Surface Modification of Poly(methyl methacrylate) - American

Anal. Chem. 2000, 72, 5331-5337

Surface Modification of Poly(methyl methacrylate) Used in the Fabrication of Microanalytical Devices Alyssa C. Henry, Tumari J. Tutt,† Michelle Galloway, Yolanda Y. Davidson,‡ C. Scott McWhorter,§ Steven A. Soper,* and Robin L. McCarley*

Choppin Laboratories of Chemistry, Louisiana State University, Baton Rouge, Louisiana 70803

We report here the chemical modification of poly(methyl methacrylate) (PMMA) surfaces by their reaction with the monoanion of r,ω-diaminoalkanes (aminolysis reaction) to yield amine-terminated PMMA surfaces. It is found that the amine functionalities are tethered to the PMMA backbone through an alkane bridge to amide bonds formed during the aminolysis of the surface ester functionalities. The distribution of the amine termini is quite uniform as judged by fluorescence micrographs. It is found that the electroosmotic flow in aminated PMMA microchannels is reversed when compared to that in unmodified channels. In addition, it is demonstrated that enzymes can be immobilized onto the amine-terminated PMMA surfaces and are effective in the restriction digestion of dsDNAs. Finally, the availability of the surface amine groups is further demonstrated by their reaction with n-octadecane-1-isocyanate to form PMMA surfaces terminated with well-ordered and highly crystalline octadecane chains. Microelectromechanical systems (MEMS) have recently entered the forefront of analytical chemistry. These systems generally take the shape of microelectrophoretic devices that are used in the separation and detection of various samples, such as DNA fragments. MEMS offer the researcher many advantages, including high sample throughput, high sample processing, minimized consumption of sample and reagent, and reduced cost.1 In recent years, MEMS have been fabricated in glass by means of wet chemical etching.1b,2 Due to the isotropic nature of the etching process with glass-based substrates, very shallow channels with low aspect ratios result. However, one of the major advantages to using glass substrates for microdevices is their ease of * To whom correspondence should be addressed. S.A.S.: (phone) (225) 3881527; (fax) (225) 388-3458; (e-mail) [email protected]. R.L.M.: (phone) (225) 388-3239; (fax) (225) 388-3458; (e-mail) [email protected]. † Summer LSU NSF-REU student from Xavier University, New Orleans, LA 70125. ‡ Currently employed at the National Institutes of Standards and Technology, Gaithersburg, MD 20899-8392. § Currently employed at the DOE-Westinghouse Savannah River Site, Aiken, SC 29808. (1) (a) Soper, S. A.; Ford, S. M.; Qi, S.; McCarley, R. L.; Kelly, K.; Murphy, M. C. Anal. Chem., in press. (b) Campana, A. M. G.; Baeyens, W. R. G.; AboulEnein, H. Y.; Zhang, X. J. Microcolumn Sep. 1998, 10, 339-355. (2) (a) Harrison, J. D.; Manz, A.; Fan, Z.; Ludi, H.; Widmer, H. M. Anal. Chem. 1992, 64, 1926-1932. (b) Harrison, J. D.; Fluri, K.; Seiler, K.; Fan, Z.; Effenhauser, C. S.; Manz, A. Science 1993, 261, 895-897. (c) Effenhauser, C. S.; Bruin, G. J. M.; Paulus, A. Electrophoresis 1997, 18, 2203-2213. 10.1021/ac000685l CCC: $19.00 Published on Web 09/27/2000

© 2000 American Chemical Society

fabrication as well as the ease of chemical modification of glass surfaces using organosilanes.3 An alternative to glass-based microdevices are those fabricated from polymers. One particular advantage to using polymer-based MEMS is the wide choice in microfabrication methods that can be selected to construct the device. For example, some of the common methods for producing such devices from polymeric materials include injection molding,4 laser ablation,5 imprinting,6 and hot embossing.7 Thus, these devices can be produced inexpensively and in large number, making their use in commercial applications particularly attractive. In addition, although very difficult to achieve with glass, high-aspect-ratio microstructure (HARMS) devices can be readily produced using plastic substrates;8 this latter characteristic is very important in the fabrication of small-footprint separation devices that exhibit minimal analyte zone broadening. Finally, the relatively low glass transition temperatures (Tg) associated with most polymers used in the construction of HARMS devices allow for the use of lowtemperature device assembly protocols. As stated above, chemical modification techniques of glass substrates have been well established using silane chemistry; however, the development of routine, simple, well-defined surface modification protocols for polymers is still in its infancy. Such modification techniques are essential to the development of MEMS technology in polymer-based substrates, such as poly(methyl methacrylate) (PMMA). In this paper, we report on a chemical modification procedure for PMMA sheets that yields a robust primary amine-functionalized chemical scaffold to which a target can be attached. The choice of PMMA stems from two specific properties associated with this polymer: the presence of a functional group for potential modification (methyl ester) and the extensive use of PMMA as a resist in lithographic applications for MEMS and microfluidic (3) Proctor, K. G.; Ramirez, S. K.; McWilliams, K. L.; Huerta, J. L.; Kirkland, J. J. In Chemically Modified Surfaces: Recent Developments; Pesek, J. J., Matyska, M. T., Abuelafiya, R. R., Eds.; The Royal Society of Chemistry: Cambridge, 1996, pp 45-60. (4) McCormick, R. M.; Nelson, R. J.; Alonso-Amigo, M. G.; Benvegnu, D. J.; Hooper, H. H. Anal. Chem. 1997, 69, 2626-2630. (5) Roberts, M. A.; Rossler, J. S.; Bercier, P.; Girault, H. Anal. Chem. 1997, 69, 2035-2042. (6) Martynova, L.; Locascio, L. E.; Gaitan, M.; Kramer, G. W.; Christenson, R. G.; MacCrehan, W. A. Anal. Chem. 1997, 69, 4783-4789. (7) Becker, H.; Deitz, W. Proc. SPIE-Int. Soc. Opt.Eng. 1998, 3515, 177-182. (8) (a) Becker, E. W.; Ehrfeld, W.; Hagman, P.; Munchmeyer, D. Microelectron. Eng. 1986, 4, 35. (b) Madou, M. Fundamentals of Microfabrication; CRC Press: Boca Raton, FL, 1986; Chapter 6.

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devices.1a,9 The amine-modified PMMA surfaces can be reacted with a variety of different compounds to yield numerous functionalities on the surface of the PMMA. Chemical modification of PMMA to yield primary amines, as well as long-chain alkyl functionalities and enzymes, will be discussed in this report. Various spectroscopic and microscopic techniques have been employed in the characterization of pristine and modified PMMA surfaces. In addition, the electroosmotic flow (EOF) properties of aminated PMMA microchannels will be reported, as well as the covalent attachment of a restriction endonuclease to the appropriately prepared PMMA surface to perform a heterogeneous microdigestion of dsDNAs. EXPERIMENTAL SECTION Preparation of N-Lithiodiaminoethane and N-Lithiodiaminopropane.10 N-Lithioethylenediamine 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, Milwaukee, WI, 2.0 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 nbutyllithium 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 (the amine anion solutions were stable for this time period). 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 2-propanol. Scanning force microscopy (SFM) studies of 2-propanol-soaked PMMA sheets demonstrate that there is no swelling of the polymer after several weeks of immersion in 2-propanol. Before modification, the machined PMMA pieces were rinsed with copious amounts of 2-propanol followed by extensive rinsing with 18 MΩ‚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-lithioethylenediamine or N-lithiodiaminopropane (transferred by cannula or syringe). After a given period of time, the reaction was quenched with 18 MΩ‚cm water. After removal from the reaction flask, the PMMA was rinsed with copious amounts of 18 MΩ‚cm water, followed by drying under a stream of nitrogen. This reaction sequence is displayed in Scheme 1. Preparation of C18H37-Modified PMMA Surfaces. Freshly prepared amine-modified PMMA pieces were placed in an airtight vessel, and the vessel was purged with nitrogen for 20 min before introduction of neat n-octadecane-1-isocyanate (Aldrich, 99%), Scheme 2. 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 demonstrate that rinsing the C18H37-modified (9) (a) Ford, S. M.; Davies, J.; Kar, B.; Qi, S. D.; McWhorter, C. S.; Soper, S. A.; Malek, C. K. J. Biomech. Eng. 1999, 121, 13-21. (b) Ford, S. M.; Kar, B.; McWhorter, C. S.; Davies, J.; Soper, S. A.; Klopf, M.; Calderon, G.; Saile, V. J. Microcolumn Sep. 1998, 10, 413-422. (10) Karandikar, B.; Puschett, J.; Matyjaszewzki, K. Polym. Prepr. (Am. Chem. Soc., Div. Polym. Chem.) 1989, 30, 250-251.

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Scheme 1

Scheme 2

PMMA with these solvents does not affect the topography, indicating that the polymer surface is not substantially swelled. Contact Angle Measurements. Sessile drop contact angle measurements utilizing 18 MΩ‚cm water were performed with a VCA 2000 contact angle system (VCA, Billerica, MA). Approximately 6 µL of 18 MΩ‚cm water was placed on the various PMMA surfaces using a syringe. Contact angle values were calculated using the software provided by the manufacturer. The left and right contact angles of the water drops were measured immediately after placement of the water droplet on the PMMA surfaces; it was found in all cases that there was no difference in the left and right contact angles. Each value reported here is 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. Reflection-Absorption Infrared Spectroscopy (RAIRS) Studies. To examine the molecular nature of pristine, NH2- and C18H37-modified PMMA surfaces, RAIRS studies were employed using Au-coated, Cr-primed glass microscope slides (Au slides).11 PMMA was spin coated onto the gold slides by means of a Specialty Coating Systems spin-coater. Commercial sheet PMMA (Goodfellow) was dissolved in dichloromethane to yield a solution with a final concentration of 0.5 mg of PMMA/mL of dichloromethane. This solution was dropped onto the spinning Au slide (2200 rpm) and was allowed to spin for 70 s. The PMMA-coated slide was then ready for chemical modification and/or analysis. RAIRS was performed using previously reported methods.11 X-ray Photoelectron Spectroscopy (XPS) Studies. Pristine PMMA and NH2-modified PMMA surfaces were prepared as described above. The two types of PMMA surfaces were analyzed with a Kratos AXIS ultra X-ray photoelectron spectrometer. (11) Peanasky, J. S.; McCarley, R. L. Langmuir 1998, 14, 113-123.

Measurements were obtained using a monochromatic Al KR X-ray source (240 W) and charge neutralization. The samples were analyzed with a 90° takeoff angle. Deconvolution of spectral peaks was performed using the Kratos software. Scanning Force Microscopy Studies. The surface topographies of pristine, NH2-, and C18H37-modified PMMA surfaces were assessed through use of a Digital Instruments Nanoscope III multimode atomic force microscope (AFM) utilizing the D scanner (13 µm × 13 µm). The microscope was operated in contact mode for all experiments. Images were flattened using the Nanoscope software. Quantification of Amines on the Surface of NH2-Modified PMMA. For all studies presented here, PMMA sheets were machined using standard milling techniques to produce pieces with dimensions 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.12,13 Restriction Endonuclease Immobilization and dsDNA Digestion As Followed through the Use of Capillary Electrophoresis. The general procedure for the attachment of the restriction enzyme to modified PMMA particles, as well as the detection of the digestion products by CE, can be found in the Supporting Information.13 Preparation of PMMA Microchannels and EOF Measurements. A 5.0 cm × 3.0 cm sheet of PMMA (Goodfellow) was subjected to bulk surface micromachining to produce a 40-mm (in length) microchannel, which was 100 µm in depth and 100 µm in width. A top plate made from PMMA 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 amination reaction described above. The diamine used in these experiments was diaminopropane. Following modification, the pieces were assembled using a thermal bonding technique previously described by us.9a EOF measurements were conducted using a method that employed a discontinuous buffer system.14 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, 5) or borate (pH ) 7, 9, 11) buffer solutions. RESULTS AND DISCUSSION I. Modification of Pristine Sheet PMMA To Yield AmineModified PMMA Surfaces. Sessile Drop Water Contact Angle Measurements of Pristine versus NH2-Modified PMMA Surfaces. The average water contact angle for pristine PMMA was found to be 66 ( 2°, which correlates well with the literature value of 67° for a highly ordered methyl ester-terminated monolayer.15 Water contact angle measurements for NH2-terminated PMMA sheets yielded an average of 33 ( 4°, a value consistent (12) (a) Ichijima, H.; Kobayashi, H.; Ikada, Y. J. Cataract Refractive Surg. 1992, 12, 395-401. (b) McGrath, R. Anal. Biochem. 1972, 49, 95-102. (c) Khan, Z.; Rafiquee, Z. A.; Aziz Khan, A. J. Indian Chem. Soc. 1996, 73, 305-306. (13) See Supplementary Information. (14) Haung, X.; Gordon, M. J.; Zare, R. N. Anal. Chem. 1988, 60, 1837-1838. (15) Ulman, A. An Introduction to Ultrathin Organic Films: From LangmuirBlodgett to Self-Assembly; Academic Press: San Diego, CA, 1991.

Figure 1. Reflection-absorption infrared spectra of pristine and NH2-modified PMMA spin-coated on Au.

with that obtained for self-assembled monolayers terminated with hydrophilic functional groups.15,16 Analysis of Pristine and NH2-Terminated PMMA Thin Films by RAIRS. Figure 1A is a representative RAIR spectrum of pristine PMMA on Au (Au-coated, Cr-primed glass microscope slides).11 This spectrum correlates well with the transmission spectrum of PMMA documented in the literature.17 Band assignments are compiled in Table 1. The most prominent band is the carbonyl stretch, ν(CdO), at ∼1733 cm-1. The band maximum position is characteristic of methyl esters, particularly those found for films of PMMA. The remaining vibrational bands observed are characteristic of the alkane and ester moieties present in the polymer. Displayed in Figure 1B is a representative RAIR spectrum of an NH2-modified PMMA thin film; the band assignments are tabulated in Table 1. The major observations that arise upon comparison of Figure 1A and B are the presence of the amide I (ν(CdO)) and the amide II (δ(N-H)) bands at approximately 1673 and 1520 cm-1, respectively, as well as substantially less intense methyl ester bandssthe ν(CdO), ν(C-O), and ν(C-OC) modessin the spectrum of the aminated PMMA film. These data point to the fact that exposure of PMMA films to a lithiated diamine results in replacement of the ester functionalities by an amide linkage, as expected (Scheme 1). In addition, a broad, illdefined band centered at ∼3314 cm-1 is present in the spectrum of the aminated PMMA film. We tentatively assign this band to the ν(N-H) transition associated with primary amines on the surface. Analysis of Pristine and NH2-Modified PMMA Sheets by XPS. Although the infrared spectroscopy results presented above point to the successful formation of amide linkages upon reaction of pristine PMMA with a lithiated diamine, these results were obtained for films of PMMA rather than as-manufactured sheets of PMMA typically used in the fabrication of MEMS devices. Thus, our specific aims for the XPS analysis of pristine and NH2-modified PMMA sheets include determining the presence and identity of nitrogen entities on sheet PMMA. (16) Bain, C. D.; Whitesides, G. M. J. Am. Chem. Soc. 1988, 111, 7155. (17) Omastova, M.; Pavlinec, J.; Pionteck, J.; Simon, F.; Kosina, S. Polymer 1998, 39, 6559-6566.

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Table 1. Infrared Band Positions and Assignments for Pristine PMMA, NH2-Modified PMMA, and C18H37-Modified PMMAa pristine PMMA position

(cm-1)

2955 2920 2850 1733 1487 1454 1436 1389 1273 1240 1189 1063

NH2-modified PMMA

assignment νa(CH3) νa(CH2) νs(CH2) ν(CdO) δ(CH2) δa(CH3) δs(CH3) δs(CH3)

position

(cm-1)

∼3314 2955 2926 2871 2856 1673 1520 1460

C18-modified PMMA

assignment

position (cm-1)

assignment

ν(N-H) νa(CH3) νa(CH2) νs(CH3) νs(CH2) ν(CdO)amide δ(N-H)amide R(CH3)/δ(CH2)

3335 2955 2920 2873 2849 1673 1622 1585 1520 1477 1468 1460 1440 1376 1288-1223 891 871 767 717

ν(N-H)urea νa(CH3) νa(CH2) νs(CH3) νs(CH2) ν(CdO)amide ν(CdO)urea δ(N-H)urea δ(N-H)amide ν(N-C-N)urea δ(CH2) R(CH3) δD(CH2) U Tx;Wx B βE/RE Px P

a These data were collected using reflection-absorption infrared spectroscopy, and all PMMA was spin-coated on Au slides. The band assignments of pristine PMMA were determined with the aid of ref 17.

The X-ray photoelectron survey spectrum of pristine PMMA displayed only two featuressa peak at 285.0 eV and another at ∼532.1 eV, indicative of C 1s and O 1s core levels, respectively. However, the survey spectrum of aminated PMMA displayed a peak at ∼400 eV in addition to the C 1s and O 1s peaks at 285.0 and 532.1 eV, respectively. The peak centered at ∼400 eV is characteristic of surfaces possessing nitrogen-containing functionalities (N 1s core level). This peak can be deconvoluted into two individual peaks, one centered at 399.4-399.5 eV and the other at 400.0-400.1 eV. The peak at lower binding energy corresponds well with that found for the N 1s core level of an amine,18 whereas the peak at higher energy can be assigned as that of an amide.18 Upon further analysis of the XPS data, it was found that the ratio of the peak area of the O 1s peak to the C 1s peak is ∼12% less for NH2-modified sheet PMMA when compared to pristine sheet PMMA. Thus, the XPS results demonstrate that not only is nitrogen present in two forms on the surface of the NH2-modified sheet PMMA but the amount of oxygen present on the surface of the NH2-modified PMMA is less than what was present on the surface of the pristine PMMA.10 This latter observation is in agreement with the infrared data (vide supra) that indicated the presence of fewer ester groups for the NH2-terminated PMMA films. Collectively, these results indicate that the amination reaction can be successfully carried out on sheet PMMA, an outcome that is important for applications requiring modification of commercially available PMMA substrates. The uniformity and accessibility of surface-terminated primary amines was investigated through the use of confocal fluorescence microscopy studies of fluorescein isothiocyanate (FITC)-labeled PMMA surfaces. Images from these studies revealed that there is an abundance of primary amine sites on the surface of the amine-modified PMMA. In addition, the homogeneity of the amine surface population is extremely high.13 (18) Briggs, D.; Seah, M. P. Practical Surface Analysis by Auger and X-ray Photoelectron Spectroscopy; John Wiley: New York, 1983.

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Quantitative Determination of NH2 Groups on the Surface of NH2-Terminated PMMA Sheets. For the reaction of sheet PMMA with N-lithiodiaminoethane, it has been found that a reaction time of 2-5 min leads to an apparent amine surface coverage of 6.85 ( 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 remains steady at ∼6 nmol cm-2 (no statistical difference in coverage with time at the 95% confidence level).13 For reaction times less than 10 min, the surface is found to be colorless and featureless by eye; however, upon exposure of PMMA sheets to the N-lithiodiamine solutions for times greater than 10-15 min, the surface of the plastic appears frosted. The apparent amine surface coverage values are approximately 6-8 times higher than the surface coverage of thiols self-assembled on Au15 or silanes on silica.3 A series of quantification studies are underway which will take into account the roughness of the sides of the PMMA sheets. Preliminary data from these studies indicate that the amine surface concentration on only the faces of the modified PMMA is roughly 5 nmol cm-2. In addition, the intrinsic roughness of the faces of the NH2-modified PMMA sheets may also contribute somewhat to the high amine surface coverage of the modified PMMA; we address this issue next. Characterization of the Surface Topography of Pristine and NH2-Terminated PMMA Sheets Using SFM. By understanding the surface topography of the PMMA sheets, it may be possible to determine the impact of face surface roughness on the surface coverage of amines. Further, we are interested in employing amine-modified PMMA in the fabrication of microanalytical separation channels (chromatographic stationary phase) and for improving adhesion between PMMA and another sheet of PMMA or between PMMA and a conductive substrate. Therefore, it was necessary to ascertain the surface topography of the pristine PMMA surfaces, as well as the amine-modified PMMA surfaces.

Figure 2. Scanning force microscopy images of (A) pristine and (B) NH2-modified sheet PMMA. The scan size is 2 µm × 2 µm and the Z-range is 10 nm.

Displayed in Figure 2A is a representative 2 µm × 2 µm contact-mode scanning force microscopy image of 3-mm Goodfellow sheet PMMA. This surface, as can be seen, is smooth and relatively free of defects; the rms roughness (entire 2 µm × 2 µm range) is 0.39 nm and the roughness factor, R, which is the surface cross-sectional distance divided by the horizontal distance, is 1.002. Shown in Figure 2B is a typical 2 µm × 2 µm scanning force microscopy image of 3-mm Goodfellow sheet PMMA that was modified with N-lithiodiaminopropane for 2 min. The modified surface is obviously much different than the pristine surface, for the surface roughness is ∼3.5 times higher (1.45 nm rms roughness, R ) 1.013) than that of the pristine surface. The rms roughness value for PMMA surfaces exposed to N-lithiodiaminoethane was determined to be 1.80, while the roughness factor was found to be 1.015. 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 reaction does not affect the surface of the PMMA in any way. Based on the SFM studies and the preliminary investigations of edge roughness, the roughness-corrected amine surface coverage is a little less than 5 nmol cm-2. This value is ∼6 times that expected for a close-packed alkane monolayer.15 This larger-thanexpected number of surface amines is most likely the result of amination reactions that occurred below the surface of the original PMMA sheet. Thus, at short reaction times, it would appear that the amination reactions occur to a depth of roughly six repeat units or ∼1.6 nm. For a solution-based polymer modification protocol, this level of penetration is actually quite low. II. Manipulation of Electroosmotic Flow in PMMA Microchannels by Amination of PMMA. The EOF has recently been determined in PMMA-based microdevices to be ∼1.4 × 10-4 cm2/ V‚s (positive value indicates EOF runs from anode to cathode) and is virtually independent of solution pH.9a To investigate the

Figure 3. EOF versus pH for modified (squares) and unmodified (circles) PMMA microdevices. In both cases, the EOF was measured from a pH range of 3-11 using acetate or borate buffers. The low concentration buffer was 10 mM, and the higher concentration buffer was 100 mM. The field strength used (150 V/cm) was selected to minimize Joule heating in the channel, which measured 100 µm × 100 µm × 4 cm.

ability to manipulate the EOF through surface modification of PMMA microdevices, a microchannel was machined into PMMA and then aminolysis of both the substrate and cover plate was performed prior to thermal bonding of 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 Figure 3. As can be seen from this figure, the EOF was reversed (negative EOF value indicates solution flow from cathode to anode) in the case of the modified PMMA microchannel, consistent with a surface with an excess positive charge on its wall. In addition, the EOF is shown to decrease as the solution pH becomes more alkaline (EOF ) -1.80 × 10-4 cm2/V‚s at pH ) 3.0; EOF ) -1.05 × 10-4 cm2/V‚s at pH ) 11.0). This decrease results from the fact that the accessible primary amines are protonated at low pH and become deprotonated with increasing pH. In the case of the unmodified PMMA microdevice, the EOF does show 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 ) 3.0; EOF ) 2.58 × 10-4 cm2/V‚s at pH ) 11.0). To probe the hydrolytic stability of the modified PMMA surfaces, we exposed our amine-modified, micromachined PMMA channels to 0.1 N NaOH for 1 h. We found that the EOF of our amine-modified channels did not change significantly (initial EOF ) -1.13 × 10-4 cm2/V‚s at pH ) 9.0 versus final EOF ) -1.15 × 10-4 cm2/V‚s at pH ) 9.0) after exposure to 0.1 N NaOH for 1 h. Recently, Morris et al. reported a 10-fold increase in the EOF of a polyester-based microdevice upon exposure to 0.1 N NaOH for 5 min.19 III. DNA Digestion with Restriction Enzymes Immobilized on PMMA. To demonstrate the application of functionalized PMMA for enzyme immobilization and reactions, an HaeIII restriction endonuclease enzyme was covalently attached through (19) Wang, S.-C.; Perso, C. E.; Morris, M. D. Anal. Chem. 2000, 72, 17041706.

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a glutaric dialdehyde linkage to amine-terminated PMMA. The HaeIII restriction enzyme recognizes a four-base sequence in dsDNA and cuts between G (guanidine) and C (cytosine) residues within this sequence. Digestion of the dsDNA phage, ΦX174-RF (5386 bp), with HaeIII produces 11 fragments and is commonly used as a standard for size determinations of linear, dsDNA from 72 to 1353 bp in length. To verify coupling of the restriction endonuclease to the functionalized PMMA and to determine whether the enzyme would retain its activity when attached to the PMMA surface, the digestion fragments produced were identified by their sizes using capillary electrophoresis. From our capillary electropherograms,13 there is no detectable difference in the DNA digestion patterns, which indicates that the recognition sequence for the enzyme was unaffected by its immobilization on PMMA. IV. Modification of Amine-Terminated PMMA Surfaces To Yield Alkyl-Terminated Surfaces. We were also interested in modifying the PMMA surface to create a microanalytical separation device that is lithographically patterned on a “chip” of PMMA and also possesses a hydrophobic phase for electrochromatography applications as has been demonstrated for glassbased microdevices.20 We thus turned our attention toward derivatizing the PMMA such that its surface is terminated in C18H37 groups (Scheme 2). After such a reaction sequence, it was found that the C18H37-modified PMMA sheet was colorless and transparent, exactly like the pristine and NH2-modified PMMA sheets. Sessile Drop Water Contact Angle Measurements of C18H37-Terminated PMMA Surfaces. As mentioned above, the water contact angle of the amine-modified PMMA surface is quite low, 33 ( 4°, as is to be expected for a relatively hydrophilic surface. The C18H37-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).15 For a closepacked methyl surface (ordered, long-chain n-alkanethiols on Au), the expected water contact angle is roughly 113°.15 The contact angle values obtained here for the C18H37-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° that is observed for n-alkanethiols on Au), such that the methylene units of the alkane chains are significantly exposed. On the basis of the infrared data (vide infra), we conclude that the chains are ordered but have a substantial tilt from the surface normal. RAIR Spectroscopy Studies. Shown in Figure 4 are representative RAIR spectra of C18H37-modified and NH2-modified PMMA. For the C18H37-modifed surface, the primary bands to note are those associated with ν(N-C-N)urea at 1477 cm-1, the ν(CdO)urea band at 1622 cm-1, the δ(N-H)urea band at 1585 cm-1, and the ν(N-H)urea band at 3335 cm-1. In addition, a large increase in the intensity of the ν(C-H) bands in the 2800-3000-cm-1 region is observed. Overall, these data indicate that the aminemodified PMMA surface has undergone a chemical reaction with the n-octacecane-1-isocyanate to form a urea linkage. Finally, because there is no band at 2200 cm-1 corresponding to ν(CdN), (20) (a) He, B.; Tait, N.; Regnier, F. Anal. Chem. 1998, 70, 3790-3797. (b) Kulter, J. P.; Jacobson, S. C.; Matsubara, N.; Ramsey, J. M. Anal. Chem. 1998, 70, 3291-3297.

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Figure 4. Reflection-absorption infrared spectra of NH2- and C18H37-modified PMMA spin-coated on Au.

there is no physisorbed n-octadecane-1-isocyanate present on the surface of the PMMA. The order (crystallinity) of the C18H37 chains bound to the PMMA surface is very high, as judged by the presence and position of certain infrared bands in Figure 4. The band positions of the νa(CH2) and νs(CH2) modes, 2920 and 2847 cm-1, indicate that the alkane chains are highly ordered.21 This is further supported by the presence of a series of bands known as the progression bands in the 1300-1200-cm-1 region, which are associated with the twisting/rocking and wagging of the methylene C-H groups (Tx and Wx modes). The position and morphology of the progression bands are characteristic of a highly crystalline alkane environment. A more detailed analysis of the infrared bands for various length alkane chains on PMMA will be reported at a later date. Scanning Force Microscopy Studies of C18H37-Terminated PMMA Surfaces. The alkane-terminated PMMA sheet surfaces exhibited a surface morphology that is slightly different from the amine-modified surface. The C18H37-modified PMMA surface is reminiscent of alkylsilane-coated silicon surfaces22 in that the C18H37-modified PMMA surface appears as if it possesses fluidlike characteristics, thus making it difficult to obtain well-resolved SFM images. A rms roughness of 2.8 nm and a roughness factor of 1.034 were obtained from SFM images13 of C18H32-modified PMMA surfaces. This is approximately 1.6-2 times greater than that of the amine-modified PMMA surfaces. CONCLUSIONS In this study, we have demonstrated the successful modification of PMMA sheets used in the manufacture of miniaturized analytical instrumentation. Specifically, we have shown that PMMA can be modified to have accessible amine sites on its surface which can be used to change the flow properties of (21) (a) Hostetler, M. J.; Stokes, J. J.; Murray, R. W. Langmuir 1996, 12, 36043612. (b) Schachtschneider, J. H. Spectrochim. Acta 1963, 19, 85-116. (c) Schachtschneider, J. H.; Snyder, R. G. Spectrochim. Acta 1963, 19, 117178. (d) Snyder, R. G. J. Chem. Phys. 1967, 47, 1316. (e) Maroncelli, M.; Qi, S. P.; Strauss, H. L.; Snyder, R. G. J. Am. Chem. Soc. 1982, 104, 7. (22) Dunaway, D. J.; McCarley, R. L. Langmuir 1994, 10, 3598-3606.

microelectrophoresis devices or can be used as a platform for further functionalization. Immobilization of a DNA restriction enzyme on amine-terminated PMMA leads to the ability to perform heterogeneous enzyme cleavage of dsDNAs. Finally, the synthesis of hydrophobic surfaces appropriate for future electrochromatography applications is demonstrated. ACKNOWLEDGMENT We acknowledge funding of this work by the National Institutes of Health (Grants HG01499 and CA84625), NSF (Grants CHE9529770, CHE-9732195), the Louisiana Education Quality Support Fund, and DARPA. In addition, we are grateful to Amy Ferryman and Professor Julia Fulghum at Kent State University for providing

us with the XPS analyses. Ronald Bouchard at LSU is acknowledged for his help with the confocal microscopy work. SUPPORTING INFORMATION AVAILABLE Experimental details and observations concerning confocal microscopy of labeled amine-terminated PMMA, as well as CE studies of dsDNA digests using immobilized restriction enzyme.13 This material is available free of charge via the Internet at http:// pubs.acs.org. Received for review June 14, 2000. Accepted August 14, 2000. AC000685L

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