NANO LETTERS
Micro/Nanowell Arrays Fabricated from Covalently Immobilized Polymer Thin Films on a Flat Substrate
2002 Vol. 2, No. 4 275-278
Mingdi Yan* and Michele A. Bartlett Department of Chemistry, Portland State UniVersity, P.O. Box 751, Portland, Oregon 97207-0751 Received November 21, 2001; Revised Manuscript Received December 21, 2001
ABSTRACT This Letter describes a new method to create micro/nanowell arrays from covalently attached polymer thin films on a silicon wafer. The immobilization chemistry utilized a photoactive cross-linker, resulting in polymer thin films of several to a few tens of nanometers. Micro/ nanowell arrays were obtained when two polymers were immobilized sequentially and the second was thicker than the first polymer. The well arrays were generated from either different polymers, or the same polymer with different molecular weights.
The development of miniaturized systems for chemical, analytical, and diagnostic applications has attracted enormous interest recently.1,2 Chip-based microwell arrays have increased the capability to perform analytical3 and biochemical reactions and assays.4,5 These microchips allow a large number of reactions and analyses to be carried out simultaneously, at much higher speed, efficiency and control than conventional methods.6,7 The small volume of the microwells significantly reduces the amount of reagents needed, resulting in reduced wastes and lower costs. Most of the microwells are silica-based that are fabricated in silicon wafers or glass slides using micromachining techniques by way of photolithography and etching into the substrate.8-10 As the surface-to-volume ratio increases dramatically for microfabricated devices as compared to conventional reaction flasks and tubes, it is important that the devices are chemically compatible with the reactions and assays taking place inside them. However, chip-based devices may not be compatible with all the reagents or biomolecules. For example, it was found that native silicon was an inhibitor of polymerase chain reaction (PCR) and amplification,11 and the PCR reactions performed in chip-based microwells had poor reproducibility.12 Recent investigations have focused on polymer-based microwells. Walt et al. developed a technique for the generation of densely packed ordered arrays of micro/ nanowells by etching the distal face of a fiber-optical bundle.13,14 A limitation is that the size and density of the microwells depend on those of the optical fiber bundle, which cannot be changed easily. Ewing and co-workers fabricated * Corresponding author. Tel: 503-725-5756. Fax: 503-725-9525. E-mail:
[email protected]. 10.1021/nl0156742 CCC: $22.00 Published on Web 02/06/2002
© 2002 American Chemical Society
picoliter microvials in polystyrene by embossing on a master mold that was created by the photolithography patterning technique.15 Recently, Whitesides16,17 and Chilkoti18 demonstrated the formation of large arrays of microwells in poly(dimethylsiloxane) (PDMS). These microwell arrays were fabricated by casting the PDMS elastomer against a master mold that was prepared by conventional photolithography. This method is particularly suitable for PMDS, but it offers few choices for alternative materials. We have developed a surface chemistry for the covalent immobilization of polymer thin films on silicon wafers using silane-functionalized perfluorophenyl azides (PFPA-silane).19,20 PFPAs are photoactive compounds that, upon photolysis, generate perfluorophenyl nitrenes that are capable of undergoing C-H and/or N-H insertion reactions with neighboring molecules. The substrate, a silicon wafer or glass, was first treated with PFPA-silane, forming a surface that was covered with a monolayer of perfluorophenyl azido groups. A polymer was then spin coated, and the film was subjected to photolysis. The nitrene intermediate that was generated upon photolysis inserted into the neighboring polymer chains. Since only the molecules adjacent to the azido groups were expected to be covalently attached, a “monolayer” of polymer resulted after the unbound polymer was removed. In this Letter, we report the generation of micro/nanowell arrays on a flat substrate with immobilized polymer thin films. In the process, a PFPA-silane derivatized silicon wafer was first patterned with a polymer via photolithography. After removal of the unattached polymer, unreacted PFPA was left in the nonirradiated areas (Figure 1). When the second polymer was spin coated and irradiated, it was
Figure 1. Schematic illustration for the fabrication of well arrays.
Figure 2. AFM image of micro/nanowell arrays created from PEOX (well bases) and PS.
installed in these regions. The topography was created due to the different thickness of the immobilized films. Therefore, when the second polymer was thicker than the first polymer, microwell arrays would result (Figure 1). Using this strategy, arrays of micro/nanowells were successfully fabricated on silicon wafers. Figure 2 is the AFM image of micro/nanowell arrays that were created with poly(ethyloxazoline) (PEOX), a hydrophilic and biocompatible polymer, and polystyrene (PS), a hydrophobic polymer. In the process, a solution of PEOX in chloroform (10 mg/mL) was spin coated at 2000 rpm for 1 min on a silicon wafer that was functionalized with PFPAsilane.20 A photomask that contains arrays of 5.5 µm circular openings was placed in direct contact with the PEOX film by applying a vacuum. The sample was then irradiated with a 450 W medium-pressure Hg lamp (Hanovia) through a 280 nm optical filter for 10 min. The unattached polymer was removed by soaking in chloroform for 30 min followed by sonication for 5 min, giving rise to arrays of circular PEOX patterns. The sample was then spin coated with a solution of PS in toluene (10 mg/mL) at 2000 rpm for 1 min, irradiated through a 280 nm optical filter for 10 min, and cleaned by sonication in toluene. Since the PS film (6.4 nm thick by ellipsometry) was thicker than PEOX (3.2 nm thick by ellipsometry), micro/nanowells of ∼5.5 µm in diameter 276
Figure 3. XPS (a) image and (b) line scan of PEOX/PS arrays. Measurements were made on a Physical Electronics Instruments Quantum 2000 Scanning ESCA Microprobe equipped with a focused monochromatic Al KR X-ray source at 1486.7 eV for excitation and a spherical section analyzer with a 16 element multichannel detection system. The collected data were referenced to an energy scale with binding energies for Cu 2p 3/2 at 932.67 ( 0.05 eV and Au 4f at 84.0 ( 0.05 eV. Low-energy electrons and argon ions were used for specimen neutralization.
and ∼3 nm in depth were created, with PEOX residing in the well bases and PS forming the well walls. The formation and chemical composition of the micro/ nanowell arrays were evaluated using X-ray photoelectron spectroscopy (XPS) and time-of-flight secondary ion mass spectrometry (TOF-SIMS). Figure 3a is the electronic image obtained by XPS for a patterned polymer array, with the light and dark areas being PEOX and PS, respectively. The X-ray beam was incident normal to the sample while the detector was set at an angle of 45° from the normal. Represented by line 17 is a 280 µm line scan of the surface using a 20 µm beam. Results of the scan, graphed as the atomic concentration versus distance for N, C, O, Si, are found in Figure 3b. The PS region was overwhelmed with C whereas a higher concentration of N was detected in the PEOX region. The Nano Lett., Vol. 2, No. 4, 2002
Figure 5. AFM image and cross-sectional profile of microwells fabricated from PS of molecular weight 280 000 (well bases) and 1 815 000. Figure 4. TOF-SIMS spectra and image of PEOX/PS arrays. The scale on the right indicates the total ion count (×10-3). The data were collected using a Physical Electronics Instruments PHI TRIFT II Model T2100 TOF-SIMS Ion Mass Spectrometer outfitted with a reflectron type 35° triple electrostatic sector time-of-flight analyzer. An isotopically pure liquid gallium metal ion gun was used with a current of 600 pA. SIMS data were acquired over a mass range of 0.5-1850 amu, with a focus on masses 57 and 91 during imaging in the positive mode. The spectral resolution was ∼4000-5000 m/δ measured at mass 26 with a spatial resolution better than 0.5 µm. Calibration was achieved using mass fragments CH3+, C2H2+, and Si+.
higher concentrations of Si and O in the PEOX region were due to the fact that the PEOX film (3.2 nm) was much thinner than the PS film (6.4 nm). Therefore the beam detected significantly more signals from the SiO2 layer on the wafer surface. TOF-SIMS employs a pulsed primary ion beam to desorb and ionize species from the sample surface. The resulting secondary ions are accelerated into a mass spectrometer, where the masses are analyzed by measuring their time-offlight from the sample surface to the detector. The image is generated by rastering a finely focused beam across the sample surface. Figure 3 are TOF-SIMS spectra and image of the PS/PEOX array at the peak intensity of 57 Da. The mass at 57 Da is the fragment resulted from PEOX (OdC+sC2H5), thus confirming that the patterned area was PEOX. The results also showed a high fidelity of the features with those on the corresponding photomask. Micro/nanowells could also be generated from the same polymer of different molecular weights. It has been observed Nano Lett., Vol. 2, No. 4, 2002
that the thickness of the immobilized film depended on the molecular weight of the polymer: the higher the molecular weight, the thicker the immobilized polymer film.20 Figure 4 is the AFM image of micro/nanowell arrays fabricated from polystyrene of molecular weight 280 000 and 1 815 000, respectively. The thickness of the higher molecular weight PS was greater than that of the lower molecular weight PS, leading to the formation of micro/nanowells. The depth of the wells obtained from the AFM image, ∼9 nm, was consistent with the ellipsometry measurements which were 6 and 15 nm for the two molecular weights PS, respectively. In conclusion, a new method has been developed for the fabrication of microwell arrays from immobilized polymer thin films on a flat silicon wafer. The topography was generated either by different polymers or the same polymer of different molecular weights. The method is versatile due to the general C-H and/or N-H insertion reactions of the photolinker.21 Therefore, the materials for well base and well wall can be tailored for specific applications. Acknowledgment. We acknowledge Portland State University and the donors of the Petroleum Research Fund, administered by the American Chemical Society, for support of this research. M.A.B. is a recipient of Oregon Laurels Education Scholarship. We thank Professor Jody House at Oregon Health and Science University and Joo Chan for their assistance on AFM. We are grateful to Mark Englehard and Dr. Daniel Gaspar, Environmental Molecular Sciences Lab at Pacific Northwest Laboratories, for their help with the attainment of XPS and TOF-SIMS data. Silicon wafers were generously donated by Wacker Siltronics Corp. 277
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NL0156742
Nano Lett., Vol. 2, No. 4, 2002
