Zhiyong Wu1

Nicolas Xanthopoulos2

Frédéric Reymond1

Joël S. Rossier1

Hubert H. Girault1

1Laboratoire d’Electrochimie,Département de Chimie

2Laboratoire deMétallurgie Chimique,Département de Matériaux,Ecole Polytechnique Fédéralede Lausanne,Lausanne, Switzerland

Polymer microchips bonded by O2-plasmaactivation

This paper presents a fabrication of polymer microchips with homogeneous materialtechnique due to surface treatment by plasma before sealing. UV laser photoablationwas used for fast prototyping of microstructures, and oxygen plasma was used as asurface treatment for both the microfabricated substrate and the polymer cover. It wasfound that with an oxidative plasma treatment, successful bonding could be achievedwithout adhesive material between polymer sheets substantially below the glass tran-sition temperature of the polymer. Homogeneous polyethylene terephthalate (PET)microstructures were characterized by scanning electron microscopy (SEM) and ana-lyzed by X-ray photoelectron spectroscopy (XPS) surface analyses after different sur-face treatments. The electroosmotic flow characteristics including the velocity and thestability over 20 days have been tested and compared to composite channels, in whichthe cover presents a polyethylene (PE) adhesive layer. Capillary zone electrophoresis inboth homogeneous and composite microanalytical devices were then performed andcompared in order to evaluate the separation efficiency. In preliminary experiments, aplate height of 0.6 �m has been obtained with homogenous microchannels. The sur-face analysis pointed out that the surface chemistry is of prime importance for theperformance of microfluidic separation.

Keywords: Double T pinched injection / On-chip electrophoresis / Photoablation / Plasma treat-ment / Polymer substrate EL 4814

1 Introduction

The field of micrototal analysis systems (�TAS) hasbecome a growing research area in the last decade andhas provided fast and parallel analytical tools applied todifferent fields such as DNA [1] or protein separation [2].Different fabrication strategies have been shown to pro-vide �TAS in prototype or industrial scale. Some applica-tions such as clinical diagnostics may need a mass pro-duction with high reproducibility and good aging proper-ties. Therefore, after the demonstration of the conceptsas well as the commercialization of the first �TAS on glasssubstrate, a lot of effort has been placed in the develop-ment of plastic chips with different technologies [3]. Plas-tics provide the advantages of high replication and diver-sity of material as well as easier bonding between thechannel plate and the cover [3]. Photoabliation [4], LIGA(LIGA = LIthographie Galvanoformung Abformung, a Ger-

man acronym for lithography, plating and molding) [5]and casting [6, 7] have been developed for prototypingmicrosystems in materials such as polymethylmethacryl-ate (PMMA), polycarbonate (PC), polystyrene (PS), poly-ethyleneterephthalate (PET) or poly(dimethylsiloxane)(PDMS). Other technologies such as injection molding[8], imprinting [9] or, more recently, plasma etching [10]have demonstrated the feasibility of mass production ofmicroanalytical devices. A common requirement of eachfabrication technology is the sealing of a cover plate overthe microstructure at the end of the fabrication. Thermalbonding is often used to seal polymers with low glasstransition temperature and melting point for example forPMMA, PC or PS [3]. Nevertheless, the contact withaggressive solvent, such as chloroform or dichloroethaneor simply acetonitrile for chip coupling to a mass spectro-meter still demand the use of stable polymer substratessuch as PET or polyimide [10]. Some applications withhigh-temperature exposure (�100�C) require higher per-formance polymers (as, e.g., PET) that have a glass tran-sition temperature between 140 and 180�C. However,sealing of microstructures with the same constitutivematerial by thermal bonding becomes difficult for thistype of polymers.

In a previous paper, PET microanalytical devices weresealed thanks to a two-layer lamination sheet, consistingof PET and polyethylene (PE), the latter acting as a ther-

Correspondence: Prof. Hubert H. Girault, Laboratoire d’Electro-chimie, Département de Chimie, Ecole Polytechnique Fédéralede Lausanne, CH-1015 Lausanne, SwitzerlandE-mail: hubert.girault@epfl.chFax: �41-21-693 36 67

Abbreviations: PC, polycarbonate; PDMS, poly(dimethyl-siloxane); PE, polyethylene; PET, polyethylene terephthalate;PMMA, polymethylmethacrylate; PS, polystyrene; XPS, X-rayphotoelectron spectroscopy; �TAS, micrototal analysis system

782 Electrophoresis 2002, 23, 782–790

ª WILEY-VCH Verlag GmbH, 69451 Weinheim, 2002 0173-0835/02/0503–782 $17.50+.50/0

mal adhesive [4]. The consequence of this technology isthat the internal surface of the microchannel is inhomo-geneous, with three walls of photoablated PET and oneof PE. This results in undesired effect on the electro-hydrodynamics of the microsystem, such as Taylor dis-persion and reduced separation efficiency [11]. Plasmatreatment is one means of modifying polymer surfacesto improve adhesion while maintaining the desirableproperties of the bulk material. This technique is mainlyused in the textile fiber industry and electronics wherehomogeneous bonding is required [12–15]. Plasma tech-nique has also been used for the bonding of PDMS micro-channels by simple contact, mainly to obtain hydropho-bic walls [16–18]. However, PDMS suffers from a limitedlife span in terms of stable electroosmotic flow (EOF),and its elastic property may also be a drawback in keep-ing a stable geometry. Also the sorptive properties ofPDMS make this polymer difficult to work with in manycases, because the sample is easily adsorbed onto thewalls of the microstructure or even diffuse into the sub-strate. Therefore, there is still an important demand formore rigid polymer substrates with longer life span, con-stant EOF, stable geometry, high reproducibility and highsolvent stability, as well as for homogeneous micro-chips (i.e. microchips where the substrate supporting themicrostructure and the cover layer are made of the samematerial).

As an answer to such requirements, this paper presents aprocedure for manufacturing homogeneous microchipswhere the surfaces of the substrate and cover plate aretreated by plasma or laser photoablation in order toenable low temperature sealing. This paper shows thebonding properties of these physical treatments and alsohighlights the EOF stability over 20 days of both homo-geneous PET chips (PET as cover plate) and compositePET-PE one (PET/PE lamination sheet as cover). Finally,separation performances of both types of chips are com-pared in simple electrophoretic separations of fluorescentprobes.

2 Materials and methods

2.1 Materials

PET sheets (100 �m thick Melinex S grade) were pur-chased from Dupont (Geneva, Switzerland). This sub-strate was used both for microstructure fabrication andas cover plate for homogeneous chips. Phosphate buffer,pH 7.0, was purchased from Fluka (Buchs, Switzerland).Fluorescein sodium salt and fluorescein biotin wereobtained from Sigma (St. Louis, MO, USA). The dyeswere dissolved in 13.4 mM phosphate buffer, and the

stock concentrations were 1.9 mM and 1.2 mM for fluores-cein and fluorescein biotin, respectively. During the elec-trophoresis, the samples were diluted with the runningphosphate buffer. Deionized water (18.2 M�) was pre-pared using a Milli-Q system from Millipore (Bedford,MA, USA).

2.2 Laser excimer for microstructure generation

The microstructures were fabricated by UV laser photo-ablation following previously described protocols (ArgonFluor Excimer Laser at 193 nm; Lambda Physik LPX2051; Göttingen, Germany) [4]. The typical channelobtained with this procedure has a trapezoidal shapewith a depth of 48 �m and a width of 58 �m at the topand of 37 �m at the bottom. The channel length was 2 cmfor EOF evaluation. For electrophoresis experiments, themain channel was 4 cm long and the arm length was1 cm.

2.3 Oxygen plasma stripper

Plasma is the fourth state of matter besides the solid,liquid and gaseous states. The main difference comparingto the gaseous state is that plasma is kept in a very highenergy level. It consists of neutral, radical and chargedparticles, and may be also accompanied with UV and visi-ble UV radiations [12, 13]. Because of this character,plasma is very reactive. Surface treatments thereforeinvolve very complex mechanisms in response to theplasma type, material, atmosphere, and operation condi-tions, resulting in different properties, which may be usedfor various purposes. Plasma treatment usually affectsonly the top layer in the nanometer range, but not thebulk. This property is especially useful for the treatmentof polymer substrates with fine microstructures. Thereare many methods of generating plasma, among whichcold type plasma is especially suitable for preventing thedeformation of the polymer substrate. The main purposesof using plasma in this paper are first to achieve bondingof two polymers sheets without any adhesive and, sec-ondly, to enhance the electrophoretic performances bychemical changes of the channel walls. Microwave-induced oxygen plasma generated by plasma stripper(Micro-Ondes 300, Carrollton, TX, US) with 2.45 GHzwas used in this work. The polymer substrates were intro-duced into the oven on a quartz plate or by means of aPyrex glass holder. Oxygen flow was 400 mL/min, witha pressure of 1 mbar, and the power was maintained at300 W during 15 s.

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Miniaturization

2.4 X-Ray photoelectron spectroscopy (XPS)characterization

Surface analyses were performed with a X-ray photoelec-tron spectrometer (Kratos Axis Ultra, Manchester, UK).Samples were rinsed with deionized water, and driedunder nitrogen flow before XPS analysis.

2.5 Sealing of the microstructures

The two goals of the present paper are to fabricate homo-geneous channels with the same PET material for themicrochannel as for the cover plate and to compare theirperformance with photoablated PET microchannels,sealed with a PET-PE laminate. Therefore, two laminationstrategies have been used: The standard PET/PE lamina-tion was performed at 130�C and 3 bar pressure (Morancepneumatic Senator Laminator, Oxford, UK) as publishedelsewhere [4, 19]. The novel lamination procedure con-sists in placing two plasma-treated PET sheets togetherimmediately after the plasma treatment without anycleaning procedure. The two sheets are then placed in-between two aluminum foils and rolled through a lamina-tion machine (RLM 419P Laminator, Switzerland) at atemperature of typically 124�C, at 20 cm/min with 24 kgforce between the rolls. Delamination tests have beenperformed by pulling the two PET plates in order to de-laminate them. The plasma treatment duration and poweras well as the lamination temperature have been opti-mized empirically until a good sealing was obtained, i.e.delamination was no longer possible, and pulling ledinstead to tearing one of the PET layers.

2.6 EOF evaluation

The measurement of EOF has been performed followingHuang’s current monitoring method [20]. The principleof the measurement is to follow the conductivity changein a channel by monitoring the current at a constantvoltage. When a solution of slightly different conductivityis pumped inside a microchannel containing a secondsolution by applying a high voltage, there is a decreaseand finally a stabilization of the current, which indicatesthe time required by the solution to run across the chan-nel. Measurements were performed with 10.7 mM and13.4 mM buffer solutions, which were electrokineticallypumped through the channel by application of a voltageof 300 V (Spellman CZE 1000R Power Supply, Haup-pauge, NY, USA). Following the technique fully describedelsewhere [4, 20], special attention has been given to thevolume of the drops laid on each channel entrance. It hasbeen indeed observed that a siphoning effect could easilyoccur during such experiments if the size of the drop is

not similar on both channel reservoirs. This phenomenonis particularly pronounced here because of the smalllength of the channel used for the present experiments.In order to consider a measurement as valid, the electro-osmotic EOFs flows were always measured in both direc-tions (starting with the lower concentration and then withthe higher concentration). The measurements were con-sidered relevant when both directions of flow gave thesame results (less than 10% variation). With this strategy,it was possible to identify artefacts that can occur fromthe above-mentioned siphoning effect. Each channelwas measured four times in both directions. It is to precisehere that the current results have not been undertaken togive a complete description of the zeta potential of thechips but are used for relative comparison between thedifferent surface states.

2.7 CZE separation with pinched injectioncontrolled with only one power supply

The performances of the homogeneous and compositemicrochannels have been studied in CZE separationexperiments. To this aim, controlled injection proceduresshould be used in order to avoid leakage of the injectedplug prior to the separation. Several approaches havebecome standard for injecting sample without leakage,among them, pinched cross [21] and double T injection[22]. The pinching consists in controlling the potential ineach reservoir of a four-branch microsystem both duringinjection and separation steps in order to minimize thedispersion of the plug by diffusion and leakage duringseparation. This technique presents the drawback to useat least three different power supplies for voltage controlduring injection and separation. If some strategies toreduce the power source number have been presented[23], the concept was not applicable to pinched double Tor cross injections. In this paper, we first demonstrate theuse of a single power supply connected to an individuallycontrolled 8-positons relay box controlled by Labviewsoftware (National Instruments Austin, TX, USA). The sys-tem consists in the application of a single potential that isdistributed to eight electrode pairs individually connectedto the switch box as shown in Fig. 1. Depending on theintensity of the high voltage to be applied in the differentreservoirs, a tunable resistance of typically 10–100 M�is connected in series with the microchannel arms. Theseresistor values represent the typical value of the filledmicrochannel. Figure 1A shows the connection patternduring the injection: a high voltage is applied directly tothe sample reservoir (S) through the relay No. 6 but isalso distributed to the buffer reservoir (B) through therelay No. 1. The tunable resistance is connected in serieswith the channel in order to decrease the potential

784 Z. Wu et al. Electrophoresis 2002, 23, 782–790

Figure 1. Schematic description of the setup used todirect the potential in the right arm during (A) injectionand (B) separation; the electric potential is applied from asingle power supply to a multirelay box that distributesthe voltage in parallel and switches any gate from open(the potential is applied to black lines) to closed position(potential is not applied to grey lines); during the injection,the potential is therefore directly applied to the samplereservoir (S) but also to the running buffer reservoir (B)through a resistance (see text for details). During theseparation, the potential is applied directly to the runningbuffer reservoir (B) and the side arms are also groundedthrough a resistance in order to avoid leakage thanks apush back effect.

applied to this reservoir. No resistance is added to thewaste reservoir (W) from the relay 8 because the channellength from the reservoir to the double T is large enough todecrease the applied potential. In this manner, a fluo-rescence image of the plug can be performed and the

resistance can be tuned until the pinching is consideredto be efficient. The optimal resistance may vary withrespect to the conductivity of the buffer used for theexperiment. The separation pattern is actuated by switch-ing the multirelay box to the distribution represented inFig. 1B, where the high voltage is now applied directly tothe reservoir through the relay No. 2. Both side arms arenow connected to the ground through relays 3 and 4,respectively, and the current is forced through two tunableresistances in order to induce a push back effect withoutinducing too high ionic current in these sections. Thewaste reservoir is now connected to the ground throughrelay No. 7. For electrophoresis, the structure included adouble T injection volume of about 600 pL and a lengthof 290 �m between the side arms. 300 V was appliedbetween the sample and the waste reservoirs with respectto the above described pinching technique and 1200 Vwas applied through the 5 cm length main channel posi-tioned under a fluorescence microscope (Axiovert 25,Lamp HPO 100/2 W, 488 nm excitation and 519 nm fluor-escence emission; Zeiss, Jena, Germany). The detectionwindow of approximately 100 �m�60 �m was chosenthanks to CCD data acquisition and recording software(CCD camera CF 8/4; Kappa, Gleichen, Germany; IMAQboard PCI-1408, National Instruments). The monitoringposition could be chosen along the separation channel.Similarly to the EOF measurements described above, aspecial attention has been given to avoid siphoning effectprior to run an experiment. 13.4 mM phosphate buffer solu-tion was used for CZE experiments. Dye probes werediluted with the same running buffer.

3 Results and discussion

3.1 XPS characterization of plasma andlaser-modified PET

Different surface treatments have been performed to thesubstrate by laser or plasma in order to either fabricatemicrostructures or to enable efficient bonding betweentwo PET sheets. After these treatments, the study ofthe surface by XPS is essential in order to understandbonding and flow characteristics obtained in the differentchannels. XPS studies give semiquantitative and qualita-tive information about the chemical composition of thesurface and, for instance, on the oxygen-to-carbon ratio(O/C) as evidenced in Fig. 2. The O/C ratios of polymersurfaces modified by oxygen plasma and further washedwith water are reported in Table 1. As expected, this ratiohas been dramatically increased by the plasma exposi-tion from 0.49 to 0.91. It is remarkable that after waterwashing of the surface the O/C ratio decreases to 0.63,suggesting that some species such as low-molecular-

Electrophoresis 2002, 23, 782–790 Polymer microchips borded by O2-plasma activation 785

Figure 2. XPS spectrum of the PET substrate (A) non-treated, (B) treated by plasma and washed. It is interest-ing to observe the O/C ratio for the different treatment(see details in text).

weight redeposited and oxidized debris may have beenremoved from the surface. This observation is similar tothe dynamic laser ablation of PETor polystyrene reportedin different previous reports [24–26]. Due to the complexconvolution in XPS spectra, it is difficult to determine,which functional groups have been created on the PETsurface. However, interpretation of the XPS results high-lights the activation effect of oxygen plasma, which willserve bonding and EOF characteristics.

As already explained, the microstructures are fabricatedby laser ablation and in previous papers it was usedwithout any further surface activation by oxygen plasma.In contrast to O2-plasma processing, laser treatmentinduces dramatic decrease of surface O/C ratio, whichwas interpreted as a reduction of the surface functionalgroups [27]. If plasma and laser activated PET have

Table 1. Summary of XPS analyses in terms of O/C massratio

No. Sample description O/C Note

1 Standard 0.40 [33]2 Sample native surface 0.49 Melinex S3 Dynamic ablated surface 0.38 From [34]4 No. 3 washed 0.22 From [34]5 Plasma-treated surface 0.91 Ready for bonding6 No. 5 washed 0.63 Final cover ready for EOF7 No. 4 plasma-treated 0.88 Channel walls and bottom8 No. 7 washed 0.54 Final channel ready for EOF

already been characterized, it was important to studythe surface state of the polymer after laser fabrication fol-lowed by plasma oxidation. The results presented inTable 1 clearly shows that the laser-treated surface, whichis strongly reduced after the fabrication can also be oxi-dized by oxygen plasma to an O/C ratio of 0.88. Washingsteps also reduced the presence of oxidized debris.Therefore, the plasma-activated photoablated surface iscomparable to the nonlaser-treated activated surface.As a conclusion, it can be observed that the channelsfabricated by laser and treated by plasma have a similarO/C content as the PET surface treated by plasma (0.88and 0.91, respectively). Furthermore, the washing stepreduces this ratio in the same order to 0.54 and 0.63,respectively. It is therefore expected that the photoablatedsurface treated by oxygen plasma is similar to a plasma-activated PETsurface without laser processing. It is actu-ally the goal of this article to seal a photoablated micro-channel in PET with a PET cover, both of these materialshaving been treated by oxygen plasma. Such a channelshould be fairly homogeneous and should support EOFwith a minimal of Taylor flow dispersion [11]. These chan-nels will then be compared with standard photoablatedchannels sealed with the standard PET/PE laminationprocedure. The situation of laser-treated surface closedwith the standard PET/PE lamination is of course differ-ent, and the very low O/C ratio of 0.22 observed in thiscase means that the surface state is very different fromthe plasma treated one. Therefore, to characterize thepreviously observed surface, different EOF measure-ments and capillary electrophoretic separations are per-formed here.

3.2 Bonding of PET-PETsheets

The standard bonding technique developed in our labora-tory for more than five years [4] in order to seal polymersheets is based on the lamination of an oxidized filmof PE by a soft lamination technique [25]. Though themechanical stability is satisfactory, Taylor dispersion and

786 Z. Wu et al. Electrophoresis 2002, 23, 782–790

Figure 3. Photographs of the photoablated microchan-nels (A) before sealing, (B) sealed with a plasma-treatedPET (called homogeneous channels) and (C) sealed witha PET/PE lamination (called composite channels). (C) Re-printed from [35], with permission.

solvent compatibility may become serious problems forhigh-performance separation devices or for coupling tomass spectrometers. The goal of the developed techni-que is to avoid any use of glue or adhesive layer but tohave a perfect sealing between two PETsheets. Becauseof the high glass transition temperature and melting pointof PET, thermal bonding is not possible in contrary to whathas been shown with PMMA, PC or PS [7, 28–30]. Never-theless, after the oxygen plasma treatment presentedabove, some sealing properties could be observed be-

Figure 4. EOF velocity of the homogeneous and compo-site PET channels; the evolution of the EOF velocity hasbeen monitored over 20 days in channels kept under dryand wet (deionized water) conditions; it is remarkable thatwith a storage under dry condition the EOF velocity staysalmost constant which is not the case when the channel isimmerged in water.

tween the two PETsheets. When the right lamination con-ditions are combined, the bonding is so strong that thedelamination of two 100 �m thick PET sheets is no morepossible. Forcing the delamination leads to tear one of thePET pieces. Cross sections of the homogeneous PET/PET channel and a standard PET/PE laminated channelare presented in Fig. 3. It is remarkable on Fig. 3B that nointerface is visible on the picture, suggesting that somecondensation reaction occurs at the interface that leadsto covalent attachment. In contrast, Fig. 3C shows thatthe interface between the two substrates is still visible.These microchannels will be used for the measurementof EOF in a comparative tests.

3.3 EOF

Surface state and homogeneity of the walls are crucialparameters for electrokinetic analyses. Furthermore, thestability of this flow with time is important in the scope toprovide analytical tools. Flow measurements presented inFig. 4 demonstrates the difference between the behaviorof both types of microchips, homogeneous and compo-site, when stored under dry or wet conditions. First, it canbe observed that the net electroosmotic mobility mea-sured in a homogeneous channel is slightly higher than

Electrophoresis 2002, 23, 782–790 Polymer microchips borded by O2-plasma activation 787

Figure 5. (A) Upper view of the photoablated double Tinjection system. (B) Microscopic fluorescence imageof the 660 pL pinched sample plug in the double T and(C) typical electropherogram of fluorescein (76 �M) de-tected after 1.0 cm of separation.

the one measured in composite channels. Two main phe-nomena could account for this difference in velocity: first,the PE layer contributes to slow down the flow in the com-posite channel that was already described to be respon-sible for Taylor dispersion [11]. Secondly, the surfaceoxidized by plasma contains significantly more oxygenfunctionalities than the laser-treated PET, which probablycontributes to an increase in charge sites and hence in thezeta potential. When the chips are kept under dry condi-tions, the EOF is stable during at least twenty days forboth homogeneous and composite channels. The valueof the electroosmotic mobility is in good agreement withprevious published values, that is about half of the valuesobtained with a glass device [4].

It is interesting to observe that if the chips are kept in wetconditions, the electroosmotic mobility decreases tomore than 50% of the original value. This decrease isprobably due to the very slow desorption of low-molecu-lar-weight charged polymer chains in contact with the sur-face that gets slowly dissolved with time. This resultshows the difficulty of controlling the events occurring onthe polymer surface with time. Nevertheless, it is quite

encouraging that keeping the chips dry for several weeksdoes not influence the EOF velocity. It must be stressedhere that this last point is one of the drawback of somePDMS chips for which some have observed dramaticdecrease of EOF in few hours [17].

3.4 Separation of fluorescent dyes

In order to study the separation efficiency of the two dif-ferent chips, fluorescein samples were injected by meansof a double T injector and separated over 1–1.5 cm in themain channel. A top view of the double T is presented inFig. 5a where a 660 pL plug can be controlled by pinchedinjection. In Fig. 5b, a fluorescence imaging of the doubleT shows that the plug is well controlled within the twoinjection arms. A pinching voltage controlled with thesame power supply prevents the diffusion of the plug inboth separation arms. Nevertheless, it must be statedthat some flow comes from the separation arms, leadingto sample dilution. The voltage is then switched and anelectropherogram of the plug can be acquired as pre-sented in Fig. 5c. For experiments performed with homo-geneous chips, a typical height of 0.6 �m has beenobtained. This means that the system sepation efficiencyis comparable to that obtained with glass chips [31, 32].It should be stressed that the dispersion due to the injec-tion length (�L = 87 �m calculated as proposed by [31])represents approx. half of the total measured variance ofthe detected peak of 160 �m. The best height of a theore-tical plate obtained with a composite channel is about4 �m, which is significantly higher than the homogeneousone. This is probably due to Taylor dispersion caused bythe PE surface that has a lower zeta potential than the restof the channel walls. It is also interesting that this value isnevertheless significantly lower than the 44 �m value pub-lished earlier by Bianchi et al. [11] in a composite PET-PEchannel. The difference probably arises from possibleexperimental errors due to siphoning effects that candramatically affect the separation efficiency. Neverthe-less, the effect of composite channels of the EOF isknown and has been observed by Ross et al. [9] whogave clear pictures of the Taylor effect taking the advan-tage of new imaging technique. They also found thatcomposite PDMS/PMMA channels had a higher plateheight than those of homogeneous channels.

The separation of two fluorescent components has thenbeen performed by the addition of biotin-fluorescein withfluorescein. In the homoegeneous plasma-treated chips(Fig. 6A), the biotin-fluorescein peak is very small andcan sometimes not be detected at all. This effect is repro-ducible over tens of injections. Amazingly, when the sameseparation is performed with the composite chip, twopeaks of the same intensity are observed (Fig. 6B). In

788 Z. Wu et al. Electrophoresis 2002, 23, 782–790

Figure 6. Electropherogam of fluorescein and fluores-cein-biotin (380 and 240 �M, respectively) in (A) homoge-neous and (B) composite �TAS; (C) separation of 380 and600 �M of fluorescein and fluorescein-biotin, respectively.

order to understand better the phenomena occuring inthe homogeneous chips, a much larger concentration ofbiotin-fluorescein has been added to the sample solutionand analyzed in homogeneous channel, resulting in abroad peak but still at a low intensity (Fig. 6C). This effect

is probably related to some kind of adsorption of the bio-tin-fluorescein that does not occur in the composite chan-nel. It must be reminded here that both surfaces aresignificantly different because one has been laser-treatedand the other plasma oxidized. The difference in O/C ratio(laser/plasma 0.22 and 0.54, respectively) observed withXPS experiments reveals the presence of very differentsurface functionalities that may have affinity to speciesto be separated, in this case fluorescein-biotin. Adsorp-tion problems are not specific to this polymer chip buthave also been observed by Ocvirk et al. [18] with PDMSsubstrates. This example shows how difficult it is todevelop an optimal polymer electrophoretic chip. Post-surface chemical processing or adding some modifier inthe buffer solution may help to overcome this effect.

It is usually assumed that the fabrication process doesnot affect the glass surface chemistry very much, andthat the zeta potential is homogeneous in glass substratechips. This is not the case for polymer substrate micro-structures, polymer surfaces being influenced both che-mically and physically during channel fabrication, or evenduring the manufacturing of the substrate itself. The pre-sent experiments show that the separation efficiencydepends on the molecules to be separated as well as onthe surface state of the polymer chips. Moreover, somephenomena such as adsorption can occur, which areunpredictable without experiment.

4 Concluding remarks

With the introduction of plasma surface treatment, bond-ing without adhesive layer was achieved for the testedpolymer substrates under the glass transition tempera-ture of the polymer. Microchannels with walls of thesame material were achieved, and the microchannels sofabricated were reproducible and stable over 20 days,and have a good efficiency in capillary electrophoresis.Theoretical plate height of sub-�m (0.6) has been ob-tained for fluorescein. Plasma-aided auto-adhesion maybecome a routine procedure in the fabrication of polymermicrochannels, and microchips of polymer substrateshould find wider application in the future. More intensivecomparative studies will be carried out in the future bythe introduction of some alternative microstructuringmethods, like imprinting, embossing, etc. followed byplasma finishing. Comparative study will also be carriedout for different substrates and for different kinds of sam-ples, especially biopolymers.

One of the authors (Wu) is grateful to the scholarship fromCSC and a research fellowship from the laboratory, toMrs Valérie Devaud and Tatiana Rohner for helpful discus-

Electrophoresis 2002, 23, 782–790 Polymer microchips borded by O2-plasma activation 789

sions, to Mr Brian Senior from CIME, EPFL for SEM mea-surements, to Prof. H. Mathieu from LMCH, EPFL for XPSanalyses, and to Mr William Baer from CMI, EPFL forplasma condition programming.

Received September 6, 2001

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