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Anal. Chem. 2010, 82, 10030–10036

Thermoswitchable Electrokinetic Ion-Enrichment/ Elution Based on a Poly(N-isopropylacrylamide) Hydrogel Plug in a Microchannel Zhiming Li,† Qiaohong He,*,† Dan Ma,† Hengwu Chen,† and Steven A. Soper‡ The Institute of Micro-analytical Systems, Department of Chemistry, Zhejiang University, Zijin’gang Campus, Hangzhou 310058, China, and Departments of Chemistry and Mechanical Engineering, Louisiana State University, Baton Rouge, Louisiana, United States This paper reports a novel protocol consisting of the thermomodulated electrokinetic enrichment, elution, and separation of charged species based upon a thermoswitchable swelling-shrinking property of a poly(N-isopropylacrylamide), PNIPAAm, hydrogel. A 0.2-1 mm long PNIPAAm hydrogel plug was photopolymerized inside a glass microfluidic channel to produce a composite device consisting of the PNIPAAm hydrogel plug and the glass microchannel (abbreviated as plug-in-channel). After voltage was applied to the composite device, anions, such as FITC, could be enriched at the cathodic end of the PNIPAAm plug when the temperature of the plug was kept below its lower critical solution temperature (LCST, ∼32 °C). The concentrated analytes could then be eluted by electroosmotic flow when the temperature of the plug was heated above the LCST. The mechanism of the thermoswitchable ion enrichment/elution process was studied with the results presented. The analytical potential of the composite device was demonstrated for the temperature-modulated preconcentration, elution, and separation of FITC-labeled amino acids. Since the first realization of micro total analysis systems (µ-TAS) in the 1990s,1 microfluidic devices have been widely applied in the fields of chemistry and biology for the rapid and fully automated, low-cost analysis and synthesis of targets.2-4 One of the compelling advantages offered by microfluidic chips is the ability to handle very small volumes of samples/reagents. Compared to conventional analysis systems, the small amount of samples/reagents required often results in placing considerable demands on the detection system. Thus, a large number of methods for on-chip analyte preconcentration have been developed to address this issue. These methods can be categorized into three general types. The first type is chromatography-based * Corresponding author. E-mail: [email protected]. † Zhejiang University. ‡ Louisiana State University. (1) Manz, A.; Graber, N.; Widmer, H. M. Sens. Actuators, B 1990, 1, 244– 248. (2) Whitesides, G. M. Nature 2006, 442, 368–373. (3) Yager, P.; Edwards, T.; Fu, E.; Helton, K.; Nelson, K.; Tam, M. R.; Weigl, B. H. Nature 2006, 442, 412–418. (4) West, J.; Becker, M.; Tombrink, S.; Manz, A. Anal. Chem. 2008, 80, 4403– 4419.

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preconcentration. 5,6 The second is size-based exclusion preconcentration.7-10 The third type is a technique11-13 that relies on the electrophoretic behavior of charged analytes to permit preconcentration, such as field-amplified sample stacking (FASS), isotachophoresis (ITP), or isoelectric focusing (IEF). Recently, electrokinetic preconcentration based on ionselective permeation induced by exclusion-enrichment effects in hybrid micro/nanofluidic devices has attracted great interest. For example, Pu et al.14 reported electrokinetic ion-enrichment and ion-depletion at the interface between microchannels and nanochannels of a hybrid micro/nanofluidic device. Wang et al.15 demonstrated a highly efficient ion-enrichment technique based on the electrokinetic trapping of ions enabled by nanofluidic filters constructed on a glass/silicon chip and reported a concentration factor as high as one million-fold for proteins. Kim et al.16 developed a simple hybrid micro/ nanofluidic device, where nanochannels were formed between a top PDMS layer and a bottom glass substrate during reversible bonding. Dhopeshwarkar et al.17,18 reported a device composed of a PDMS-glass hybrid microchannel and a short in-channel synthesized nanoporous poly(2-hydroxyethyl methacrylate-co-acryl acid), poly(HEMA-co-AA), gel for the preconcentration of charged species. Zhou et al.19 developed a threelayer micro/nanofluidic device where a PET nanoporous (5) Stroink, T.; Paarlberg, E.; Waterval, J. C. M.; Bult, A; Underberg, W. J. M. Electrophoresis 2001, 22, 2374–2383. (6) Saavedra, L.; Barbas, C. J. Biochem. Biophys. Methods 2007, 70, 289–297. (7) Hatch, A. V.; Herr, A. E.; Throckmorton, D. J.; Brennan, J. S.; Singh, A. K. Anal. Chem. 2006, 78, 4976–4984. (8) Khandurina, J.; Jacobson, S. C.; Waters, L. C.; Foote, R. S.; Ramsey, J. M. Anal. Chem. 1999, 71, 1815–1819. (9) Foote, R. S.; Khandurina, J.; Jacobson, S. C.; Ramsey, J. M. Anal. Chem. 2005, 77, 57–63. (10) Song, S.; Singh, A. K.; Kirby, B. J. Anal. Chem. 2004, 76, 4589–4592. (11) Sueyoshi, K.; Kitagawa, F.; Otsuka, K. J. Sep. Sci. 2008, 31, 2650–2666. (12) Breadmore, M. C. Electrophoresis 2007, 28, 254–281. (13) Osbourn, D. M.; Weiss, D. J.; Lunte, C. E. Electrophoresis 2000, 21, 2768– 2779. (14) Pu, Q. S.; Yun, J. S.; Temkin, H.; Liu, S. R. Nano Lett. 2004, 4, 1099–1103. (15) Wang, Y. C.; Stevens, A. L.; Han, J. Y. Anal. Chem. 2005, 77, 4293–4299. (16) Kim, S. M.; Burns, M. A.; Hasselbrink, E. F. Anal. Chem. 2006, 78, 4779– 4785. (17) Dhopeshwarkar, R.; Sun, L.; Crooks, R. M. Lab Chip 2005, 5, 1148–1154. (18) Dhopeshwarkar, R.; Crooks, R. M.; Hlushkou, D.; Tallarek, U. Anal. Chem. 2008, 80, 1039–1048. (19) Zhou, K.; Kovarik, M. L.; Jacobson, S. C. J. Am. Chem. Soc. 2008, 130, 8614–8616. 10.1021/ac101768j  2010 American Chemical Society Published on Web 11/24/2010

membrane was sandwiched between two PDMS slabs with microchannels and observed sample stacking near the interface of nanopores and microchannels. Additional work on this topic can be referred to in recently published reviews.20-23 Poly(N-isopropylacrylamide), PNIPAAm, is a thermalresponsive polymer that exhibits a reversible phase transition at around 32 °C (known as the lower critical solution temperature, LCST).24 At a temperature below its LCST, the polymer chains swell and the polymer becomes hydrophilic, whereas at the temperature above the LCST, the chains shrink and the polymer becomes hydrophobic. Recently, PNIPAAm has been employed to prepare microvalves in microfluidic channels for the manipulation of fluids25-30 and the enrichment and separation of proteins via surface adsorption/desorption in a microfluidic chip.31 To the best of our knowledge, no work has been reported concerning the electrokinetic enrichment and elution of ions by exploiting the thermoswitchable swelling-shrinking behavior of PNIPAAm. Here, we report our recent work on this topic. EXPERIMENTAL SECTION Chemicals and Reagents. NIPAAm, N,N′-methylenebisacrylamide (BIS), and 3-methacryloxypropyltrimethoxysilane, MPTMS, were purchased from Acros Organics (Morris Plains, NJ). 2-Hydroxy-2-methylpropiophenone, Darocure-1173, and FITC were obtained from Sigma-Aldrich (St. Louis, MO). Alanine (Ala) and glycine (Gly) were from Kangda Amino Acid Works (Shanghai, China). All reagents were analytical grade or better and were used as received with the exception of NIPAAm, which was purified by recrystallization with n-hexane before use. Apparatus. An in-house constructed computer-programmed power supply was used for electrokinetic ion-enrichment, elution, and capillary electrophoresis. An inverted fluorescence microscope (Eclipse TE2000-S, Nikon, Japan) equipped with a built-in mercury lamp, a spectral filter set for FITC, and a digital camera was used to observe the ion transport and take fluorescence micrographs. The confocal laser-induced fluorescence detection system (LIF) including the signal processing system was constructed as described in ref 32. A Peltier heating-cooling device (4 mm × 6 mm, Jingie Industry and trading Co. Ltd., Tianjin, China) was used to control the temperature of the plug, and the switch of the Peltier was conducted by commercial relays with Siemens Logo Module.30 (20) (21) (22) (23) (24) (25) (26) (27) (28) (29) (30) (31) (32)

Holtzel, A.; Tallarek, U. J. Sep. Sci. 2007, 30, 1398–1419. Park, S.; Chung, T. D.; Kim, H. C. Microfluid Nanofluid 2009, 6, 315–331. Napoli, M.; Eijkel, J. C. T.; Pennathur, S. Lab Chip 2010, 10, 957–985. Kim, S. J.; Song, Y. A.; Han, J. Chem. Soc. Rev. 2010, 39, 912–922. Gil, E. S.; Hudson, S. M. Prog. Polym. Sci. 2004, 29, 1173–1222. Saitoh, T.; Sekino, A.; Hiraide, M. Anal. Chim. Acta 2005, 536, 179–182. Idota, N.; Kikuchi, A.; Kobayashi, J.; Sakai, K.; Okano, T. Adv. Mater. 2005, 17, 2723–2727. Yu, C.; Mutlu, S.; Selvaganapathy, P.; Mastrangelo, C. H.; Svec, F.; Frechet, J. M. J. Anal. Chem. 2003, 75, 1958–1961. Hisamoto, H.; Funano, S.; Terabe, S. Anal. Chem. 2005, 77, 2266–2271. Li, Z. M.; Chen, H. W.; Ma, D. Chem. J. Chin. Univ.-Chin. 2009, 30, 32– 36. Li, Z. M.; He, Q. H.; Ma, D.; Chen, H. W. Anal. Chim. Acta 2010, 665, 107–112. Huber, D. L.; Manginell, R. P.; Samara, M. A.; Kim, B. I.; Bunker, B. C. Science 2003, 301, 352–354. He, Q. H.; Fang, Q.; Du, W. B.; Fang, Z. L. Electrophoresis 2007, 28, 2912– 2919.

Figure 1. Schematic of the microfluidic chip with immobilized PNIPAAm hydrogel plug (not to scale). The channel was 40 µm in depth and 180 µm in top-width. B, buffer reservoir; S, sample reservoir; P, PNIPAAm hydrogel plug; DC, CCD-image capture region for estimation of enrichment factors; DB and DF, LIF detection points for backward and forward elution modes, respectively; LB and LF, migration distances of the concentrated band during backward and forward elution, respectively. The dash-lined rectangle represents the cavity (on the back of the glass chip) where the Peltier was positioned.

Preparation of the Microchip with a PNIPAAm Hydrogel Plug in the Microchannel. Glass chips were fabricated using procedures described elsewhere.33 Preparation of the PNIPAAm hydrogel plug inside the glass microchannel was as described in our recently published work.30 Briefly, after the microchannel was vinylized with MPTMS, a monomer solution [14.3% (w/v) NIPAAm, 0.14% BIS, and 2% Darocure-1173 in water-ethanol with dissolved oxygen being removed] was injected into the microchannel. A photo mask with the pattern of the hydrogel plug was aligned to the channel. After being placed on a heat sink of ice-water, the mask-covered glass chip was exposed to UV light emitted from a UV-illuminator for 1 min. The microchannel was then gently flushed with warm water (40 °C) to remove unreacted monomer components from the channel. The microfluidic chip with immobilized PNIPAAm plug is schematically illustrated in Figure 1. Operation Procedure for the Electrokinetic Ion-Enrichment and Elution/Separation. Before each experiment, the microchannel was flushed with 50 µM borate buffer solution with the PNIPAAm plug heated above its LCST to open (shrink status) the fluidic pathway and then allowed to equilibrate for 30 min at room temperature. Afterward, a 50 µM borate solution was infused into reservoir B, while a FITC solution prepared in 50 µM borate buffer was infused into reservoir S. Two platinum electrodes connected to a high voltage supply were immersed into reservoirs B and S. The microchip was then aligned to the optical system of either a fluorescence microscope or the LIF system. Afterward, a program for control of the bias voltage and Peltier function (cooling or heating) was triggered to perform the ion-enrichment and elution or CE separation (Table 1). During the enrichment and elution/ separation process, fluorescence images were captured with the microscope-equipped digital CCD camera or fluorescence intensity-time profile was recorded using the LIF detector. After an enrichment-elution cycle finished, a between-run washing of the channel network with the running buffer was carried out at around 45 °C. (33) Meng, F.; Chen, H. W.; Dou, Y. H.; Fang, Z. L. Chem. J. Chin. Univ.-Chin. 2004, 25, 844–846.

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Table 1. Program for Control of Bias Voltage and Peltier Function voltage (V) step enrichment elution/separation forward backward a

duration (s)

Peltier status/plug status

sample reservoir

buffer reservoir

0-170 171-173

cooling /closed preheating/closed

0 0

+800 +800

174-280

heatingb/open

+800 0

0 +800

a

During cooling, the plug temperature was around 25 °C. b During heating, the plug temperature was around 45 °C.

Figure 2. Fluorescence micrographs obtained during enrichment and elution of FITC with the microfluidic device shown in Figure 1. B and S refer to the reservoirs shown in Figure 1. (a) The hydrogel plug was at room temperature while no bias voltage was applied; (b) same as panel a, but 172 s after a bias voltage of +700 V was applied to reservoir B; (c and d) 5 and 17 s after the hydrogel plug was heated to open (at around 45 °C) and the polarity of the bias voltage was reversed, respectively. The red arrows in panels c and d indicate the moving direction of the concentrated band. (a′, b′, c′, and d′) Schematics that illustrate conditions corresponding to the micrographs shown in panels a-d. A 20 µM FITC solution in 50 µM sodium tetraborate buffer was filled in the channel before the test started. A 1 mm length PNIPAAm hydrogel plug was synthesized near the center of a 55 mm length glass channel.

RESULTS AND DISCUSSION Thermoswitchable Electrokinetic Enrichment and Elution. In our previous works,29,30 PNIPAAm hydrogel plugs that were prepared inside glass channels served as thermally actuated open/close microvalves to manipulate hydrodynamic flows. It was very interesting to notice in the present work that if the PNIPAAm hydrogel plug inside a glass channel was exploited to modulate an electrokinetically driven ion and solution fluxes, the plug acted as, in addition to an open/close valve, an effective ion-concentrator. Figure 2 shows the 10032

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PNIPAAm-plug-modulated electrokinetic ion-enrichment and elution process. Before an enrichment process, a FITC solution prepared in 50 µM sodium tetraborate buffer was filled into the entire channel network. At this point, dim but uniform fluorescence could be observed in both the open glass channel and the hydrogel plug (Figure 2a). After an electric field was applied across the microchannel (reservoir S being the cathode) while the hydrogel plug was kept at room temperature (below the LCST, the plug swelled to be closed), the fluorescence intensity at the cathodic end of the plug gradually increased

while that at the anodic end decreased (Figure 2b), implying that the negatively charged FITC dye molecules were enriched at the cathodic end and depleted at the anodic end of the hydrogel plug. This step was referred to as the ion-enrichment step. At the end of the ion-enrichment step, the hydrogel plug was heated to around 45 °C (above the LCST, the plug shrank to be open), followed by reversing the polarity of the bias voltage. Thus, the enriched FITC band moved toward reservoir B (cathodic electrode) and penetrated through the hydrogel plug (Figure 2c) and finally entered the cathodic channel (channel P-B on the left of the plug as shown in Figure 1) due to an electroosmotic effect (Figure 2d). This step was termed the elution step. Video images of the ion-enrichment and elution process can be found in the Supporting Information (Video S-1). We hypothesized that ion enrichment and depletion that occurred in the cathodic and anodic regions, respectively, of the PNIPAAm hydrogel plug resulted from the electrokinetic ionpermselective effect of the swelled hydrogel plug. For the slightly basic conditions used in this work, a small percentage of the polymer amide bonds may be hydrolyzed,7 leaving negatively charged carboxylic groups on the PNIPAAm chains. Below the LCST, the cross-linked PNIPAAm network swelled, reducing the pore size, and consequently, nanosized pores were formed in the hydrogel plug. When an appropriate voltage was applied across the swelled hydrogel plug, the negatively charged plug with nanopores acted as a cation-selective membrane due to co-ion electrostatically exclusion or the electric double layer (EDL) overlap within the swelled plug. Thus, cations (counterions) migrated into the hydrogel plug while the anions were excluded. This resulted in ion-enrichment near the cathodic end of the plug while ion-depletion occurred near the anodic end. At this low temperature, average EOF would be diminished due to the high electrical and hydraulic resistance of the closed hydrogel plug. When the temperature of the hydrogel plug was heated to around 45 °C, the PNIPAAm network shrank. This rendered the pores enlarged and the plug opened. Upon application of an electrical field of reversed polarity, the EOF supported by either the glass microchannel or the micropores in the plug delivered the enriched band, penetrating the plug and moving into the cathodic channel (channel P-B on the left of the plug); the enriched band was eluted by the EOF. This hypothesis was supported by the following experiments. First, the profile of the dc current passing through the microchannel with the hydrogel plug over time could be an excellent indicator of the continuity of the electrolyte medium filling the plug-in-channel composite. As shown in Figure 3, at a temperature of 45 °C (above LCST), a steady current was maintained after a voltage was applied to the plug-in-channel composite. The current was slightly lower than that passing through a glass microchannel of the same size, but without the hydrogel plug (called blank channel), implying that the hydrogel plug acted as a pure electrical resistor. At the temperature of 25 °C (below LCST), however, not only was the absolute current reduced due to the decrease in the electrolyte conductivity at the lower temperature, but also the current no longer was constant with time. In the initial time-interval (10-20 s) after the voltage was applied to the channel, the electric current exponentially

Figure 3. Current-time (I-t) curves obtained when a bias voltage was applied to glass microchannels with or without a 1-mm long PNIPAAm hydrogel plug at different temperatures. The microchannel was filled with 0.1 mM Na2B4O7, and a bias voltage of 1.2 kV was applied. Glass microchannel dimensions were 40 µm (depth) × 180 µm (top-width) × 25 mm (length).

declined and then reached a steady state. This phenomenon observed at 25 °C was quite similar to previous works18,34 for micro/nanofluidic devices, and exponential decline in current has been ascribed to electrokinetically induced concentration polarization in the bulk solution adjacent to the micro/nanofluidic interfaces. Thus, the dc current-time profile supported by the present plug-in-channel composite at 25 °C supports the existence of the nanopores in the hydrogel plug when the plug was in a swelled status, and the ion-enrichment demonstrated by the device at 25 °C can result from concentration polarization effects. Second, a test on the influence of buffer concentration on the enrichment efficiency verified the above suggested mechanism. As the ion-permselectivity behavior of nanopores is relevant to the thickness of the EDL inside the nanopores and because the EDL thickness depends on the electrolyte concentration, the buffer concentration should impact the enrichment efficiency.14 Tests showed that a significant reduction in the enrichment efficiency was observed as the borate buffer concentration increased stepwise from 0.05 mM to 10 mM (see Figure S-1 of the Supporting Information). Therefore, the ion-enrichment observed with the present plug-in-channel composite was induced by the ion-permselectivity behavior of the PNIPAAm nanopores under the conditions of the temperature below the LCST and low ion strength. Third, an average EOF of 2.7 × 10-4 cm2/V · s (RSD 2.6%, n ) 10) directing to the cathode was supported by the plug-inchannel network at pH 8.2 (50 µM sodium tetraborate solution) at a temperature of 45 °C. Therefore, the elution of the enriched band resulted from the EOF. We were unable to measure the EOF at room temperature with the electric current method35 because concentration polarization would be induced by the applied voltage. Nevertheless, visual tests showed that at room temperature, FITC in the sample reservoir could not be electrokinetically driven into the plug-in-channel composite (34) Kim, S. J.; Han, J. Y. Anal. Chem. 2008, 80, 3507–3511. (35) Huang, X. H.; Gordon, M. J.; Zare, R. N. Anal. Chem. 1988, 60, 1837– 1838.

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filled with a blank borate buffer if a +700 bias voltage was applied to the sample reservoir (anode terminal) and the buffer reservoir was grounded. This observation indicated that at room temperature the velocity of the EOF (directing toward cathode) was less than the electrophoretic velocity of FITC (directing to anode), and the electrophoresis transport dominated in the plug-in-channel composite. As can be seen in Figure 2b, a portion of FITC was trapped at the front bed of the hydrogel plug during the ion-enrichment step. Since the pore-size inside the polymer network was not as uniform as nanochannels fabricated in glass or a silica wafer via MEMS technology, the nonuniformity of the pore-size might cause the existence of mixed micro- and nanopores inside the threedimensional architecture of the polymeric network. Thus, there would be local adjacent concentration polarization zones contacting or even overlapping each other inside the PNIPAAm network.20 Especially, at the axial edge regions of the plug, the average pore size would be larger than that in the central parts of the plug due to edge effects occurring during photopolymerization. Therefore, significant local ion enrichment may occur in the front bed of the cathodic end of hydrogel plug. Based on the above observations, it can be concluded that the thermoregulated ion-enrichment and elution process originates from the thermoswitchable behavior of PNIPAAm that modulates the plug-in-channel composite from an electrophoretic ionconcentrator below the LCST to an electroosmotic deliverer above the LCST. Enrichment Factors. Preliminary tests on the degree of photobleaching showed that the fluorescence intensities of FITC solutions decreased by less than 5% within 3 min, the photobleaching being not dramatic due to the low photon fluence of the microscope excitation source. Thus, we kept the shutter of the microscope open during the entire observation period. Figure 4 shows the profiles of the average fluorescence intensities against the enrichment time obtained with two FITC solutions of different concentrations. Compared to the stagnant reference FITC solutions (the horizontal dashed lines in Figure 4), enrichment factors of 17-22 were achieved for the two tested solutions within 3 min. The enrichment factors were poorer than those reported for fluorescein with a hybrid micronano-micro channel system fabricated on a glass chip,14 but were comparable to that achieved with a poly(HEMA-co-AA) hydrogel microplug in a PDMS/glass channel.17 The relatively poorer enrichment factors observed in the present work could be ascribed partially to the nonuniformity of the pore-size within the PNIPAAm network and to the heterogeneity of the pore and channel surfaces. As can be seen in the Figure 2, while the fluorescence intensity of the FITC ions entrapped at the solution-plug (cathodic end) interface increased with longer times, the concentrate band gradually widened; consequently, the enrichment factor leveled. The widening of the concentrated band was caused by the dynamic redistribution of the applied voltage over the entire plug-in-channel composite, i.e., gradual decrease of the local electrical field strength in the ionenrichment region in conjunction with the gradual increase of the local field strength in the ion-depletion region. This observation was quite similar to what that discussed for the poly(HEMA-coAA) hydrogel microplug in a PDMS/glass channel for enrichment of fluorescein dye anions.18 Furthermore, the discontinuity of the 10034

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Figure 4. The fluorescence intensity versus the enrichment time. The fluorescence images captured by the microscope-equipped digital camera at different enrichment time intervals were transferred to fluorescence intensity using ImageJ software. FITC test solutions of 100 nM and 5 µM were prepared in a 50 µM borate buffer. A voltage of +800 V was applied to the buffer reservoir while the sample reservoir was grounded. The horizontal dashed lines represent the fluorescence intensity of the stagnant FITC solution of the indicated concentrations filled in the microchannels but subjected to no ionenrichment. The insert is a curve for 100 nM FITC plotted with a finer ordinate scale.

electric field and the heterogeneity of the surface property could cause discontinuity of the local EOFs that might lead to generation of pressure flow (parabolic flow profiles could be noticed in the images of the video in the Supporting Information) even recirculating flows in the microchannel, consequently disturbing the preconcentration. Further works on details of the fluid and ion flow regime in the present system are currently underway in our group. Forward and Backward Elution. After ion-enrichment, the preconcentrated band elution commenced when the hydrogel plug was raised to around 45 °C. Depending on the applied voltage polarity, the elution could be conducted in either the backward or forward direction. For backward elution, the bias voltage applied for ion-enrichment was maintained, so that reservoir S always acted as the cathode (as shown in Figure 1 and Table 1). Under this condition, the concentrated band quickly moved in the direction toward reservoir S due to the cathodic EOF. Profile I in Figure 5 shows the fluorescence-time profile for backward elution. During the time interval of 0-170 s, the hydrogel plug was kept at room temperature and ion-enrichment was carried out. In the first 20 s for Profile I, a steady baseline for the borate buffer filled-channel was recorded until the front of the FITC anion flux reached the detection point. At 171 s, the temperature of the PNIPAAm gel plug was increased. As soon as the plug was heated above its LCST (it took 3 s30), the plug opened and the EOF delivered the FITC solution remaining in the cathodic channel (channel P-S on the right of the hydrogel plug as shown in Figure 1) and the FITC-enriched band moved toward reservoir S. As the FITC concentrate reached detection point DB at about 200 s, a large fluorescence peak was recorded. It is noticed that a significant shoulder appeared in the tail of the peak, which caused a tailing problem for backward elution. This was

Figure 5. Fluorescence-time traces of ion-enrichment and elution cycles monitored with an LIF detector. Profile I was recorded at detection point DB (LB ) 5 mm, referred to Figure 1) for backward elution, while Profile II was recorded at DF (LF ) 5 mm) for forward elution. The bias voltages and Peltier set temperatures used are listed in Table 1. The dotted line indicates the open/closed status of the PNIPAAm hydrogel plug, and the blue-colored, vertical arrow denotes the time when the polarity of the applied voltage was switched in the forward elution test. A 5 nM FITC solution was prepared in 100 µM borate buffer solution and was used for these experiments. A 1 mm long PNIPAAm hydrogel plug was synthesized inside the glass microchannel.

rationalized by the fact that the preconcentrated band was half in free solution and half within the hydrogel plug (see Figure 2b). Thus, while the portion of FITC ions enriched in the free solution was quickly delivered by the EOF to move downstream, some of the FITC ions trapped within the hydrogel plug network would suffer from adsorption effects to the collapsed PNIPAAm network. The reproducibility of the total ion-enrichment and backward elution was favorable (RSD for peak-appearing time was 2.6% and that for the peak area was 7.4%, n ) 3). The recorded traces of fluorescence signal versus time for three parallel preconcentration and backward elution operations can be found in Figure S-2(a) of the Supporting Information. Another way to elute the concentrated band from the hydrogel plug was the use of forward elution. With this elution mode, the polarity of the applied voltage was reversed once the hydrogel plug was heated to place in the open condition. Thus, the EOF delivered the concentrated band toward the buffer reservoir (reservoir B, see Figure 1). The fluorescence-time profile recorded at DF (see Figure 1) is shown as Profile II in Figure 5. Compared to the signals obtained with the backward elution, the peak height significantly decreased with a corresponding reduction the peak area. The reduction in peak area could partially be ascribed to that portion of the FITC molecules absorbed to the hydrogel plug due to its hydrophobic nature at temperatures above its LCST. It was interesting that the peak width at this condition was narrower than that observed in the backward elution process [2.32 ± 0.13 s, n ) 3, for the forward elution versus 2.97 ± 0.09 s for backward elution] despite the fact that the preconcentrated band for the forward elution process should penetrate the PNIPAAm hydrogel plug before reaching detection point DF. At this point, we cannot exactly explain these results. Nevertheless, we suspect it may be related to reversion of voltage polarity that may partially destroy the discontinuity of the electrolyte medium along the channel. One significant merit

Figure 6. Electropherogram of preconcentrated FITC-labeled amino acids detected at the forward detection point with LF ) 6.5 mm. FITClabeled Ala and Gly, at a concentration of 3 nM for each, were subjected to preconcentration for 173 s, forward elution, and finally a CE separation. A 0.2 mm long PNIPAAm hydrogel plug was synthesized in the glass channel and used for preconcentration. Other conditions are as described in Figure 5.

of forward elution over backward elution lies in the absence of peak-tailing. With forward elution, the RSD for the peakappearing time was 0.71% and the peak area RSD was 5.5% (n ) 3) (see Figure S-2(b) of the Supporting Information). The unique advantage of the present electrokinetic ion enrichment/elution system over the previously reported ion-enrichment systems lies in that relying on the thermoswitched open/close action of the hydrogel plug, the present system not only permits the concentrated band to be backward eluted to perform CE separation15,16 but also allows the concentrated band to penetrate forward through the hydrogel plug for following operations. Especially, the forward elution mode is somewhat similar to the process that ionic or molecular species penetrate through the nanopores of bio- and artificial membranes. Thus, it might be promising for the present ion-enrichment/elution system to be adopted in the bio- and medical sciences for such research and applications as drug release, ion transport in cell biology, etc. Electrophoretic Enrichment and CE Separation of Amino Acids. This thermoswitchable ion-enrichment and forward elution process was used for the online preconcentration and CE separation of FITC-labeled amino acids. Figure 6 shows a typical fluorescence-time trace for the preconcentration, elution, and CE separation. After 173 s for ion-enrichment and 100 s for the elution and separation, FITC derivates of Ala and Gly (starting concentration ) 3 nM) produced two peaks that were partially resolved. Several reasons may be responsible for the two amino acids not being baseline separated: (i) a relatively long preconcentrated band was subjected to CE. (ii) The separation medium in both the plug and channel were not continuous, consequently producing extra band broadening due to slightly different EOFs that might produce some laminar flow (even recirculating flow). (iii) The separation conditions were not purposely optimized. Even so, the preliminary data demonstrate that the potential of the thermomodulated, electrokinetic ion-enrichment and elution/ separation protocol is viable for sample pretreatment using a microchip. Analytical Chemistry, Vol. 82, No. 24, December 15, 2010

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CONCLUSION The thermoresponsive PNIPAAm hydrogel plug synthesized inside a glass microchannel showed ion-selective permeation behavior at a temperature below its LCST and electroosmotic flow supporting behavior at a temperature above its LCST. This thermoswitchable behavior resulted from the thermoswitchable change in its polymeric network structure. At a temperature below the LCST, some nanopore structures formed in the polymer network due to swelling of the PNIPAAm hydrogel network. Consequently, the PNIPAAm plug served as a cation-selective membrane that can lead to concentration polarization occurring near the nanopore and microchannel interface. At a temperature above the LCST, larger pore structures dominated the network due to shrinking of the hydrogel network, which allowed the hydrogel plug to support an EOF. Although a few problems were encountered, such as the preconcentrated band showing broadening and loss of analytes due to adsorptive behaviors, these preliminary investigations have demonstrated the potential of the composite PNIPAAm plug in a glass microchannel to perform as an online thermoswitchable ion-concentration/CE separation or assay device for lab-on-chip systems. In addition, it is known that

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there exists nano- or micropores in biomembranes, which play a key role in transmembrane mass transport. As the LCST (32 °C) of the PNIPAAm is close to normal body temperature, the composite PNIPAAm hydrogel plug in a microchannel might be used as a biomimetic device in the bio- and medical sciences, for applications such as drug release, ion transport in cell biology, and tissue engineering. ACKNOWLEDGMENT This work was funded by the National Natural Science Foundation of China (project nos. 20775068 and 20890020) and the National Basic Research Program of China (973 Program, project no. 2007CB714502). SUPPORTING INFORMATION AVAILABLE Additional information as noted in the text. This material is available free of charge via the Internet at http://pubs.acs.org. Received for review July 13, 2010. Accepted November 2, 2010. AC101768J