DEPLETION ZONE ISOTACHOPHORESIS: A NEW MICRO/NANOFLUIDIC ELECTROKINETIC METHOD J.W. Quist1,2, K.G.H. Janssen1,2, J. Lit, H.J. van der Linden1,2 and T. Hankemeier1,2*

1 Division of Analytical Biosciences, LAC;J)R, Leiden University, the Netherlands

2Netherlands Metabolomics Centre, Leiden University, the Netherlands

ABSTRACT We present our discovery that in a micro/nanofluidic concentration device a depletion zone can induce an

isotachophoretic separation. This new method, which we coined depletion zone isotachophoresis (dzITP), has been demonstrated to be well suited for the concentration and separation of ionic analytes in ultrasmall complex biological samples.

KEYWORDS: Depletion zone isotachophoresis, Analyte focusing, Purification, Bioanalysis

INTRODUCTION The comprehensive analysis of low-abundance analytes in ultrasmall volumes of complex biological matrices

- including samples from single cells - is one of the main challenges of today's science and technology. To address this challenge, efficient on-chip concentration and separation techniques are critically needed. For ionic analytes, isotachophoresis (ITP) proves a powerful candidate, as demonstrated by reports on millionfold sample stacking [1] and the analysis of RNAs from cell lysate [2] on chip. Another exciting example is the micro/nanofluidic concentration device which was first presented by Wang et ai., who reported concentration factors up to 108 and showed coupling to a capillary electrophoresis separation [3].

During experiments performed on a similar concentration device, we discovered that dependent on analyte concentration, a transition can take place to a focusing and separation regime that shares crucial properties with ITP.

THEORY Within nanochannels, ion distribution of counter- and co-ions (defined relative to the surface charge) may be affected

significantly by wall surface charge. When an electric fidd is applied over such a nanochannel, the current carries electrolyte counter- and co-ions asymmetrically. This leads to a phenomena called concentration polarization. For glass channels having a negative surface charge, this results in ion depletion at the anodic entrance of the nanochannel, and ion enrichment at the cathodic entrance [4].

In devices consisting of two parallel microchannels connected by a nanochannel (figure 1), a bulk flow through the microchannel promotes the growth of the depletion zone downstream to the nanochannel, while upstream its growth is limited. This bulk flow might be an electro-osmotic flow -100 iJm

(EOF), a pressure-driven flow, or a combination of these. Figure 1: Micrograph of a part of a chip, where two Under appropriate experimental conditions, the upstream microchannels are connected by a nanochannel border of the depletion zone can have a very stable position over time.

The border of the depletion zone is a concentration gradient and therefore gives rise to a gradient in electric field. On this gradient, co-ions (which are anions in the case of glass channels) can be focused efficiently [3],[5]. Given that the upstream border of the depletion zone has a stable position over time, the focusing of a co-ion i occurs at a position on the gradient where its net velocity becomes zero. This net velocity Vi. net is the result of a sum of bulk flow and opposite electrophoretic transport:

(1)

where Vhulk is the bulk flow velocity (which naturally is independent of local electric field) , Jii the electrophoretic mobility of co-ion i, and E is the electric field. Thus, a co-ion will focus at a location where the electric field is such, that its electrophoretic velocity equals the bulk flow velocity (Vi,net = 0 ). Co-ions with different mobilities will focus at different positions on the electric field gradient (figure 2, left).

During focusing, analytes may reach concentrations which are dominant compared to the background electrolyte concentration. This results in a transition to a different regime, which we have named depletion zone isotachophoresis (dzITP). In this regime further focusing results in broadening plateaus (figure 2, right), until all co-ions are ordered in adjacent zones according to their mobility.

This process has many similarities to the process of isotachophoresis (ITP). In ITP, analytes are sandwiched between a high-mobility leading electrolyte and a low-mobility trailing electrolyte. An ITP-separation also results in the focusing of analytes towards a plateau concentration, and an ordering in adjacent zones according to ionic mobility. However, in dzITP the depletion zone replaces the trailing electrolyte.

978-0-9798064-3-8/~TAS 201 0/$20©201 0 CBMS 1634 14th International Conference on Miniaturized Systems for Chemistry and Life Sciences

3 - 7 October 2010, Groningen, The Netherlands

- + - + - -

jf --------------~--------------position position

Figure 2: Gradientfocusing (left) and dzITP (right) in a hybrid microlnanofluidic device. Blue and green bands indicate anionic analytes with higher and lower mobility respectively. A: depletion zone; B: background electrolyte; C: nanochannel. + and - indicate the polarities of the potential differences . In the graphs, the solid lines indicate electrophoretic velocities and the dashed line indicates the bulk flow velocity, which has opposite direction.

EXPERIMENTAL Chips were fabricated in Pyrex glass using standard microfabrication techniques after UV lithography. Micro- and

nanochannels were made by BHF etching. Two parallel microchannels (depth: 2 )..Lm, width: 20 )..Lm, length: 20 mm) were connected in the middle by a nanochannel (depth: 50 nm, width: 20 )..Lm, length: 50 )..Lm) (figure 1). Powderblasted wells were situated at both ends of each microchannel. Electrodes were connected to each of the wells. Positive voltages were applied to the electrodes connected to the microchannel in which the dzITP process was to be performed, with 50-100 V applied on the well from which the sample was injected and 0-10 V on the other well. The electrodes connected to the other microchannel were kept at ground. The dzITP process was monitored using fluorescence microscopy.

dzITP was first discovered in an exploratory experiment in which an electrolyte of 5.0 mmol/L NaCI at pH 9.3 contained 0.50 mmol/L dis odium fluorescein. An accidental contamination with carbonate ions contributed to the discovery that an ITP-like process was going on. This contamination resulted from the fact that the solution was in contact with air for several minutes. Therefore, CO2 could dissolve, resulting in an increase in concentration of carbonate ions (to ~0 . 5 mmol/L) and a decrease in pH (to pH ~8 . 3) . Only 2 )..LL of solution was pipetted into the wells, therefore measurement of the exact contribution of carbonate ions to the experiment (via pH and conductivity) was difficult. An adjusted chip-to-world interface and standard operation protocol is being developed for sufficient control over atmospheric influences.

As proof of principle of the applicability to complex biological sample, dzITP was performed using hydrolyzed yeast extract. 0.40 mg of hydrolyzed yeast extract (Sc(N)-AU1) was labeled at a 1:1 weight ratio with fluorescein isothiocyanate (FITC) by an overnight incubation in 2 mL of 50% acetone. The solvent was evaporated and the residue of labeled yeast extract was redissolved in 50 )..LL water and centrifuged. The supernatant was lOx diluted in 5.0 mmol/L HEPES and spiked with 50 )..Lmol/L FITC-phenylalanine and FITC-glutamic acid. dzITP was performed with dilutions ranging from 10 to 200 times of the spiked sample in 8.0 mrnoliL NaCI, pH 9.5.

RESULTS AND DISCUSSION 1200

S' S 800 . £

VJ .: B .5 400

0 A B C

500 1000 1500

Figure 3 shows dzITP with an electrolyte of 5.0 mmoliL NaCI containing 0.50 mmoliL fluorescein and ~0.5 mmol/L of carbonate contamination. Distinct zones are formed . Fluorescein has the lowest mobility and therefore focuses closest to the nanochannel. Carbonate has a somewhat higher mobility, and therefore focuses next to the fluorescein zone. Although carbonate itself is not fluorescent, fluorescein coming from the sample well passes through the carbonate zone at elevated concentration, acting as a so-called distance from nanochannel ()l m)

D

2000

underspeeder [6] and enabling visualization of Figure 3: Fluorescence intensity profile showing a proof of principle of the zone. The resulting intensity profile dzITP Several zones are forming: A) depletion zone; B) concentrated appears as a typical isotachophoretic fluorescein ; C) concentrated carbonate + fluorescein; D) electrolyte + separation. However, there is a depletion zone carbonate + fluorescein. The left border of zone B is close to the right instead of a trailing electrolyte (hence the name angle in the upper microchannel (see figure 1). Inset: the fluorescence depletion zone isotachophoresis). micrograph that corresponds to the intensity profile.

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Figure 4 shows the time evolution of dzITP of a sample of FITC-Iabeled yeast extract at 200x 100

dilution. This sample is very complex and might E contain hundreds of anionic species in low :::1.

concentrations, of which few are fluorescent and ~ 200 c

therefore visible in the experiment. jg Although there is efficient focusing (>103

), g most analytes could not be observed to reach ~

plateau concentrations. Three bright bands are ~ formed, which likely to belong to FITC ~ (remaining from the labeling procedure) and ~

E! FITC-phenylalanine and FITC-glutamic acid rn i:5

(with which the sample was spiked). A fourth faint fluorescent band can be observed staying at a stable position, indicating the border of the depletion zone. Possibly this band consists of

300

400

500

600

low-mobility FITC-Iabeled peptides. The rapidly Time (5)

..

broadening non-fluorescent zone adjacent to this Figure 4: Time evolution of dzITP of FITC-labeled yeast extract. band probably mainly consists of HEPES, which Colors indicate fluorescence intensity, which is plotted against time was present in the sample at relatively high and axial position in the microchannel. The red arrow indicates the concentration. The weakly fluorescent zones depletion zone border, which has a stable position from t = 200s. between the three bands with bright fluorescence possibly consist of non-labeled amino acids from the samples.

During these experiments, there was continuous transport of sample anions from the sample well towards the nanochannel. However, similar separations also could be obtained by filling only the micro channel with sample, resulting in dzITP of 400 pL of (biological) sample. This volume can easily be downscaled by filling smaller parts of the channel. Thus, dzITP has the potential to become an important tool in single cell analysis.

An interesting feature is that compounds within defined windows of ionic mobility can be selectively eluted in upstream direction. For example, if a high concentration of an ion with a mobility of 32 V lm2s·1 is injected, all ions with mobility > 32 Vi m2s· 1 will be driven out of the channel. A subsequent injection of an ion with a somewhat lower mobility (say, 30 V lm2s· l

) would then selectively elute all ions with mobilities between 30 and 32 V lm2s· 1 from the channel. Note that during elution the sample ions will remain focused.

Thus, dzITP allows the selection or the removal of specific classes of compounds, including proteins and metabolites. Because uncharged molecules are carried away with the bulk flow, the method also enriches charged compounds over neutral compounds. Therefore, dzITP can be used for purification strategies based on electrokinetic properties. The combination of analyte focusing, separation and purification is expected to greatly enhance detection limits in electrochemical detection, fluorescence spectroscopy, mass spectrometry and many other techniques.

CONCLUSION The newly discovered method of dzITP has been demonstrated to combine concentration, separation and purification

of picoliter-scale complex biological samples in a micro/nanofluidic device. This technique holds great potential for single-cell analysis which is a current 'hot topic' in the life sciences.

REFERENCES [1] B. Jung et aI., On-chip million-fold sample stacking using single-interface isotachophoresis, Analytical Chemistry,

vol. 78, pp. 2319-2327, (2006). [2] R.B. Schoch et aI., Rapid and selective extraction, isolation, preconcentration, and quantitation of small RNAs

from cell lysate using on-chip isotachophoresis, Lab on a Chip, vol. 9, pp. 2145-2152, (2009). [3] yc. Wang et aI., Million-fold preconcentration of proteins and peptides by nanofluidic filter, Analytical Chemistry,

vol. 77, pp 4293-4299, (2005). [4] Q. Pu et aI., Ion-enrichment and ion-depletion effect ofnanochannel structures, Nano Letters, vol. 4, pp 1099-1103,

(2004). [5] lG. Shackman and D. Ross, Counter-flow gradient electrofocusing, Electrophoresis, vol. 28, pp 556-571 , (2007). [6] R.D. Chambers and lG. Santiago, Imaging and quantification of isotachophoresis zones using nonfocusing

fluorescent Tracers, Analytical Chemistry, vol. 81 , pp. 3022-3028, (2009).

CONTACT *T. Hankemeier, tel: +31-71-527-4226; hankemeier@lacdr.leidenuniv.nl

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