Surface modification of polymer-based microfluidic devices

Analytica Chimica Acta 470 (2002) 87–99

Surface modification of polymer-based microfluidic devices Steven A. Soper∗ , Alyssa C. Henry, Bikas Vaidya, Michelle Galloway, Musundi Wabuyele, Robin L. McCarley1 Choppin Laboratories of Chemistry, Department of Chemistry, Louisiana State University, 232 Choppin Hall, Baton Rouge, LA 70803-1804, USA Received 16 November 2001; received in revised form 11 April 2002; accepted 22 April 2002

Abstract We report the chemical modification of poly(methyl methacrylate) (PMMA), and poly(carbonate) (PC) surfaces for applications in microfluidic systems. For PMMA, a reaction of the surface methyl ester groups with a monoanion of ␣,␻-diaminoalkanes (aminolysis reaction) to yield amine-terminated PMMA surfaces will be described. Furthermore, it was found that the amine functionalities were tethered to the PMMA backbone through an alkane bridge to amide bonds formed during the aminolysis of the surface ester functionalities. The electro-osmotic flow (EOF) in aminated-PMMA microchannels was reversed when compared to that in unmodified channels. Finally, the availability of the surface amine groups was further demonstrated by their reaction with n-octadecane–1-isocyanate to form PMMA surfaces terminated with well ordered and highly crystalline octadecane chains, appropriate for performing reverse-phase separations. Examples of reverse-phase separations of ion-paired double-stranded DNAs in electric fields (capillary electrochromatography (CEC)) will be demonstrated using a PMMA-based fluidic chip. For PC, sulfonation of the surface with SO3 will be described; this sulfonation makes the surface very hydrophilic. EOF studies of the sulfonated-PC surfaces indicated changes in the pH-dependent profile when compared to unmodified PC. © 2002 Elsevier Science B.V. All rights reserved. Keywords: Surface modification; Polymer microdevices; MEMS; DNA electrophoresis

1. Introduction Microelectromechanical systems (MEMS), have recently entered the forefront of instrumentation development in analytical chemistry. MEMS offer the researcher many advantages, including high sample throughput, high sample processing rates, minimized consumption of sample and reagent, reduced manufacturing cost and system integration [1,2]. ∗ Corresponding author. Tel.: +1-225-578-1527; fax: +1-225-578-3458. E-mail addresses: [email protected] (S.A. Soper), [email protected] (R.L. McCarley). 1 Co-corresponding author. Tel.: +1-225-578-3239; fax: +1-225-578-3458.

In recent years, analytical MEMS have been fabricated in glass by means of wet chemical etching [2–5]. Due to the isotropic nature of the etching process in glass, very shallow channels with low aspect ratios result. This can be overcome using deep reactive ion etching to create high-aspect-ratio microstructures (HARMs), but the overhead of fabrication makes it prohibitive for mass production applications. However, one of the major advantages of using glass for microdevices are the established chemical modification procedures of its surface using organosilanes [6]. An alternative to glass-based MEMS devices are those fabricated from polymers [1,2]. One particular advantage to using polymers is the wide choice in microfabrication methods that can be selected to

0003-2670/02/$ – see front matter © 2002 Elsevier Science B.V. All rights reserved. PII: S 0 0 0 3 - 2 6 7 0 ( 0 2 ) 0 0 3 5 6 - 2

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Table 1 Listing of the physical properties of two representative thermoplastics commonly used for MEMS-based devices

Tg (K) Dielectric strength (V/cm) Thermal conductivity (W m/K) Effects of strong acids/bases Effects of organic solvents Clarity (UV–VIS) Fluorescence background

Glass

PMMA

PC

963 11.8 × 104 0.732 Attacked, bases only Insoluble Opaque/transparent Excellent

377 17.7 × 104 0.193 Attacked, acids only Soluble Opaque/transparent Fair

430 3.8 × 103 0.193 Attacked Soluble Opaque/transparent Poor

For comparison purposes, the physical properties of glass, a common substrate used for MEMS devices, is shown as well.

construct the device. For example, some of the common methods for producing such devices from polymeric materials includes injection molding [7], laser ablation [8], imprinting [9], or hot embossing [10]. Thus, devices can be produced inexpensively and in large number when using polymeric materials, making their use in commercial applications particularly attractive. In addition, although very difficult to achieve with glass, HARMs can be readily patterned on polymer substrates [11–14]; this latter characteristic is very important in the fabrication of small-footprint devices. Finally, the relatively low glass transition temperatures (Tg ) associated with most polymers used in the construction of HARMs devices allow for the use of low-temperature device assembly protocols [13,14]. A listing of some of the physical properties of two thermoplastics (poly(methyl methacrylate) (PMMA) and poly(carbonate) (PC)) is shown in Table 1 along with the chemical structure of each polymer. Also, the physical properties of glass are shown for comparison purposes. As can be seen, glass tends to have excellent optical properties compared to PMMA and PC, providing the opportunity for ultrasensitive detection when fluorescence is used for transduction. Also seen from the physical properties listed in this table is that these polymers are not compatible with most organic solvents and in some cases, strong bases or acids. In contrast, glass displays minimal compatibility issues with these solvent systems. As stated, chemical modification techniques of glass have been well established using silane-based chemistry; however, the development of routine, sim-

ple, well-defined surface modification protocols for polymers is still in its infancy [15]. Such modification techniques are essential to the development of MEMS technology in polymer-based substrates, like PMMA, or PC. In this paper, we will report on general modification procedures for PMMA and PC. In the case of PMMA, a robust primary amine-functionalized chemical scaffold is produced to which a target can be attached. The amine-modified PMMA surfaces can be reacted with a variety of different compounds to yield numerous functionalities on the surface of the PMMA. For PC, sulfonation of the surface using fuming sulfuric acid produces a more hydrophilic surface that displays a chemical composition and surface morphology different from that of unmodified PC. Various spectroscopic and microscopic techniques were employed to characterize the unmodified and modified PMMA and PC surfaces. In addition, the electro-osmotic flow (EOF) properties of aminated-PMMA and sulfonated-PC microchannels will be reported.

2. Experimental 2.1. Preparation of N-lithiodiaminoethane and N-lithiodiaminopropane [16] N-lithiodiaminoethane and N-lithiodiaminopropane were synthesized by first placing 6 mmol of dry diamine (Aldrich) in a round bottom flask. The diamine was purged with nitrogen for 20 min before the introduction of 1 mmol of n-butyllithium (Aldrich,

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Fig. 1. Synthetic scheme for (A) the amination of the PMMA surface and (B) the conversion of aminated-PMMA surface into a C18 surface.

Milwaukee, WI, 2 M in cyclohexane). Upon introduction of the n-butyllithium, a dark purple product, in the case of N-lithiodiaminoethane, was observed. Similarly, a yellow–brown product was evident upon the addition of n-butyllithium to the purged diaminopropane. Each product was stirred for 3 h before use and was kept in the purged, sealed vessel for no longer than 1 week. 2.2. Preparation of NH2 -modified PMMA surfaces Commercial PMMA sheets were obtained from Goodfellow. The PMMA sheets were machined (on edges) to a given size and then cleaned by soaking

in isopropanol. Scanning force microscopy (SFM) studies of isopropanol-soaked PMMA sheets demonstrated that there was no swelling of the polymer after several weeks of immersion in isopropanol as noticed by no change in surface topography. Before modification, the machined PMMA pieces were rinsed with copious amounts of isopropanol followed by extensive rinsing with 18 M
cm water (Barnstead, Dubuque, IA). The PMMA pieces were then dried under a stream of nitrogen. Following a 20 min nitrogen purge in a sealed vessel, the PMMA pieces were exposed to N-lithiodiaminothane or N-lithiodiaminopropane (transferred by cannula or syringe). After a given period of time, the reaction was quenched with 18 M
cm

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water. After removal from the reaction flask, the PMMA was rinsed with copious amounts of 18 M
cm water, followed by drying under a stream of nitrogen. This reaction sequence is displayed in Fig. 1A. 2.3. Preparation of carboxylated PMMA surfaces Freshly-prepared NH2 -modified PMMA (ATPMMA) sheets, AT-PMMA thin films spin coated on the Au, or AT-Au slides were placed in a solution of 5 mM non-anedioic acid (Aldrich) and 95 mM dicyclohexylcarbodiimide (DCC) (Aldrich), in purged ethanol for at least 3 h. The modified surfaces were then rinsed with copious amounts of ethanol and dried in a stream of N2 . 2.4. Preparation of C18 H37 -modified PMMA surfaces [15]

2.6. Contact angle measurements Sessile drop contact angle measurements utilizing 18 M
cm water were performed with a VCA 2000 Contact Angle System (VCA, Billerica, MA). Approximately 6 ␮l of 18 M
cm water were placed on the various PMMA or PC surfaces using a syringe. Contact angle values were calculated using the software provided by the manufacturer. Each value reported was the average of at least five separate drops of water on a given substrate; values were found not to vary significantly among a set of substrates from a given preparation. 2.7. Reflection–absorption infrared spectroscopy (RAIRS) studies

Freshly-prepared NH2 -modified PMMA pieces were placed in an airtight vessel and the vessel purged with nitrogen for 20 min before introduction of neat n-octadecane–1-isocyanate (Aldrich, 99%; Fig. 1B). After 10 min exposure to n-octadecane–1-isocyanate, the PMMA pieces were quickly rinsed with copious amounts each of hexanes, toluene, and acetone and then dried under a stream of N2 . SFM studies indicated that rinsing the C18 H37 -modified PMMA with these solvents does not affect the topography, indicating that the polymer surface was not substantially swelled.

In order to examine the molecular nature of pristine, NH2 - and C18 H37 -modified PMMA and pristine and sulfonated-PC surfaces, RAIRS studies were employed using Au-coated, Cr-primed glass microscope slides (Au slides) [17]. PMMA or PC was spin coated on the Au slides by means of a ‘Specialty Coating Systems spin-coater’. Commercial sheet polymers were dissolved in dichloromethane to yield a solution with a final concentration of 0.5 mg polymer/ml. This solution was dropped onto the spinning Au slide (2200 rpm) and allowed to spin for 70 s. The coated slide was then ready for chemical modification and/or analysis. RAIRS was performed using previously reported methods [17].

2.5. Preparation of sulfonated-PC surfaces

2.8. Scanning force microscopy studies

Bisphenol-A–PC sheets were purchased from Goodfellow and MSC Industrial Supplier and were mechanically machined to appropriate-sized pieces. The PC pieces or hot embossed microchips were soaked in an HPLC grade (Aldrich) isopropanol bath and further rinsed with isopropanol before rinsing with copious amounts of deionized water, then dried in a stream of high purity nitrogen. PC substrates were sulfonated by placing them in a container filled with SO3 vapor generated from fuming sulfuric acid at 50 ◦ C for 10 min. The sulfonated-PC samples were then rinsed with water and dried in a stream of nitrogen.

The surface topographies of pristine, NH2 -, and C18 H37 -modified PMMA surfaces were assessed through the use of a Digital Instruments Nanoscope III Multimode atomic force microscope (AFM) utilizing the D scanner (13 ␮m × 13 ␮m). The microscope was operated in contact mode for all experiments. Images were flattened using the Nanoscope software. 2.9. Quantification of amines on the surface of NH2 -modified PMMA PMMA sheets were machined using standard milling techniques to produce pieces with dimensions

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of 15 mm × 15 mm × 3 mm. The total number of diamine molecules present on a given NH2 -modified PMMA sheet was determined using a variation of the ninhydrin method in conjunction with the method described by Ichijima et al. and others [18–20]. 2.10. Preparation of PMMA/PC microchannels and EOF measurements A 5 cm ×3 cm sheet of PMMA or PC was subjected to bulk surface micromachining to produce a 4 cm (in length) microchannel, which was 100 ␮m in depth and 100 ␮m in width. A top plate made from the appropriate polymer was also machined such that it completely covered the microchannel with holes drilled in this top plate to accommodate solution reservoirs. Prior to thermal bonding the top plate to the substrate, both pieces were subjected to the appropriate modification chemistry described above. Following modification, the pieces were assembled using a thermal bonding technique previously described by us [13]. EOF measurements were conducted using a method, which employed a discontinuous buffer system [21]. Briefly, this technique involves filling the microchannels with a low ionic strength buffer and then, emptying one well and filling it with a higher ionic strength buffer. The electrodes are then placed in the two wells flanking the microchannel and the current monitored as a function of time. The current increases due to filling of the microchannel with the higher ionic strength buffer. The EOF is calculated by determining the time it takes for the current to plateau and dividing by the applied field strength. The electric field was supplied by a Spellman high-voltage power supply (CZ1000R, Plainview, NY) and was run at a field strength of ∼150 V/cm. The EOF was measured at various pH values using either acetate (pH = 3 and 5) or borate (pH = 7, 9 and 11) buffer solutions. The PMMA or PC microchips used for electrophoresis were hot embossed from methods developed in our laboratory [22]. Basically, a molding die fabricated in Ni (electroform) was fabricated using LIGA techniques, which resulted in a master that was the negative of the final microparts and defined the topography of the fluidic chips. The embossing machine was a PHI press (City of Industry, CA), which was equipped with an in-house constructed vacuum

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chamber. The polymer pieces to be embossed were baked at 85 ◦ C for 12 h to remove residual water and then, rinsed with IPA. For PMMA, the platens on the embossing machine were set at 185 ◦ C and for PC at 210 ◦ C. Embossing was done at 1000 lb for 4 min. Following embossing, the chips were assembled with a cover plate of the same material to enclose the fluidic channels by clamping together the cover plate and substrate and placing it in a convection oven that was set at a temperature slightly above the glass transition temperature of the polymer. The microelectrophoresis chip consisted of a cross with a separation channel length of 4 cm (effective length = 3 cm), a channel depth of 80 ␮m and width of 20 ␮m. 2.11. DNA analysis in PC and PMMA microchips For the electrophoretic separations in these polymer-based microdevices, X174 DNA was digested with the restriction enzyme Hae III (Amersham Pharmacia Biotech, Piscataway, NJ) and subsequently diluted in 1X TBE buffer (40 mM Tris, 40 mM boric acid, 1 mM EDTA, pH 8). The fragments were labeled with the mono-intercalating dye, TOPRO5 (747/767 nm, Molecular Probes, Eugene, OR) with a final dye concentration of 1 ␮M. Methyl cellulose (MC, Aldrich Chemicals, Milwaukee, WI) was used as the DNA sieving matrix and was prepared in 1× TBE buffer at a concentration of 1% (w/v). The MC solution was degassed and then loaded into the microchannel device using a syringe. The sample was loaded into the sample reservoir and electrokinetically injected into the cross channel for 5 s at an electric field of 133 V/cm. After injection, the electrodes were then switched to the separation channel reservoirs and an applied voltage of 67 V/cm was used to separate the DNA fragments. The fluorescence detection point was 3 cm from the T-junction injector. Fluorescence detection was performed using a laser-induced fluorescence system configured in an epi-illumination format, which consisted of a 750 nm diode laser (Melles Griot, Irving, CA, 4 mW) and a 20X, NA = 0.45 microscope objective. The resulting emission was filtered with a 780 nm band pass filter (Omega Optical, Brattleboro, VT) and imaged onto a single photon avalanche diode (SPAD).

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Capillary electrochromatography (CEC) separation of the double-stranded oligonucleotides was carried out using an ion-pair reverse-phase chromatography method developed in our laboratories with conductivity detection (CD) [23]. Briefly, the carrier electrolyte consisted of 25% acetonitrile and 75% water containing 50 mM triethylammonium acetate (TEAA) serving as the ion-pairing agent. The oligonucleotides analyzed were a low mass ladder (Gibco-BRL, Gaithersburg, MD), which consisted of 100, 200, 400, 800, 1200 and 2000 bp fragments. Injection was accomplished as described above with the field strength used for the separation equal to 150 V/cm. The contact conductivity detector was operated at a frequency of 5 kHz and a pulse amplitude of ±0.5 V. CEC was performed in open-channels of the PMMA microfluidic devices, whose surfaces had been modified with –C18 H37 as described above.

3. Results and discussion 3.1. Sessile drop water contact angle measurements The average water contact angle for pristine PMMA was found to be 66◦ ± 2◦ , which correlated well with the literature value of 67◦ for a highly ordered methyl ester-terminated monolayer [24]. Water contact angle measurements for NH2 -terminated PMMA sheets yielded an average of 33◦ ± 4◦ , a value consistent

with that obtained for self-assembled monolayers terminated with hydrophilic functional groups [24,25]. In the case of the PC and sulfonated-PC materials, the sessile water contact angles were determined to be 85◦ ± 2◦ and 42◦ ± 2◦ , respectively. 3.2. Analysis of pristine and modified PMMA or PC thin films by reflection–absorption infrared spectroscopy (RAIRS) Fig. 2B is a representative RAIR spectrum of pristine PMMA on Au (Au-coated, Cr-primed glass microscope slides) [17]. This spectrum agreed well with the transmission spectrum of PMMA documented in the literature [26]. The most prominent band was the carbonyl stretch, ν(C==O), at approximately 1733 cm−1 . The band maximum position was characteristic of methyl esters, particularly those found for films of PMMA. The remaining vibrational bands observed were characteristic of the alkane and ester moieties present in the polymer. Displayed in Fig. 2A is a representative RAIR spectrum of an NH2 -modified PMMA thin film. The major observations that arose upon comparison of Fig. 2A and B were the presence of the amide I (ν(C==O)) and the amide II (δ(N–H)) bands at approximately 1673 and 1520 cm−1 , respectively as well as substantially less intense methyl ester bands—the ν(C==O), ν(C–O), and ν(C–O–C) modes—in the spectrum of the aminated-PMMA film. These data point to the fact

Fig. 2. RAIRS of NH2 -modified PMMA (A) and pristine PMMA (B) spin coated on the Au slides.

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Fig. 3. RAIRS of pristine (A) and modified PC (B), which was spin coated on the Au slides.

that exposure of PMMA films to a lithiated diamine results in replacement of the ester functionalities by an amide linkage as expected. Typical RAIR spectra of pristine and sulfonated PC on Au are presented in Fig. 3. The presence of new bands at ∼1100 and 625 cm−1 in Fig. 3B correspond to the symmetric stretch of the SO2 group (ν s SO2 ) and the SO stretch (νSO) and confirms the presence of sulfonate groups on the surface. The asymmetric SO2 stretch (∼1300 cm−1 ) overlaps with the C–O stretches of the PC group. 3.3. Quantitative determination of NH2 groups on the surface of NH2 -terminated PMMA sheets [15] For the reaction of sheet PMMA with N-lithiodiaminoethane, it was found that a reaction time of 2–5 min leads to an apparent surface coverage of

6.85 nmol cm−2 ±0.60 nmol cm−2 . As for the reaction of sheet PMMA with N-lithiodiaminopropane, it was found that the apparent surface coverage of amines remained steady at approximately 6 nmol cm−2 (no statistical difference in coverage with time at the 95% confidence level). 3.4. Characterization of the surface topography of pristine and NH2 -terminated PMMA sheets using scanning force microscopy (SFM) Displayed in Fig. 4A is a representative 2 ␮m × 2 ␮m contact-mode SFM image of sheet PMMA. This surface, as can be seen, was smooth and relatively free of defects; the RMS roughness (entire 2 ␮m × 2 ␮m range) was 0.39 nm. Shown in Fig. 4B is a typical 2 ␮m × 2 ␮m SFM image of sheet PMMA that was modified with N-lithiodiaminopropane for 2 min. The

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Fig. 4. SFM images of (A) pristine; (B) NH2 -modified sheet PMMA and (C) C18 H37 -modified PMMA. The scan size is 2 ␮m × 2 ␮m and the Z-range is 10 nm.

modified surface was obviously much different than the pristine surface and the surface roughness was approximately 3.5 times higher (1.45 nm RMS roughness) compared to that of the pristine surface. The RMS roughness value for PMMA surfaces exposed to N-lithiodiaminoethane was determined to be 1.80. The observed increase in surface roughness may be due to a slight swelling/dissolution of the PMMA by the lithiated diamine because it has been found that the cyclohexane solvent used in the aminolysis re-

action does not affect the surface of the PMMA in any way. 3.5. Electro-osmotic flow in PMMA and PC microchannels The EOF has recently been determined in PMMA-based microdevices and was found to be nearly independent of solution pH [13]. In order to investigate the ability to manipulate the EOF through

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surface modification of PMMA, a microchannel was machined into PMMA and then amination of both the substrate and cover plate was performed prior to thermal bonding the cover plate to the machined PMMA sheet. The EOF values as a function of pH for both unmodified and modified PMMA microdevices are shown in Fig. 5A. As can be seen from this figure, the EOF was reversed (negative EOF value indicates solution flow from cathode to anode)

Fig. 5. (A) EOF vs. pH for unmodified (squares), NH2 -modified (down triangles), C18-modified (diamonds) and –COOH modified (circles) PMMA microdevices and (B) pH-dependent EOF profiles for PC that was unmodified (circles) or sulfonated (squares). The EOF was measured from a pH range of 3 to 11 for PMMA and 4 to 10 for PC using acetate or borate buffers. The low concentration buffer was 1 mM and the higher concentration buffer was 2 mM. The field strength used (150 V/cm) was selected to minimize Joule heating in the channel, which measured 100 ␮m × 100 ␮m × 4 cm for PMMA and 50 ␮m × 100 ␮m × 4 cm for PC.

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in the case of the modified PMMA microchannel, consistent with a surface with an excess positive charge on its wall. In addition, the EOF was shown to decrease as the solution pH became more alkaline (EOF = −1.80 × 10−4 cm2 /(V s) at pH = 3; EOF = −1.05 × 10-4 cm2 /(V s) at pH = 11). This decrease results from the fact that the accessible 1◦ amines were protonated at low pH and become deprotonated with increasing pH. In the case of the unmodified-PMMA microdevice, the EOF showed a slight increase in magnitude at higher pH values, but not to the same degree as seen for the modified device (EOF = 2.17 × 10−4 cm2 /(V s) at pH = 3; EOF = 2.58 × 10−4 cm2 /(V s) at pH = 11). Also shown in Fig. 5A is an EOF pH profile for an amine-terminated PMMA surface that was chemically converted to a –COOH surface. As can be seen, at low pH, the EOF runs from cathode to anode and has an absolute value slightly smaller than the original amine-terminated surface. However, at pH values >5, the EOF reverses in direction (anode to cathode), indicative of a surface with excess negative charge. We attribute these observations to a surface with both amine and carboxylic functionalities. At low pH, the amine and carboxyl groups are protonated, producing a surface with excess positive charge. At pH values above 5, the amines and carboxyls are deprotonated, producing a surface with a negative charge, causing reversal of the EOF. The EOF in both modified and unmodified PC exhibited a flow that traveled from anode to cathode, indicative of a surface with excess negative charge at the pH values investigated. The EOF profiles of modified and unmodified PC as a function of pH are shown in Fig. 5B. EOFs measured in sulfonated-PC microchannels showed significantly higher values at pH values below 8 compared to the unmodified PC. We attribute this increase in EOF at lower pH to differences in acid/base properties of the sulfonate groups compared to the unmodified PC surface. Because the pKa of sulfonic acids are much lower than 4, they can be expected to be fully deprotonated at pH above 4, while the native PC surface contains functional groups that apparently have a higher pKa value resulting in higher charge density for the sulfonated-PC surface at these pH values, which produces a higher EOF. Interestingly, the EOF of both PC surfaces above pH = 7 were similar indicating a surface charge density that was comparable.

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3.6. Modification of amine-terminated PMMA surfaces to yield alkyl-terminated surfaces We were also interested in modifying the PMMA surface so as to create a microanalytical separation device that was patterned on a “chip” of PMMA and also possessed a hydrophobic phase for electrochromatography applications as has been demonstrated for glass-based microdevices [27]. We, thus, turned our attention toward derivatizing the PMMA such that its surface was terminated in –C18 H37 groups. After such a reaction sequence, it was found that the C18 H37 -modified PMMA sheet was colorless and transparent, exactly like the pristine and NH2 -modified PMMA sheets. As mentioned above, the water contact angle of the amine-modified PMMA surface was quite low, 33◦ ± 4◦ , as is to be expected for a relatively hydrophilic surface. The C18 H37 -terminated PMMA surface yielded a water contact angle of 103◦ ± 10◦ , a value that is consistent with a smooth poly(ethylene) surface (exposed methylene units) [25]. For a close-packed methyl surface (ordered, long-chain n-alkanethiols on Au), the expected water contact angle is roughly 113◦ [25]. The contact angle values obtained here for the C18 H37 -terminated PMMA would seem to indicate that the chains are either highly disordered or that the chains have a substantial tilt from the surface normal (>30◦ is observed for n-alkanethiols on Au), such that the methylene units of the alkane chains are significantly exposed. From IR data (not shown) the alkane chains of the C18 H37 -modified PMMA surfaces are highly ordered as indicated by the position of the symmetric and asymmetric methylene bands (2847 and 2920 cm−1 ) and the presence of bands (Tx and Wx bands) which are only noted in highly crystalline, ordered alkane systems. An AFM image of the C18 H37 -modified PMMA surface is shown in Fig. 4C. As can be seen, the alkaneterminated PMMA sheet exhibited a surface morphology that was slightly different from the amine-modified surface. The C18 H37 -modified PMMA surface is reminiscent of alkylsilane-coated silicon surfaces [28], in that the C18 H37 -modified PMMA surface appears as if it possesses fluid-like characteristics, thus, making it difficult to obtain well-resolved SFM images. A RMS roughness of 2.8 nm was obtained from SFM images of C18 H37 -modified PMMA surfaces. This is

approximately 1.6–2 times greater than that of the amine-modified PMMA surfaces. In order to investigate the effects of the –C18 H37 modification on the EOF properties of the PMMA surface, we measured the pH-dependent EOF of this modified PMMA surface, the results of which are shown in Fig. 5A. As can be seen, the EOF was found to travel from cathode to anode as was observed for the NH2 -modified PMMA chip, with reduced values as the pH was increased. This result indicates that there are some unreacted amine groups that produce a wall that has cationic character at low pH, with the charge density decreasing at higher pH due to deprotonation of the surface amine groups. At the high pH values, the EOF for the C18 H37 surface at pH = 11 was significantly smaller (in magnitude) than that of the NH2 -modified PMMA due to less charge density on this chip’s surface. 3.7. Capillary electrochromatography of dsDNAs To demonstrate the ability of this modified surface to behave as a hydrophobic stationary phase for reverse-phase separations, an ion-paired electrochromatographic separation was performed on double-stranded DNA fragments with TEAA serving as an ion-pairing agent. Fig. 6A shows the separation of a DNA ladder using the unmodified-PMMA walls with contact CD used for transduction while Fig. 6B shows the same separation, but using a PMMA-based microchip that possessed the C18 surface layer. For the native PMMA surface, only one broad band appeared for the DNA fragments and in addition, the background signal from the CD was very unstable. In addition, the instability of the CD response was due to dissolution of the PMMA surface by the acetonitrile organic modifier used in the mobile phase. This observation was supported through inspection of blank PMMA sheets subjected to this mobile phase, which significantly altered the appearance of the polymer, producing a non-transparent surface with severe roughening and swelling. In the case of the C18-modified PMMA chip, the individual DNA fragments were resolved and the stability of the CD signal evident. We noticed no dissolution of the PMMA surface upon continued exposure to this mobile phase. Apparently, the C18 layer protects the underlying PMMA from acetonitrile. Also, the peaks observed

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Fig. 6. CEC separation of a DNA sizing ladder in an unmodified (A) or C18-modified PMMA microdevice (B), which was prepared via hot embossing. The separation was performed in a PMMA device with an effective channel length of 3 cm. The mobile phase consisted of 25% acetonitrile and 75% water, which also contained TEAA (50 mM) as the ion-pairing agent. Transduction was accomplished using contact CD with Pt wires.

for the oligonucleotides were negative due to the lower conductivity of the DNA bands compared to the background carrier electrolyte, consistent with our previous data for this type of separation with CD [29]. 3.8. Capillary electrophoresis of DNAs in PMMA and PC microchips Fig. 7 shows the separation of 5 ␮g/ml of a X174 DNA digested with Hae III in PMMA and PC microdevices. As can be seen from this data, qualitatively, the separation in the unmodified-PMMA-based device appears to be more efficient and provides better resolution for the fragments comprising this ladder

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Fig. 7. Microelectrophoretic separation of X174 DNA digested with Hae III restriction enzyme. The electrophoresis was carried out using methyl cellulose as the sieving matrix at a field strength of 150 V/cm. The effective length of the separation channel was 3 cm. The detection of the double-stranded DNA fragments was accomplished using LIF (λex = 750 nm; 4 mW average power). The DNA fragments were stained on-column using a TOPRO5 nuclear staining dye that was loaded into the running buffer used for the electrophoresis (1 ␮M). The separation was carried out on unmodified-PMMA (A); unmodified-PC (B) or sulfonated-PC (C) microdevices that were prepared using hot embossing.

(total number of fragments = 11) compared to the unmodified-PC device. Indeed, the chromatographic efficiency as determined from calculation of the plate numbers for the 603 bp fragment indicated more plates for the PMMA-based device, 3.1 × 105 m−1 for PMMA and 9.3 × 104 m−1 for PC.2 We suspect that the reduced efficiency and poor resolution in the PC device is due to solute–wall interactions. In the case of PMMA, these types of interactions are absent, resulting in higher plate numbers and, thus, better electrophoretic resolution. We attempted to minimize solute–wall interactions in PC devices by sulfonating the PC surface using our modification chemistry. Fig. 7C shows the separation of this same 2 The plate numbers (N) were calculated using the standard chromatographic formula (on a per meter basis), N = 16(tmig /w b )1/2 , where tmig is the migration time and w b is the width of the peak at the base.

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sizing ladder that was carried out in a sulfonated-PC device. When we calculated the plate number for the 603 bp fragment, the value was determined to be 1.4 × 105 m−1 , a slight improvement to that observed for the unmodified-PC device. Increasing the negative charge density on the wall of the device reduced solute–wall interactions via electrostatic considerations. It was also noticed that the migration time for the sulfonated-PC device for the 603 bp fragment was longer than that observed for the unsulfonated-PC device, which most likely resulted from the increased EOF at this pH for the sulfonated device. In our electrophoresis examples, the EOF opposes the electrophoretic mobility of the DNA and as such, devices with higher EOFs will lengthen the migration time.

4. Conclusions In this study we have demonstrated the successful modification of PMMA and PC sheets used in the manufacture of miniaturized analytical devices. Specifically, we have shown that PMMA can be modified so as to have accessible amine sites on its surface, which can be used to change the flow properties of microelectrophoresis devices or as a scaffold for further functionalization. In addition, sulfonation of PC can also be easily carried out, which also modifies its EOF properties. The synthesis of hydrophobic surfaces on PMMA also produced devices that were appropriate for electrochromatography applications. And finally, sulfonation of PC surfaces was found to reduce solute–wall interactions improving electrophoretic resolution of DNAs separated in gel matrices. The attractive feature of polymer-based devices is that low temperatures required for device assembly allowed modification of the microchannels and cover plate before heat annealing. While the results of the EOF measurements and chromatographic separations indicated that the monolayers remain viable following assembly, minor changes in the film topography or chemical composition were not investigated.

Acknowledgements We would like to acknowledge funding of this work by the National Institutes of Health (HG01499

and CA84625), NSF (CHE-9529770, CHE-9732195), the Louisiana Education Quality Support Fund, and DARPA.

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