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Technical Note
Current monitoring in microchannel with repeated constrictions
for accurate detection of sample location in isotachophoresis
Merav Karsenty, Tally Rosenfeld, Khaled Gommed, and Moran Bercovici
Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/ac5036346 • Publication Date (Web): 25 Nov 2014
Downloaded from http://pubs.acs.org on November 28, 2014

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2 Current monitoring in microchannel with repeated constrictions for
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4 accurate detection of sample location in isotachophoresis
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6 Merav Karsenty, Tally Rosenfeld, Khaled Gommed, and Moran Bercovici
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8 ABSTRACT
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10 We present a new method for accurate detection of sample location in peak mode
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isotachophoresis (ITP). The technique is based on the design of a microchannel with multiple
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13 constrictions, and on detecting the passage of the ITP interface through these constrictions. We
14 achieve this by monitoring the electric current across the channel, which exhibits sharp decreases
15 as the ITP interface moves more rapidly through the higher current density constrictions. We
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17 show that cross-correlation between the electric current signal and a pre-defined step function is
18 an effective method for detecting changes in the slope of the electric current curve in real-time,
19 and is robust to changes in the composition of the buffers. We demonstrate the use of the
20 technique to deliver sample to a designated location in the channel with an accuracy as low as
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22 50 µm. Importantly, the method does not require the use of any optics, and thus can be used to
23 monitor the location of unlabeled species for application in a variety of ITP assays.
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25 INTRODUCTION
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27 Isotachophoresis (ITP) is an electrophoretic technique allowing focusing and separation of
28 sample molecules based on their effective electrophoretic mobility. ITP uses a discontinuous
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30 buffer system consisting of a leading electrolyte (LE), having a high electrophoretic mobility
31 leading ion, and a terminating electrolyte (TE), having a low electrophoretic mobility trailing
32 ion. In peak mode ITP, sample ions are focused at a sharp electric field gradient formed at the
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34
interface between the two buffers.1 The ability of ITP to significantly focus trace analytes, from
35 volumes of the order of microliters into well confined picoliter volumes, established it as an
36 important method in separation science with applications ranging from medical diagnostics to
37 environmental monitoring.2
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In recent years, the ability to leverage the high concentrations obtained in ITP toward
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41 accelerating the reaction kinetics of co-focused species has been studied in detail.3–8 Acceleration
42 of reactions is particularly important in detection of biomolecules, such as nucleic acids and
43 proteins, where hybridization or binding rates often pose a bottleneck in assay sensitivity. In the
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past year, at least three groups have further expanded this direction and began exploring the use
46 of ITP for acceleration of surface-based reactions; our group demonstrated the use of ITP for
47 accelerating the hybridization between focused DNA sequences and immobilized DNA probes,
48 leading to two orders of magnitude improvement in limit-of-detection (LoD) compared to a
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50 standard flow assay.9 Han et al. have, in parallel, studied a similar system, and further expanded
51 it to multiple reaction sites in a DNA microarray format;10 Khnouf et al. provided a proof of
52 concept where ITP was used for improving the sensitivity of protein detection in a surface
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immunoassay.11 These advancements, together with its robustness and ease of implementation in
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55 microfluidic devices, position ITP as a promising technology for lab-on-chip applications, and in
56 particular rapid and sensitive point-of-care diagnostics.
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2 These assays, while presenting a significant improvement in the LoD of surface reactions assays,
3 are still limited by a short reaction time (of the order of 1 s) corresponding to the time it takes the
4 focused sample to traverse the functionalized surface. Further advancement in ITP-based surface
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6 reactions relies on the ability to accurately determine the location of the (typically unlabeled)
7 focused sample and deliver it to a designated reaction site. While this could, in principle, be
8 achieved by visual means if the sample is labeled (i.e. human inspection or image processing),
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there is no existing solution for accurately monitoring the location of an unlabeled sample.
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11 Furthermore, aiming to utilize this technology for simple and low-cost point-of-care diagnostics,
12 automating the process and eliminating the need for manual controls are of key importance.
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14 Reading of the electric current during ITP has been long demonstrated as a useful tool for
15 monitoring the velocity and progress of electrophoresis processes. Most notably, Huang et al.
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17 demonstrated the use of current monitoring for measuring the electroosmotic flow in channels.12
18 Specifically for ITP, Reinhoud et al. developed an analytical model relating the current
19 measurement to the location of the ITP front in a straight channel. They then used the model to
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design an automated feedback system for counterflow, in order to hold the focusing region
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22 stationary.13 More recently, Harrison et al. developed a similar model relating the electric current
23 to the location of the front for constant applied voltage, and have validated it in counterflow ITP
24 performed in a large (preparative) scale electrophoresis apparatus. The method provided an
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26 accuracy of approximately 10% of the channel’s length (several centimeters in a 29 cm long
27 channel).14 While this is sufficient for maintaining a stationary ITP interface by counterflow, it is
28 far from the accuracy required in order to deliver a focused sample to a designated order 10-100
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µm reaction site. Furthermore, any changes to the conductivities of the buffers (e.g. due to the
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31 contents of the sample) modify the relation between location and current, further reducing
32 accuracy.
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34 We here present a novel method for precise and automatic detection of the ITP interface location
35 in a microchannel by current monitoring alone. Our technique uses geometrical constrictions in
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37 the microchannel to obtain a unique profile of the current signal during ITP. We first provide an
38 overview of the principles of the method, then present an analytical model for predicting the
39 electric current profile, and finally present experimental results demonstrating the method is
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robust to significant variations in buffer conditions and can deliver the sample to a designated
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42 location with an accuracy of 50 µm.
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44 PRINCIPLE OF THE METHOD
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46 ITP uses a discontinuous buffer system consisting of low mobility buffer (TE), and high mobility
47 buffer (LE). As ITP propagates, the low conductivity TE displaces the high conductivity LE in
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the channel, resulting in gradual increase in the overall resistance in the channel and a decrease
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50 in current (for an applied constant voltage). When adding geometrical constrictions to the
51 channel (Figure 1a), narrow sections have higher resistance and hence higher electric field. As a
52 result, the ITP interface travels at a higher velocity in these sections, resulting in what appears as
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54 a sharp ‘step’ in the current reading (Figure 1b). Video S1 in Supplementary Information
55 presents a movie of ITP electromigration in the channel together with its electric current trace.
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2 While the exact magnitude of the current still depends on buffer conditions, the sharp electric
3 field gradient is associated with a specific spatial location and can be used to accurately detect
4 the location of the interface. We developed an algorithm for detecting these changes in electric
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6 current, which is based on a cross-correlation of the electric current signal with a pre-defined
7 step function (Figure 1c). The algorithm works in real time and indicates the exact time in which
8 the ITP interface enters the constriction (Figure 1d). The computer can then be set to deliver the
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sample to a desired location and stop the electric field.
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31 Figure 1. Experimental results demonstrating the use of channel constrictions for accurate
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determination of ITP plug location. (a) Overlay of four raw fluorescence images showing the
34 location of the sample at different times during electromigration. (b) Corresponding electric
35 current measurement vs. time. Electric current monotonically decreases as the low mobility TE
36 displaces the high mobility LE. When the ITP interface enters the constriction, a rapid and
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38 significant drop in current occurs. (c) We detect the transitions in real time by cross-correlation
39 of the signal with a predefined step function. Maximum correlation signal is expected when the
40 step function overlaps with the step in the current signal. Red circles indicate the points in time
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in which the algorithm detects the transition (i.e. local maxima in the cross-correlation). (d)
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43 Correlation signal resulting from applying the algorithm to the electric current signal. The
44 method is robust to noise and peak values (red circles) indicate entrance to the constriction
45 regions. Peaks are recognized by the computer in real time and the ITP plug can be stopped at a
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47 designated location.
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49 THEORY
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51 We here present a simple algebraic model which enables prediction of such electric current
52 curves as a function of the buffers properties and channel geometry.
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54 As illustrated in Figure 2a, we consider a channel consisting of N identical constrictions, each of
55 length L2, separated by N-1 wide regions of length L3. The constrictions region is connected to
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57 reservoirs on either side through wide sections of lengths L1 and L4. Applying constant voltage
58 across the channel, the ITP interface moves along the different regions of the channel. We define
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2 xp (t ) as the time dependent axial position of the ITP interface along the channel, measured from
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the entrance of the TE reservoir. To obtain an algebraic expression, we choose a discrete
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5 approach and indexed each of the wide regions by i, ranging from 1 to N+1.
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7 At any given location of the ITP interface, the total electrical resistance of the channel can be
8 obtained by modeling each section as an individual resistor, and summing over all resistors in
9 series. The electric current is then given by
10
11
V
12 I (t ) = , (1)
13 Rr ,i ( x p (t ))
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15 where Rr ,i ( x p (t )) is the overall resistance of the channel. This resistance is a function of the
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17 interface location along the channel, x p (t ) , the channel section in which it’s located, i, and
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whether this section is wide or narrow (represented by the index r). We apply an Ohmic model
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20 in each region, and assume peak mode ITP, where the characteristic length of the ITP interface is
21 small compared to the characteristic geometrical dimensions. In a wide region i, the total
22 resistance can be expressed as
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24
1  1 1  L (i − 1)  1 1  1  L1 + L2 (i − 1) + L3 ( N − 1) + L4 L2 ( N + 1 − i) 
25 Rw,i ( x p (t )) =  −  x p (t ) + 2  − +  +  + Rres ,(2)
26 Aw  σ T σ L  σ T  An Aw  σ L  Aw An  3
14 4244 3 1444444444444444 2444444444444444
27 aw bw.i
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29 and in the narrow region i as
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31 1  1 1  ( L1 + L3 (i − 1))  1 1  1  L3 ( N − 1) + L4 L1 + L2 ⋅ N + L3 (i − 1) 
32 Rn ,i ( x p (t )) =  −  x p (t ) +  − +  +  + Rres ,(3)
An  σ T σ L  σT  Aw An  σ L  Aw An  3
33 144244 3 1444444444444444 2444444444444444
an bn,i
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36 where the wide and narrow cross-section areas are denoted by Aw and An respectively, and the
37 conductivities of the adjusted TE and LE solutions are denoted by σ T and σ L respectively. Rres
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39 represents the resistance of both reservoirs (assumed to be constant in time)15. Thus, the
40 generalized total resistance can be expressed as
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42 Rr ,i ( x p (t )) = ar x p (t ) + br ,i , (4)
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45 where ar and br,i are the regional index-dependent coefficients.
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47 The ITP velocity, dxP / dt , is given by the product of the leading ion mobility, µL , and the
48 electric field in the LE region, EL , and can be expressed as
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dx p µ L I (t )
51 = µ L EL = , (5)
52 dt σ L Ar
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54 where Ar is the cross-section area of region r, occupied by the ITP interface. Substituting eq.(1)
55 and eq.(4) into eq.(5), and integrating both sides we obtain,
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xp (t ) t
2 µ LV
3 ∫ ( ar x + br ,i ) dx = ∫σ Ar
dτ , (6)
x (t r ,i ) t L
4 p r ,i

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6 where tr ,i is the time in which the interface reaches region r in section i (e.g. tw,2 ), and xp (tr ,i ) is
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8 the location of the interface at the exact same time. The integration yields the quadratic equation
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µLV
ar ( x p (t )2 − x p (tr ,i )2 ) + br ,i ( x p (t ) − x p (tr ,i ) ) = ( t − tr ,i ) .
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(7)
11 σ L Ar
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14 This equation is readily solved analytically for xp (t) and its result can be substituted into eq.(1)
15 to obtain the electric current trace in time.
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38 Figure 2: Schematic illustration of our channel, consisting of N constrictions, and demonstration
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40 of experimental validation of the model in a typical microchannel, consisting of four
41 constrictions. (a) Four characteristic lengths are denoted by L1, L2, L3, and L4. The wide and
42 narrow cross-section areas are denoted by Aw and An respectively. Each wide region is indexed
43 by i, ranged between 1 to N+1. The axial position of the ITP interface along the channel is
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45 defined by xp(t). (b) Model results showing a time trace of the electric current for a geometry
46 consisting of N=4 constrictions. We analytically solved eq.(7) for xp(t), and substitute it into
47 eq.(1) to obtain the current at any point in time. Four ‘steps’ in the current are registered,
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corresponding to times at which the ITP interface passes through the constrictions.
50 (c) Comparison of model predictions (solid orange line) with experimental results (black dots)
51 for the constrictions region. Results are in good agreement in both step magnitude and length
52 (duration). The times at which the ITP interface spends in each of the wide and narrow regions
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54 are indicated by w,i and n,i respectively. The model parameters are: LE is 100 mM HCl and
55 200 mM BisTris; TE is 10 mM Tricine and 20 mM BisTris. The applied voltage is 400V;
56 σL=0.757 S/m, σT= 0.064 S/m, Rres=2×107 Ω, An=25×10-6 m; Aw=10-4 m; L1=7.4×10-3 m;
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L2=5×10-4 m; L3=5×10-4 m; L4=2.2×10-2 m.
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2 EXPERIMENTAL
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4 Channel design
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6 We designed a 40 mm long microchannel which contains four constrictions, forming three wide
7 chambers (Figure 1a). The width of the wide sections of the channel was set to 100 µm while the
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width of the narrow sections, in the constriction regions, was set to 25 µm. The first constriction
10 was located 10 mm from the entrance to the channel, allowing enough time for ITP to stabilize
11 and for the sample to sufficiently accumulate. Each constriction’s length was set to 500 µm,
12 while the wide chambers between constrictions were 250 µm long. Using a mylar mask
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14 reflecting this geometry, a 20 µm thick SU8 mold was fabricated at Stanford Microfluidic
15 Foundry (Stanford University, CA, http://www.stanford.edu/group/foundry/) using standard
16 lithography. Using this mold, the microfluidic chips were fabricated in house from PDMS and a
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cross linker to monomer ratio of 1:10, and using standard protocols.16
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20 Experimental setup
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22 We performed all ITP experiments at a constant voltage of 400 V, using a high voltage source
23 meter (model 2410, Keithley Instruments, Cleveland, OH) controlled by MATLAB (R2011b,
24 Mathworks, Natick, MA). We obtained images using an inverted epifluorescent microscope
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(Ti-U, Nikon, Tokyo, Japan) equipped with a metal halide light source (Intensilight, Nikon
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27 Japan), a 4X (NA=0.13, WD=17.2 mm) Nikon PlanApo objective and a Chroma 49006 filter-
28 cube (620/60 nm excitation, 700/75 nm emission and 660 nm dichroic mirror). Images were
29 captured using a 16 bit, 2560x2160 pixel array CMOS camera (Neo, Andor, Belfast Ireland)
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31 cooled to -40ºC and processed with MATLAB. The camera was controlled by NIS Elements
32 software (v.4.13, Nikon, Tokyo, Japan). We measured buffer conductivities in the reservoirs
33 using a conductivity meter (PC700, Eutech Instruments, The Netherlands).
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Isotachophoresis assay
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We performed the ITP experiments using LE buffer composed of 100 mM HCl, 200 mM BisTris
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39 and 1% 1.3 MDa poly(vinylpyrrolidone) (PVP) for suppression of electroosmotic flow (EOF).17
40 For the contamination experiments (Figure 4) we spiked the LE with NaOH at concentrations of
41 10 mM, 20 mM, 40 mM and 50 mM. We used Dylight 650 (Thermo Fisher Scientific, Waltham,
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43 MA) as our sample, due to its known photostability. Tricine, BisTris, and PVP were obtained
44 from Sigma-Aldrich (St. Louis, MO). HCl was obtained from Merck (Darmstadt, Germany), and
45 NaOH from Bio-Lab Ltd (Jerusalem, Israel). All buffer stock solutions were prepared in 20 mL
46 glass bottles and kept at room temperature. All buffer solutions were prepared using deionized
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48 water (DI) from Sigma-Aldrich (St. Louis, MO).
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50 We filled the LE reservoir with 20 µL of LE solution and filled the channel by applying vacuum
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from the other reservoir for 2 min. We then rinsed the TE reservoir with DI and filled it with
53 18 µL of TE solution and 2 µL of 10 nM Dylight. We turned on the electric field and set the
54 algorithm to turn off the electric field when the ITP plug reaches the second chamber.
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2 Location detection algorithm
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4 As shown in Figure 1 and discussed in the theory section, as the ITP plug enters the
5 constrictions, a rapid and sharp decrease in the current occurs, resulting as a step pattern in the
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current signal. Our algorithm is designed to detect these steps in real time, indicating the exact
7
8 time of entrance into the constriction. In each experiment, the electric current is normalized by
9 its initial value (Figure 4a). This guarantees that a single set of parameters could be used
10 regardless of the specific conductivities of the buffers. The normalized signal is smoothed in real
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time using a seven neighbors median filter. The change of slope in the electric current could, in
13 principle, be detected by numerically computing the local gradient. However, this method is very
14 sensitive to noise. Instead, as illustrated in Figure 1c, we define a backward facing step function
15 whose magnitude is 0.5 and spans over 11 points. At each point in time, this step function is
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17 centered on the electric current value and the two functions are cross-correlated with one
18 another. As shown in Figure 1d, the resulting correlation signal is characterized by distinct
19 peaks, whose maxima corresponds to the point in time in which the interface enters the
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constriction (red circles in Figure 1b and Figure 1d). As can be seen in Figure 1d, the peaks have
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22 a high signal to noise ratio, and based on several experiments, we determined a threshold value
23 (set to 0.04) that distinguishes between the true peaks and the noise. The location of the interface
24 can then be determined based on the number of peaks counted and the ITP interface can be
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26 stopped at the desired chamber. While the algorithm detects the time at which the ITP plug
27 enters into the constriction, a short pre-programed time delay can be used to stop the plug in the
28 following wide chamber. In doing so, one must account for an inherent time delay when the
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control program is implemented in software. Video S2 in Supplementary Information presents a
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31 dynamic illustration of the correlation process.
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33 RESULTS AND DISCUSSION
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35 Model validation
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37 Figure 2b represents the model results showing a time trace of the electric current for a geometry
38 consisting of four constrictions. Figure 2c presents a comparison between our model prediction
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40 and the experimental results. We analytically solved eq.(7) for xp (t ) , using MATLAB, and
41 substitute it into eq.(1) to obtain the electric current at any point in time. We obtained all model
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parameters (lengths of the sections, conductivities of the buffers) from direct measurements and
44 used PeakMaster18 and Spresso19 to calculate the adjusted TE conductivity. The only free-
45 parameter in the system is the resistance of the reservoirs, Rres. We choose its value
46 (Rres=2×107 Ω) such that the first step in the model and in the experimental results coincide in
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48 time. This value is consistent with estimates provided by Persat et al. for the resistance of the
49 reservoir.20 Results show that the model accurately predicts both the magnitude and duration of
50 the electric current steps.
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52 Sample delivery accuracy
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54 Figure 3a presents a sequence of raw fluorescence images of ITP electromigration, using 1 nM
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56 of Dylight to indicate the location of the interface. As the ITP interface enters a constriction, the
57 automatic tracking algorithm detects the ITP plug location and turns off the electric field as it
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2 enters the second chamber of the channel (after the second constriction). Figure 3b shows the
3 resulting stop locations of five repeats of the experiment. We define the stop location as the
4 location of the maxima of the width-averaged intensity signal. The red dashed lines represent the
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6 range of 95% confidence on the mean location, assuming a Student’s t-distribution. Results show
7 that the algorithm is able to stop the ITP plug with an accuracy of 50 µm. Video S3 in
8 Supplementary Information presents a movie in which a focused sample is delivered to a
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designated chamber.
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Figure 3. (a) Raw fluorescence images showing the delivery of an ITP plug to a desired
39 location. Upon arrival to the second chamber, the electric field is turned off and the sample is
40 allowed to diffuse. (b) Experimental results demonstrating the accuracy of the technique. Blue
41 circles represent the stop locations of five experiments, in which the algorithm was set to turn off
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43 the electric field after the ITP plug exits the second constriction. We define the stopping point as
44 the location of the maximal signal of the width averaged intensity. Red dashed lines represent
45 the range of 95% confidence on the mean location, and show a 50 µm accuracy in sample
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delivery.
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49 Robustness to contamination
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51 In many practical applications (e.g. in urine or blood analysis) samples may have a significant
52 ion concentration which affects the conductivity, pH, or both. This in turn may affect the ITP
53 velocity and the electric current trace. Figure 4a presents the electric current vs. time, for the
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same LE buffer spiked with 10 mM, 20 mM, 40 mM and 50 mM NaOH (final concentrations in
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56 the reservoir). As expected, the additional sodium concentration results in overall higher electric
57 current values as well as a higher ITP velocity. For this reason, standard current monitoring is
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2 not sufficiently accurate (i.e. the same current value corresponds to drastically different locations
3 at different conductivities). As our method is based on detecting changes in the slope of the
4 current, it is far less sensitive to buffer conditions. Figure 4b presents the stopping locations
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6 obtained for different sodium concentrations in the LE (three repeats for each condition, and five
7 repeats for the pure system). All cases were run using the exact same algorithm parameters. Red
8 dashed lines represent a 95% confidence on the mean, accounting for all cases and repeats.
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Results show that the algorithm worked well for all cases and is robust to significant
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11 contaminations of the LE, with a total accuracy of 70 µm.
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Figure 4. Experimental results demonstrating the robustness of the methods to significant
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37 contamination of the LE. (a) Electric current vs. time for ITP performed using LE spiked with
38 NaOH concentrations between 0 and 50 mM. As expected, the absolute values of the current as
39 well as its rate of change vary significantly between experiments. The electric current value thus
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cannot be used to robustly detect the location of the ITP interface. (b) Experimental results
42 showing the automatically obtained stop locations, with the LE spiked with NaOH concentration
43 between 0 and 50 mM (five repeats of pure LE case and three repeats for all other cases). Red
44 dashed lines represent a 95% confidence on the mean, accounting for all cases and repeats.
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46 Results show the algorithm is insensitive to such contaminations and can deliver the focused
47 sample to a desired location with an accuracy of 70 µm.
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51 CONCLUSIONS
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53 We demonstrated the use of the technique to deliver sample to a designated location in the
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channel with an accuracy of 50 µm (pure LE) or 70 µm (LE ‘contaminated’ with as much as
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56 50 mM of NaOH). The direct use of this technique is in allowing longer reaction times between
57 focused samples and immobilized surface probes in ITP-based surface reactions.9 Clearly, the
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2 delivery accuracy could be further optimized based on the specific requirement and constraints
3 of each implementation. The analytical model we provided could be used to predict the electric
4 current curves for arbitrary constriction geometries and applied voltages, and thus assist in such
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6 optimizations. For example, having smaller constrictions would result in sharper features in the
7 electric current curve, which would be easier to detect. However, this would also result in faster
8 transition of the interface through the constriction, which make precise stopping of the voltage
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more challenging. Furthermore, a major source of inaccuracy in our current implementation is
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11 likely due to the use of a Matlab code as the controller (for both sampling the current and
12 commanding the voltage stop). Implementation of the controller in hardware would allow higher
13 accuracy and faster response times.
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15 The technique may also be useful for other purposes such as control of light shutters before
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17 passing through a detection location (e.g. to avoid photobleaching), or switching of the electric
18 field from one reservoir to another (e.g. to initiate transient ITP). In general, it could be used for
19 sequencing a chain of events occurring as the ITP interface passes through the channel. We
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believe that the relatively high accuracy of the method, together with its simplicity would make
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22 it a useful tool in ITP-based diagnostic assays.
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24 ACKNOWLEDGMENTS
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26 We gratefully acknowledge funding from the Israel Science Foundation Grant no. 512/12 and
27 1698/12, and the Israel Ministry of Economy, as part of the NOFAR program no. 50660.
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29 SUPPORTING INFORMATION AVAILABLE
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31 S1: Video showing the electric current trace as the ITP interface electromigrates through
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33 multiple constrictions. S2: Video illustrating the operation of the cross-correlation algorithm for
34 detecting the location of the ITP interface. S3: Video demonstrating the delivery of a focused
35 sample to a desired site. This information is available free of charge via the Internet at
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http://pubs.acs.org/.
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38
39 REFERENCES
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