MINIATURISATION FOR CHEMISTRY, BIOLOGY & BIOENGINEERING A flexible sample introduction method for polymer microfluidic chips using a push/pull pressure pump Zhiyong Wu, Henrik Jensen, Jean Gamby, Xiaoxia Bai and Hubert H. Girault* Laboratoire d’Electrochimie Physique et Analytique, Ecole Polytechnique Fe´de´rale de Lausanne, CH-1015 Lausanne, Switzerland. E-mail: [email protected]; Fax: 141.21.693.36.67; Tel: 141.21.693.31.51 Received 21st July 2003, Accepted 18th May 2004 First published as an Advance Article on the web 22nd July 2004 A push/pull sample introduction method based on push/pull pressure flow for microfluidic systems (cross, double T and multichannel structures) is presented. This leads to well-defined and controllable sample plugs even when dealing with long channels. By tuning the relative push/pull pressure, it is shown that the size of the sample plug can effectively be controlled. Good signal reproducibility upon continued sample introduction and subsequent chip electrophoresis employing fluorescence detection is demonstrated for different chip geometries (i.e. short channels and long channels). Since the performance of the method is relatively insensitive to chip geometry, it is particularly useful for polymeric prototype microchips as tedious optimization is not required. Furthermore, the push/pull sample introduction is extended to multichannel chips thus demonstrating the possibilities of applying the methodology for realizing single chip high throughput sample analysis. Instrumentation Introduction The advent of micro Total Analysis Systems (mTAS) in analytical chemistry has received a lot of attention in recent years.1,2 Although much progress has been achieved in terms of general chip design, such as chip electrophoresis for sample separation and detector integration, a reliable and flexible sample introduction remains as a challenging task. Most of the current methods rely on electrodynamic injection, although in some cases, this procedure has proven to be problematic, since flooding or leakage may occur.3,4 Meanwhile, a certain sample bias (depending on the analyte mobility) is introduced in response to the high electric field used for the sample introduction.3 This problem is particularly noticeable for polymeric microfluidic chips as the surface stability and properties are difficult to control. One solution to remove this injection bias is to use the double-T injection design together with a long enough injection time.5 Another effective solution could be based on the pressure injection which is insensitive to the channel surface and the sample properties.6–11 In this paper, a pressure induced sample introduction procedure is presented. Basically the non-biased sample plugs are introduced in a separation channel by applying a push and pull pressure simultaneously at respectively the sample and sample waste reservoirs. It is demonstrated that for polymer chips the new method is comparable to the existing methods in terms of reproducibility and furthermore the sample dilution by the buffer is avoided. Finally, the setup is extended to the multichannel chips which are suitable for general high throughput sample analysis. Experimental DOI: 10.1039/b308405a Chemicals and materials 512 The PET chip substrate (100 mm, Melinix S grade) was purchased from DuPont (Switzerland). A 20 mM (pH ~ 7) phosphate buffer (Fluka, Buchs Switzerland) was used as the running buffer. Fluorescein sodium salt and fluorescein biotin were obtained from Sigma (St. Louis, MO, USA). Deionized water (18.2 MV) was prepared using a Milli-Q system from Millipore (Bedford, MA, USA). Lab Chip, 2004, 4, 512–515 Axiovert 200 (Zeiss) equipped with a CCD camera CF 8/4 (Kappa, Gleichen, Germany) was used for fluorescence detection with a UV excitation source Lamp HPO 100/2 W (Mercury lamp) and the filter was set for fluorescein (495/ 519 nm). A high voltage source (Spellman CZE 1000R Power Supply, Hauppauge, NY, USA) was used for electrophoresis. Acquisition of fluorescence using an IMAQ PCI-1409 card (National Instruments) and the high voltage was computer controlled using a homemade LabView program (National Instruments). The dual syringe pump (Model 260) was from Kd Scientific (USA). The switch valve with plastic pump body (CHEMINERT2) was from VICI (Valco Instruments, Switzerland). Inner diameter of the tube for the peristaltic pump was 0.13 mm. The internal diameter of the plastic connection tube was 0.25 mm. The volume of the glass syringe (Hamilton) was 100 ml. Fabrication of the microchips The microchip of PET substrate was fabricated using an excimer laser (Argon Fluor Excimer Laser operating at 193 nm (200 mJ, 50Hz) from Lambda Physik LPX 2051, Go¨ttingen, Germany). The fabrication process is similar to that reported elsewhere.12 At first, a main separation channel (longitudinal) was photoablated with two cavities (reservoirs) at both ends. Then, two transversal sample channels (1 cm long), upward and downward with respect to the main channel, are photoablated vertically with a typical center-to-center distance of 200 mm between them to form the double-T injection design. In order to reduce flow resistance (back pressure), the sampling channels are 4 times wider than the main channel. In the case of chip with multi separation channels, the upward sample channel was aligned with respect to the bottom separation channel, while the downward sample channel to the top one. It has to be pointed out that in this case, the junctions between the separation channels and the sample channels have a double depth. However, no negative effect of this depth difference on the separation has been observed. Finally, the microchannel is thermally bonded with PE using a lamination apparatus (Morane pneumatic Senator Laminator, Oxford, UK). This journal is ß The Royal Society of Chemistry 2004 Fig. 1 Sample introduction by pressure-induced push/pull flow using a switch valve. The solid line connection on the valve represents the sampling state and the dotted line the stand-by state. Sampling process First the sample flow in the sampling loop was initiated to make sure the double-T region in the main channel is filled. Then the flow in the separation channel was started immediately after the sampling loop was switched to the stand-by position (Fig. 1). The polymer electrophoresis chip was fixed with plastic blocks through which the plastic tubes were connected with the valve and the pump. Safety warning: attention should be paid to the sampling loop since high voltage leakage may occur through the sample solution and connection tubes even though the valve body and connection tubes are all made of plastic. Results and discussion The advantages of using synchronized push and pull pressures is that sample flooding in the separation channel can be minimized and the sample dilution by the running buffer is avoided. When a well-defined sample plug has been formed (Fig. 2a), the injection valve is switched to equalize the pressure on both sides of the double-T and a high voltage field is applied along the separation channel. An electroosmotic flow is established which drives the sample plug along the separation channel (Fig. 2b and c). It can be observed that a substantial dilution takes place in the sample waste channel following the separation step; this phenomenon is caused by a pressure gradient between the open reservoir in the separation channel and the waste reservoir in the sample introduction channel. An important parameter for the shape of the sample plug is the push/pull flow ratio, r. As shown schematically in Fig. 3 three situations can be encountered. For r v 1 the plug size is reduced as part of the running buffer in the main channel is pulled out of the channel (Fig. 3a). In Fig. 3b the push and pull pressures are equal (r ~ 1) and the buffer in the separation channel is left unchanged. Finally, for r w 1 (Fig. 3c) the sample plug increases over time as more sample is pushed into the channel than pulled out of it. Depending on the value of r, different sample amounts are introduced into the separation channel. It is thus in principle possible to effectively control sample shape and amount by tuning the push/pull pressure ratio. In practice, it is usually advantageous to work with a small degree of self-pinching (r v 1) to prevent for sample Fig. 2 Images of sample introduction by push/pull flows. a, b and c show the injection and switch process respectively. The sample flow direction is from bottom to top and migrated to the right. The microchannels are 50 mm wide and 50 mm deep. Fluorescein was used as indicator dye. Fig. 3 Three sample plug patterns corresponding to the push/pull sample introduction at different flow ratios. Sample flow direction from down to top; a: r v 1; b: r ~ 1; c: r w 1, where r is the flow rate ratio of push and pull flows respectively. flooding into the separation channel and thereby a time independent sample plug. Electro- and push/pull sample introduction were compared under similar experimental conditions (i.e. using the same chip and separation voltage). The separation of fluorescein and fluorescein biotin present in the sample was carried out. For electroinjection, the injection voltage is 400 V. For both injection methods, the injection time was 60 s and a separation voltage of 800 V is applied. The EOF in the channel was in the direction of the electric field (i.e. positive to negative along the separation channel) fluorescein therefore reaches the detection point after fluorescein biotin since their electrophoretic migration and the EOF have opposite directions. The migration time, t, is give by eqn. (1): t~ L L ~ vep zveof (uep zueof )E (1) where vep and veof are the electrophoretic velocity and the electroosmotic velocity of the sample respectively, uep and ueof are the electrophoretic and the electroosmotic mobility respectively, E is the electric field strength and L is the detector position. The push/pull sample introduction (the flow rate was 0.4 ml min21 in the sampling channel) results in well-defined peaks illustrating an effective separation of fluorescein and fluorescein biotin as shown in Fig. 4. For six sequential injections of 2 mM fluorescein, a relative standard deviation (RSD) of 4.8% and 1.5% have been obtained corresponding to the peak height and the migration time of the signal respectively, which implies a satisfactory reproducibility. Meanwhile, the same experiment using the electroinjection Fig. 4 Fluorescein biotin and fluorescein electropherograms obtained by chip capillary electrophoresis with a push/pull flow rate of 0.4 ml min21 (syringe pump). The running buffer was a phosphate buffer (20 mM, pH ~ 7); separation voltage is 800 V over 22 mm long channel (100 mm wide, 45 mm deep; double-T injector with a center-tocenter distance of 200 mm long). Lab Chip, 2004, 4, 512–515 513 Fig. 5 Electrophoregram of fluorescein and fluorescein biotin obtained in a long microchannel (13 cm long with a width of 50 mm and a depth of 50 mm). The applied voltage was 4 kV over 13 cm long channel with an effective separation channel length of 11 cm (a). The ‘‘Swiss roll’’ structure is shown in (a) and the separation of fluorescein and fluorescein biotin (both 0.3 mM in 20 mM phosphate buffer of pH ~ 7) is shown in (b). has shown a lower signal sensitivity due to the sample dilution effect and a poor reproducibility (i.e. RSD of migration time up to 20%). The difference in migration times can be ascribed to differences in the EOF, which could be due to different degree of surface oxidation in the laser ablation process.13,14 The reproducibility in the case of electroinjection can, at least partially, be related to a high voltage induced sample bias during injection, but other factors also plays a role.7 For instance electroinjection is also dependent on the nature of the channel surface (i.e. the zeta potential), non-constant Laplace pressures, siphoning effects and solvent evaporation from the sample reservoirs.7 One of the advantages of photoablation microfabrication is that the length of the separation channel can be increased without significantly increasing the fabrication costs. Fig. 5b shows the separation of fluorescein and fluorescein biotin using the push/ pull sample introduction method in a long channel, i.e. with a total length of 13 cm. In this case, the setup is a bit different as the flexible polymer substrate is bend in a roll structure so that the inlet and outlet are fixed on the same base as shown in Fig. 5a. The three-dimensional roll structure is practical when the separation chip is to be incorporated into an actual device. Upon six repeated experiments, a RSD of 11.5% and of 2.4% are obtained corresponding to the fluorescein signal height and to its migration time respectively, which illustrates that the push/pull sample introduction procedure is also feasible in combination with a long separation channel. The main point to emphasize is that even for the relatively long separation time, no sample leakage into the separation channel was observed. In order to explore the possibilities of using the push/pull method in a high throughput format, a separation chip featuring multi-parallel channels was fabricated (Fig. 6a; the parallel separation channels are separated by 200 mm). By loading the sample across the channel array, well-defined plugs could be formed as demonstrated by the fluorescent image in Fig. 6b. Similar to that is shown in Fig. 2c, when the valve is switched, the sample is injected in the multichannel and then separated, as shown in Fig. 6c. The array channels shared the same inlet and outlet reservoirs; the same electrical field for separation was thus applied for all the channels. As for the single channel polymer microchips, effective separation of fluorescein and fluorescein biotin has been observed with a RSD of 2.8% (peak height) and 6.0% (migration time) between the four separation channels. The multichannel chip thus demonstrates that simultaneous parallel analysis is in principle possible using the present sample injection method. In a further developed system, it can be envisioned that each channel is optimized to separate and analyze a specific analyte. In this way a complete sample analysis can be conducted employing only one microchip thus significantly reducing analysis time and required sample amount. In conclusion, a new sample introduction method based on push/pull pressure induced flow has been developed and tested on polymeric microfluidic chips. The new method has a number of advantages compared with the more conventional procedure Fig. 6 Channel-array chip with a double-T injector structure for push/pull pressure flow injection; the 4 separation channels share the same inlet and outlet reservoir through which the separation voltage was applied (a). (b) shows the fluorescent image of the sample loading employing the pushpull procedure, and (c) represents the image of the separation. 514 Lab Chip, 2004, 4, 512–515 based on electro-injection. First of all, no sample bias due to charge discriminations is introduced during sample injection and no disturbance of the buffer in the separation channel occurs. Due to the use of simultaneous push and pull flows, the sample dilution, which is observed for pinch injection mode, is avoided. The quality of the signal is independent of injection time and the reproducibility upon repeated sample injections is comparable to the results obtained using electroinjection under similar conditions. In addition, it is possible to control the plug size and introduce sample pinching by tuning the push/pull pressure ratios. When using a long separation channel, the advantages of using a flexible polymeric substrate is clearly illustrated as the chip can be bend without affecting the separation performance. The present method was also extended to a novel microchip design featuring a series of parallel separation channels, which could be utilized for studying complex (for instance biological) samples. The advantage is that only one sample introduction is needed for carrying out simultaneous independent multianalyte detection, thus reducing dead volume (and thereby required sample amount) as well as the analysis time. Although the present investigation has only focused on electroosmotic flow in the separation channel, it is easy to envisage that the method will also work for pressure-induced flow as encountered in flow injection analysis or chip chromatography.8 It can thus be stated that the flexible sample introduction method described in this paper has the potential to find wide general applications for analytical devices incorporating microfluidic chips. Acknowledgements Dr Jacques Josserand (EPFL) and Dr Joel S. Rossier (Diagnoswiss) are thanked for fruitful discussions and Valerie Devaud is gratefully acknowledged for technical support. The EU is acknowledged for financial support through the project ‘‘MicroProteoMics’’ (grant no. QLG2-CT-2001-01903 / OFES 01.0182-2). 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