic acid, protein and small molecule analytes. Here we present a general procedure for the fabrication and use of such sensors.

As medicine is currently practiced, doctors send specimens to a central laboratory for testing and thus must wait hours or days to 
receive the results. ...

Lensfree On-chip Tomographic Microscopy Employing Multi-angle Illumination and Pixel Super-resolution

Tomographic imaging has been a widely used tool in medicine as it can provide three-dimensional (3D) structural information regarding objects of different size scales. In micrometer and millimeter scales, optical microscopy modalities find increasing use owing to the non-ionizing nature of visible light, and the availability of a rich set of illumination sources (such as lasers and light-emitting-diodes) and detection elements (such as large format CCD and CMOS detector-arrays). Among the recently developed optical tomographic microscopy modalities, one can include optical coherence tomography, optical diffraction tomography, optical projection tomography and light-sheet microscopy. 1-6 These platforms provide sectional imaging of cells, microorganisms and model animals such as C. elegans, zebrafish and mouse embryos.
Existing 3D optical imagers generally have relatively bulky and complex architectures, limiting the availability of these equipments to advanced laboratories, and impeding their integration with lab-on-a-chip platforms and microfluidic chips. To provide an alternative tomographic microscope, we recently developed lensfree optical tomography (LOT) as a high-throughput, compact and cost-effective optical tomography modality. 7 LOT discards the use of lenses and bulky optical components, and instead relies on multi-angle illumination and digital computation to achieve depth-resolved imaging of micro-objects over a large imaging volume. LOT can image biological specimen at a spatial resolution of &lt;1 &mu;m x &lt;1 &mu;m x &lt;3 &mu;m in the x, y and z dimensions, respectively, over a large imaging volume of 15-100 mm3, and can be particularly useful for lab-on-a-chip platforms.

Lensfree optical tomography is a three-dimensional microscopy technique that offers a spatial resolution of &lt;1 &mu;m &times; &lt;1 &mu;m &times; &lt;3 &mu;m in x, y and z dimensions, respectively, over a large imaging-volume of 15-100 mm3, which can be particularly useful for integration with lab-on-a-chip platforms.

Tomographic imaging has been a widely used tool in medicine as it can provide three-dimensional (3D) structural information regarding objects of different ...

On-Chip Endothelial Inflammatory Phenotyping

Atherogenesis is potentiated by metabolic abnormalities that contribute to a heightened state of systemic inflammation resulting in endothelial dysfunction. However, early functional changes in endothelium that signify an individual's level of risk are not directly assessed clinically to help guide therapeutic strategy. Moreover, the regulation of inflammation by local hemodynamics contributes to the non-random spatial distribution of atherosclerosis, but the mechanisms are difficult to delineate in vivo. We describe a lab-on-a-chip based approach to quantitatively assay metabolic perturbation of inflammatory events in human endothelial cells (EC) and monocytes under precise flow conditions. Standard methods of soft lithography are used to microfabricate vascular mimetic microfluidic chambers (VMMC), which are bound directly to cultured EC monolayers.1 These devices have the advantage of using small volumes of reagents while providing a platform for directly imaging the inflammatory events at the membrane of EC exposed to a well-defined shear field. We have successfully applied these devices to investigate cytokine-,2 lipid-3, 4 and RAGE-induced5 inflammation in human aortic EC (HAEC). Here we document the use of the VMMC to assay monocytic cell (THP-1) rolling and arrest on HAEC monolayers that are conditioned under differential shear characteristics and activated by the inflammatory cytokine TNF-&alpha;. Studies such as these are providing mechanistic insight into atherosusceptibility under metabolic risk factors.

Microfluidic flow chambers etched by photolithography and fabricated from PDMS are applied to probe functional outcomes associated with EC dysfunction and inflammation. In a representative experiment, the ability of differential shear stress to modulate monocytic cell adhesion to cytokine activated EC monolayers is demonstrated.

Atherogenesis is potentiated by metabolic abnormalities that contribute to a heightened state of systemic inflammation resulting in endothelial ...

Dry Film Photoresist-based Electrochemical Microfluidic Biosensor Platform: Device Fabrication, On-chip Assay Preparation, and System Operation

In recent years, biomarker diagnostics became an indispensable tool for the diagnosis of human disease, especially for the point-of-care diagnostics. An easy-to-use and low-cost sensor platform is highly desired to measure various types of analytes (e.g., biomarkers, hormones, and drugs) quantitatively and specifically. For this reason, dry film photoresist technology - enabling cheap, facile, and high-throughput fabrication - was used to manufacture the microfluidic biosensor presented here. Depending on the bioassay used afterwards, the versatile platform is capable of detecting various types of biomolecules. For the fabrication of the device, platinum electrodes are structured on a flexible polyimide (PI) foil in the only clean-room process step. The PI foil serves as a substrate for the electrodes, which are insulated with an epoxy-based photoresist. The microfluidic channel is subsequently generated by the development and lamination of dry film photoresist (DFR) foils onto the PI wafer. By using a hydrophobic stopping barrier in the channel, the channel is separated into two specific areas: an immobilization section for the enzyme-linked assay and an electrochemical measurement cell for the amperometric signal readout.
The on-chip bioassay immobilization is performed by the adsorption of the biomolecules to the channel surface. The glucose oxidase enzyme is used as a transducer for electrochemical signal generation. In the presence of the substrate, glucose, hydrogen peroxide is produced, which is detected at the platinum working electrode. The stop-flow technique is applied to obtain signal amplification along with rapid detection. Different biomolecules can quantitatively be measured by means of the introduced microfluidic system, giving an indication of different types of diseases, or, in regard to therapeutic drug monitoring, facilitating a personalized therapy.

A microfluidic biosensor platform was designed and fabricated using low-cost dry film photoresist technology for the rapid and sensitive quantification of various analytes. This single-use system allows for the electrochemical readout of on-chip-immobilized enzyme-linked assays by means of the stop-flow technique.

In recent years, biomarker diagnostics became an indispensable tool for the diagnosis of human disease, especially for the point-of-care diagnostics. An ...

Fabrication and Validation of an Organ-on-chip System with Integrated Electrodes to Directly Quantify Transendothelial Electrical Resistance

Organs-on-chips, in vitro models involving the culture of (human) tissues inside microfluidic devices, are rapidly emerging and promise to provide useful research tools for studying human health and disease. To characterize the barrier function of cell layers cultured inside organ-on-chip devices, often transendothelial or transepithelial electrical resistance (TEER) is measured. To this end, electrodes are usually integrated into the chip by micromachining methods to provide more stable measurements than is achieved with manual insertion of electrodes into the inlets of the chip. However, these electrodes frequently hamper visual inspection of the studied cell layer or require expensive cleanroom processes for fabrication. To overcome these limitations, the device described here contains four easily integrated electrodes that are placed and fixed outside of the culture area, making visual inspection possible. Using these four electrodes the resistance of six measurement paths can be quantified, from which the TEER can be directly isolated, independent of the resistance of culture medium-filled microchannels. The blood-brain barrier was replicated in this device and its TEER was monitored to show the device applicability. This chip, the integrated electrodes and the TEER determination method are generally applicable in organs-on-chips, both to mimic other organs or to be incorporated into existing organ-on-chip systems.

This publication describes the fabrication of an organ-on-chip device with integrated electrodes for direct quantification of transendothelial electrical resistance (TEER). For validation, the blood-brain barrier was mimicked inside this microfluidic device and its barrier function was monitored. The presented methods for electrode integration and direct TEER quantification are generally applicable.

Organs-on-chips, in vitro models involving the culture of (human) tissues inside microfluidic devices, are rapidly emerging and promise to provide useful ...

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 ...

Preparation, Purification, and Use of Fatty Acid-containing Liposomes

Liposomes containing single-chain amphiphiles, particularly fatty acids, exhibit distinct properties compared to those containing diacylphospholipids due to the unique chemical properties of these amphiphiles. In particular, fatty acid liposomes enhance dynamic character, due to the relatively high solubility of single-chain amphiphiles. Similarly, liposomes containing free fatty acids are more sensitive to salt and divalent cations, due to the strong interactions between the carboxylic acid head groups and metal ions. Here we illustrate techniques for preparation, purification, and use of liposomes comprised in part or whole of single chain amphiphiles (e.g., oleic acids).

Liposomes containing single-chain amphiphiles, particularly fatty acids, exhibit distinct properties compared to those containing diacylphospholipids due to the unique chemical properties of single chain amphiphiles. Here we describe techniques for the preparation, purification, and use of liposomes comprised in part or whole of these amphiphiles.

Liposomes containing single-chain amphiphiles, particularly fatty acids, exhibit distinct properties compared to those containing diacylphospholipids due ...

Simultaneous Quantification of Selected Kynurenines Analyzed by Liquid Chromatography-Mass Spectrometry in Medium Collected from Cancer Cell Cultures

The kynurenine pathway and the tryptophan catabolites called kynurenines have received increased attention for their involvement in immune regulation and cancer biology. An in vitro cell culture assay is often used to learn about the contribution of different tryptophan catabolites in a disease mechanism and for testing therapeutic strategies. Cell culture medium that is rich in secreted metabolites and signaling molecules reflects the status of tryptophan metabolism and other cellular events. New protocols for the reliable quantification of multiple kynurenines in the complex cell culture medium are desired to allow for a reliable and quick analysis of multiple samples. This can be accomplished with liquid chromatography coupled with mass spectrometry. This powerful technique is employed in many clinical and research laboratories for the quantification of metabolites and can be used for measuring kynurenines.
Presented here is the use of liquid chromatography coupled with single quadrupole mass spectrometry (LC-SQ) for the simultaneous determination of four kynurenines, i.e., kynurenine, 3-hydroxykynurenine, 3-hydroxyanthranilic, and xanthurenic acid in the medium collected from in vitro cultured cancer cells. SQ detector is simple to use and less expensive compared to other mass spectrometers. In the SQ-MS analysis, multiple ions from the sample are generated and separated according to their specific mass-to-charge ratio (m/z), followed by the detection using a Single Ion Monitoring (SIM) mode.
This paper draws the attention on the advantages of the reported method and indicates some weak points. It is focused on critical elements of LC-SQ analysis including sample preparation along with chromatography and mass spectrometry analysis. The quality control, method calibration conditions and matrix effect issues are also discussed. We described a simple application of 3-nitrotyrosine as one analog standard for all target analytes. As confirmed by experiments with human ovary and breast cancer cells, the proposed LC-SQ method&#160;generates reliable results and can be further applied to other in vitro cellular models.

Described here is a protocol for the determination of four different tryptophan metabolites generated in the kynurenine pathway (kynurenine, 3-hydroxykynurenine, xanthurenic acid, 3-hydroxyanthranilic acid) in the medium collected from cancer cell cultures analyzed by liquid chromatography coupled with a single quadrupole mass spectrometry.

The kynurenine pathway and the tryptophan catabolites called kynurenines have received increased attention for their involvement in immune regulation and ...

Self-Assembly of Gamma-Modified Peptide Nucleic Acids into Complex Nanostructures in Organic Solvent Mixtures

Current strategies in DNA and RNA nanotechnology enable the self-assembly of a variety of nucleic acid nanostructures in aqueous or substantially hydrated media. In this article, we describe detailed protocols that enable the construction of nanofiber architectures in organic solvent mixtures through the self-assembly of uniquely addressable, single-stranded, gamma-modified peptide nucleic acid (&#947;PNA) tiles. Each single-stranded tile (SST) is a 12-base &#947;PNA oligomer composed of two concatenated modular domains of 6 bases each. Each domain can bind to a mutually complimentary domain present on neighboring strands using programmed complementarity to form nanofibers that can grow to microns in length. The SST motif is made of 9 total oligomers to enable the formation of 3-helix nanofibers. In contrast with analogous DNA nanostructures, which form diameter-monodisperse structures, these &#947;PNA systems form nanofibers that bundle along their widths during self-assembly in organic solvent mixtures. Self-assembly protocols described here therefore also include a conventional surfactant, Sodium Dodecyl Sulfate (SDS), to reduce bundling effects.

This article provides protocols for the design and self-assembly of nanostructures from gamma-modified peptide nucleic acid oligomers in organic solvent mixtures.

Current strategies in DNA and RNA nanotechnology enable the self-assembly of a variety of nucleic acid nanostructures in aqueous or substantially hydrated ...

On-Chip Octanol-Assisted Liposome Assembly for Bioengineering

Microfluidics is a widely used tool to generate droplets and vesicles of various kinds in a controlled and high-throughput manner. Liposomes are simplistic cellular mimics composed of an aqueous interior surrounded by a lipid bilayer; they are valuable in designing synthetic cells and understanding the fundamentals of biological cells in an in vitro fashion and are important for applied sciences, such as cargo delivery for therapeutic applications. This article describes a detailed working protocol for an on-chip microfluidic technique, octanol-assisted liposome assembly (OLA), to produce monodispersed, micron-sized, biocompatible liposomes. OLA functions similarly to bubble blowing, where an inner aqueous (IA) phase and a surrounding lipid-carrying 1-octanol phase are pinched off by surfactant-containing outer fluid streams. This readily generates double-emulsion droplets with protruding octanol pockets. As the lipid bilayer assembles at the droplet interface, the pocket spontaneously detaches to give rise to a unilamellar liposome that is ready for further manipulation and experimentation. OLA provides several advantages, such as steady liposome generation (&#62;10 Hz), efficient encapsulation of biomaterials, and monodispersed liposome populations, and requires very small sample volumes (~50 &#181;L), which can be crucial when working with precious biologicals. The study includes details on microfabrication, soft-lithography, and surface passivation, which are needed to establish OLA technology in the lab. A proof-of-principle synthetic biology application is also shown by inducing the formation of biomolecular condensates inside the liposomes via transmembrane proton flux. It is anticipated that this accompanying video protocol will facilitate the readers to establish and troubleshoot OLA in their labs.

The present protocol describes octanol-assisted liposome assembly (OLA), a microfluidic technique to generate biocompatible liposomes. OLA produces monodispersed, micron-sized liposomes with efficient encapsulation, allowing immediate on-chip experimentation. This protocol is anticipated to be particularly suitable for synthetic biology and synthetic cell research.