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Mesoporous Membrane Device for Asymmetric Biosensing Zhigang Wang,† Richard T. Haasch,‡ and Gil U. Lee*,† School of Chemical Engineering, Purdue University, West Lafayette, Indiana 47907-2100, and Center for Microanalysis of Materials, University of Illinois, Urbana, Illinois 61801 Received September 10, 2004. In Final Form: December 1, 2004 An array of micron size reactors has been designed and fabricated on a mesoporous membrane to create a platform for asymmetric biochemical sensing. Fabrication of this device required that a technique be developed to integrate the mesoporous alumina membrane with a polymeric layer that maintains the integrity of the membrane surface and permeability. This device was used to control an enzyme reaction at the surface of the membrane through the diffusion of the substrates from the opposite sides of the membrane. Asymmetric reactions promise new modes of sensing, enhanced stability of delicate biomolecular systems, and enhanced sensitivity and speed in sensing.
Biosensors are being employed in a large and rapidly increasing number of important medical and environmental applications.1,2 Mechanically, biosensors can be classified as affinity,3 catalytic,4 membrane protein,5-7 and cell based molecular receptors.8 The affinity and catalytic sensors dominate commercial biosensing, while membrane protein and cell based sensors have been largely limited to laboratory use. An important technical advantage of the affinity and catalytic sensors is that they can be performed on glass, polymer, and cellulose surfaces,9 allowing rapid solid-phase extraction of the analyte from the sample. For example, the enhanced sensitivity and speed of affinity sensing executed on nitrocellulose membranes, often referred to as dot-blot or dipstick assays,10 is due to the enhanced surface area and wicking properties of the membrane, which increase the rate of transport of the analyte to the surface of the membrane and thus result in an increased rate of reaction. Microfabrication is enabling new modes of biosensing due to the precise control it provides over fluid handling and temperature.11-14 These so-called lab-on-a-chip (LOC) technologies are rapidly gaining commercial acceptance now that standard fluidic components have been developed and plastic micromachining techniques have been implemented to reduce costs. Sampling of analytes in complex * To whom correspondence should be addressed. E-mail: gl@ ecn.purdue.edu. † Purdue University. ‡ University of Illinois. (1) Rodriguez-Mozaz, S.; Marco, M. P.; de Alda, M. J. L.; Barcelo, D. Pure Appl. Chem. 2004, 76, 723-752. (2) Turner, A. P. F.; Magan, N. Nat. Rev. Microbiol. 2004, 2, 161166. (3) Morgan, C. L.; Newman, D. J.; Price, C. P. Clin. Chem. 1996, 42, 193-209. (4) Clark, L. C.; Lyons, C. Ann. N.Y. Acad. Sci. 1962, 102, 29-45. (5) Thompson, M.; Krul, U. J.; Worsfold, P. J. Anal. Chim. Acta 1980, 117, 121-132. (6) Cornell, B. A.; BraachMaksvytis, V. L. B.; King, L. G.; Osman, P. D. J.; Raguse, B.; Wieczorek, L.; Pace, R. J. Nature 1997, 387, 580583. (7) Bieri, C.; Ernst, O. P.; Heyse, S.; Hofmann, K. P.; Vogel, H. Nat. Biotechnol. 1999, 17, 1105. (8) Brousse, L. Sens. Acutuators, B 1996, 34, 270-275. (9) Kurstak, E.; Tijssen, P.; Kurstak, C.; Morisset, R. Ann. N.Y. Acad. Sci. 1975, 254, 369-384. (10) Leary, J. J.; Grigati, D. J.; Ward, D. C. Proc. Natl. Acad. Sci. U.S.A. 1983, 80, 4045-4049. (11) Weigl, B. H.; Bardell, R. L.; Cabrera, C. R. Adv. Drug Delivery Rev. 2003, 55, 349-377. (12) Chow, A. W. AIChE J. 2002, 48, 1590-1595. (13) Mouradian, S. Curr. Opin. Chem. Biol. 2002, 6, 51-56. (14) Figeys, D.; Pinto, D. Anal. Chem. 2000, 72, 330A-335A.
biological environments remains a major challenge for LOC devices. Inorganic mesoporous membranes composed of alumina15 and silicon16 show excellent potential for LOC sampling devices because they have high pore densities and can be processed under standard microfabrication conditions. This has led to the incorporation of nanoporous alumina membranes in microfabricated silicon devices.17 These membranes have also been employed as sensors and sampling devices in non-LOC applications.18-21 In this letter, we demonstrate that biochemical reactions can be executed on alumina mesoporous membranes that have been integrated into polymer microfluidic layers for the controlled delivery of specific reagents to the front and back of the membrane. A process for integrating inorganic membranes in polymeric microfluidic devices which maintains the surface properties and permeability of the membrane is described. A model protein system, horseradish peroxidase, was used with the mesoporous membranes to demonstrate that asymmetric reactions can be executed at the membrane surface. The rate of reaction of the enzyme with its substrates was detected colorimetrically and was determined to be controlled by the diffusion of the substrate through the membrane. Asymmetric biochemical reactions promise new modes of sensing with improved sensitivity, response time, and stability. Membrane reactors make it possible, in principle, to sense asymmetric protein reactions that take place in many membrane proteins. These membrane reactors also promise to enhance the sensitivity and response time of solid-phase affinity and catalytic reactions through the active transport of reagents. That is, the rate of advection and diffusion of analytes and reagents through the membrane can be actively controlled through the pressure differential across the membrane and the thickness of the membrane, respectively. An important advantage of mesoporous membranes over nitrocellulose ones is that the pore size of mesoporous membranes can be controlled to achieve selective filtration in biomolecular systems. The stability of molecular receptors, catalysts, and cells (15) Furneaux, R.; Rigby, W.; Davidson, A. Nature 1989, 337, 147. (16) Matthias, S.; Muller, F. Nature 2003, 424, 53-57. (17) Toh, C. S.; Kayes, B. M.; Nemanick, E. J.; Lewis, N. S. Nano Lett. 2004, 4, 767-770. (18) Dalvie, S. K.; Baltus, R. E. J. Membr. Sci. 1992, 71, 247-255. (19) Hovijitra, N.; Lee, S. W.; Shang, H.; Wallis, E.; Lee, G. U. SPIE Defense and Security Symposium, Orlando, FL, April 2004. (20) Lee, S. B.; Mitchell, D. T.; Trofin, L.; Nevanen, T. K.; Soderlund, H.; Martin, C. R. Science 2002, 296, 2198-2200. (21) Martin, C. R. Science 1994, 266, 1961-1966.
10.1021/la0477340 CCC: $30.25 © 2005 American Chemical Society Published on Web 01/15/2005
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Table 1. Relative Atomic Compositions of the Microreactor Surface as Measured by XPSa sample
O 1s (532.2 eV)
C 1s (286.9 eV)
C 1s (285 eV)
Al 2p (76.3 eV)
ozone-cleaned alumina membrane surface of microreactor surface of microreactor after Ar/O2
53.3 35.0 62.7
2.1 14.3
20.7 48.1 6.1
23.9 2.8 31.2
a The data were collected using a monochromatized Al KR source (15 kV, 300 W) at a take-off angle of 60° with respect to the surface. High-resolution spectra were acquired using a pass energy of 40 eV, producing an energy resolution of 0.2 eV. For charge neutralization, the low hybrid mode was used with a 200 µm aperture size and 150 W X-ray power. Typical operating pressures were 10-9 Torr.
Figure 1. Optical micrographs of a microreactor array: (A) top view with reactors of 200, 300, and 400 µm diameter; (B) side view of a reactor array in which water drops have been placed in the microreactors.
may be improved through the delivery of specific reagents or nutrients to the membrane surface from either the reactor or supporting sides. Figure 1 presents optical micrographs of a top and side view of an array of circular microreactors that have been constructed on a mesoporous alumina membrane. In the top view, Figure 1A, the dark areas in this figure are alumina membrane, while the light areas are a 30 µm thick polymer film that forms the microfluidic layer. The microreactors were fabricated on 60 µm thick aluminum membranes (Whatman Company, Clinton, NJ) with a nominal pore size of 200 nm. The polymer layer is constructed from SU-8, an epoxy based negative photoresist used to create high aspect ratio polymeric microfabricated devices.22-24 The large size of the reactors in this device was chosen to allow each reactor to be addressable with an inkjet arrayer. Water drops have been added to the microreactor array in the side view, Figure 1B. The SU-8 microreactors were initially created using six processing steps: spin coat, soft bake, expose, post-exposure bake, develop, and hard bake. To obtain maximum process reliability, residual materials were removed from the mesoporous membranes using an ozone gas (Jetlight Co., Irvine, CA) that is capable of rapidly permeating the (22) Shaw, J. M.; Gelorme, J. D.; Labianca, N. C.; Conley, W. E.; Holmes, S. J. IBM J. Res. Dev. 1997, 41, 81-94. (23) Malek, C. G. K. Microelectron. J. 2002, 33, 101-105. (24) Lee, K. Y.; Labianca, N. C.; Rishton, S. A.; Zolgarnain, S.; Gelorme, J. D.; Shaw, J.; Chang, T. H.-P. J. Vac. Sci. Technol., B 1995, 13, 3012-3016.
membrane. Before coating, the mesoporous membranes were clean baked at 90 °C for 15 min to ensure all residual moisture was removed. The membranes were then taped to a polymer-coated paper template and placed on the vacuum chuck of the spin coater (Headway Research Inc, Garland, TX). This impervious layer prevents the photoresist from being drawn through the pores of the membrane during the spin-coating step. The SU-8 (MicroChem Corp., Newton, MA) spin coating was carried out at 1500 rpm for 50 s to create 20-40 µm thick films, as determined by profilometry in postprocessing (Tencor Alpha Step 200 Profilometer, Milpitas, CA). After coating the membrane, the template was removed and the device was soft baked at 65 °C for 5 min and then 95 °C for 10 min on a digital hot plate (Barnstead/Thermolyne, Dubuque, IA). Pattern transfer of the mask of the microreactors was carried out in a Suss MJB-3 mask aligner (SUSS MicroTec Inc, Garching, Germany) at a 1:1 image transfer ratio using a 365 nm light source of 23 mW/cm2 intensity for 12 s. After exposure, the device was baked at 65 °C for 5 min and then at 95 °C for 10 min. The device was then developed and rinsed with reagent-grade isopropyl alcohol (Mallinckrodt Baker Inc., Paris, KY). A hard bake at 120 °C for 10 min was found to be essential for creating durable membrane bonding to the SU-8 film. The structure of the microreactors and membrane was characterized using profilometery, atomic force microscopy (Nanoscope IV, Digital Instruments, Santa Barbara, CA), and electron microscopy (JSM-840, JEOL, Peabody, MA). The SU-8 microstructure did not appear to cause strain or stress within the alumina membrane to a level that could be measured with these techniques. This probably results from the three baking steps for SU-8, which release the stress in the polymer film and the relatively high elastic modulus of alumina. The chemical properties of the membrane surface in the microreactors were characterized with X-ray photoelectron spectroscopy (XPS). XPS measurements were conducted using an imaging X-photoelectron spectrometer (Axis ULTRA, Kratos, Chestnut Ridge, NY) equipped with a charge neutralization system. Table 1 summarizes the results of high-resolution XPS scans from an ozone-cleaned alumina membrane, a membrane surface in a microreactor, and a membrane surface in a microreactor after cleaning with a radio frequency (rf) plasma. Oxygen, aluminum, and carbon are the three primary species observed on the ozone-cleaned membrane surfaces. The rather high concentration of carbon and oxygen on this surface suggests a thin layer of organic contamination remains on the membranes even after ozone cleaning. After patterning with SU-8, the abundance of carbon increased significantly and the aluminum signal decreased significantly. In fact, the amounts of both the saturated and unsaturated carbon at 286.9 and 285 eV, respectively, were observed to increase. These trends suggest that a film of SU-8 remains on the membrane surface after development but that its thickness does not exceed 10 nm. The thin film of SU-8 was removed from the membranes by making several modifications to the microfabrication
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Langmuir, Vol. 21, No. 4, 2005 1155 Table 2. Permeability Measurements on Alumina Membranes with a Nominal Pore Diameter of 200 nm development time (min)
bare membrane SU-8 device SU-8 device SU-8 device SU-8 device
1 4 4 4
Ar/O2 plasma reactor conditions
nitrogen permeability (×10-6 m/Pa‚s)
water permeability (×10-8 m/Pa‚s)
200 W, 5 min 250 W, 5 min 300 W, 5 min
4.29 0.13 2.41 3.56 4.24
2.14 0.11 0.73 1.18 1.37
process. First, the development time of the SU-8 film was increased from 1 to 4 min. Second, the device was exposed to an Ar/O2 RF plasma (Branson Series 3000 Barrel Etcher, Branson International Plasma Corporation, Hayward, CA). Care was taken in selecting a plasma treatment that minimized the decomposition of the SU-8 microstructure and maximized the permeability of the membrane as described below. It was found that optimum reaction time was ∼5 min at a power of 300 W. Optical micrographs of the SU-8 surface after Ar/O2 plasma treatment under these conditions, such as Figure 1, indicate that the SU-8 surface is roughened but there is no delamination of the SU-8 from the membrane. The total carbon measured on the surface of the microreactor surface with XPS after the Ar/O2 plasma treatment decreased to 6.1%, while the alumina increased to 31.2%. The measurements of both carbon and aluminum suggest that the membranes are cleaner after RF plasma treatment than before they are coated with SU-8. The permeability of a membrane is a sensitive measure of pore diameter, density, and chemistry. The permeability was measured with an instrument composed of a pressure driven fluid, pressure transducer, flow meter, and membrane holder, which has been described previously.19 The permeability, Lp, measured the flow through the membrane at a defined pressure
∆F Lp ) A∆P where ∆F is flow, A is the area of the membrane, and ∆P is pressure. A unique feature of this instrument is that the permeability is measured across an unsupported membrane. The results of the nitrogen and water permeability measurements on a 200 nm nominal pore size membrane are shown in Table 2. The permeability of the membrane after treatment with SU-8 was found to be significantly lower than that of the bare membranes, which confirmed that a thin layer of SU-8 coated the surface of the membrane. Treatment with the RF plasma appears to remove a significant amount of SU-8, which is evidenced by the fact that the nitrogen permeability of the membranes is indistinguishable from that of the bare membranes after treatment with an Ar/O2 plasma at 300 W for 5 min. However, the fact that the water permeability of the membranes after plasma cleaning remains lower than that of the bare membrane suggests the pores may still be modified. We hypothesize that the surface of pores may be hydrophobic, which is supported by the observation that the Ar/O2-plasma-treated SU-8 has a contact angle of 15°, which is slightly higher than the bare alumina membrane. The bottom surface of the microreactor was also characterized by atomic force microscopy (AFM) before and after treatment with the Ar/O2 plasma. Figure 2A presents an AFM image of the surface of an alumina membrane after ozone treatment in which a rough membrane surface and several large pores are observed. After patterning with SU-8, Figure 2B, the surface of the membrane in the microreactor was completely flattened
Figure 2. Tapping mode AFM images of the surface of the alumina membrane and microfabricated reactor: (A) 200 nm pore size alumina membrane after ozone cleaning; (B) microfabricated reactor surface after SU-8 development; (C) microfabricated reactor surface after 5 min of 300 W Ar/O2 cleaning. Scheme 1
and the pores could not be seen. This is consistent with the presence of a thin SU-8 film on the membrane and suggests that a significant amount of SU-8 may have entered the pores. After the microreactor surface was cleaned with the Ar/O2 plasma, Figure 2C, structures characteristic of pores were observed once again, but the edges of the pores also appear to be much smoother. Asymmetric biochemical reactions were demonstrated in the mesoporous microreactors using the horseradish peroxidase (HRP) enzyme as a model catalyst.25 Peroxidases are a class of enzymes known to decompose two (25) Hosoda, H.; Takasaki, W.; Oe, T.; Tsukamoto, R.; Nambara, T. Chem. Pharm. Bull. 1986, 34, 4177-4182.
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molecules of hydrogen peroxide into water through a superoxide ion pathway. HRP has been used as an immunohistochemical label, as its specificity for the second molecule of hydrogen peroxide is low, and other electron donors can be substituted. This allows HRP to be used as a catalyst for chemiluminescent26 and colorimetric27 substrates. In this work, the colorimetric reaction shown in Scheme 1 was used to detect the presence of the enzyme. For the reaction, the 4-chloro-1-naphthol (4-CN) and N,N′diethyl phenylenediamine dihydrochloride (DEPDA) substrates react to form a water insoluble product that has a deep blue color. In the asymmetric assay, the back of the membrane device was first exposed to the substrate solution, which was composed of 1.3 mM 4-CN (Sigma), 0.23 mM DEPDA (Sigma), and 4.4 µM H2O2 in 10 mM phosphate buffer (PB: 5 mM Na2HPO4 and 5 mM NaH2PO4) at pH 7.0. HRP was then introduced onto the microreactor’s membrane surface as 10 µg/mL HRP-labeled streptavidin (KPL, Gaithersburg, MD) in a phosphate buffered saline solution (PBS: 5 mM Na2HPO4, 5 mM NaH2PO4, 5.4 mM KCl, and 0.12 M NaCl) at pH 7.0. The HRP reaction was run until a uniform blue-colored dye could be detected by the eye, which took ∼60 s. Figure 3 presents an optical micrograph of a microreactor array in which an asymmetric reaction has been conducted. This image was prepared by running the asymmetric HRP reaction in an Ar/O2-cleaned microreactor array as described above, rinsing the array with water, and drying the array with nitrogen. A deep blue color was immediately observed in the bottom of the microreactors, while no color change was detected in areas where the SU-8 photoresist coated the membrane surface. The deposition of the blue dye product on the reactor surface clearly indicates that HRP, 4-CN, DEPDA, and H2O2 were present at high concentrations at the membrane surface. No color change took place in microreactors when only HRP was added to the front surface, but a slow color change was observed when only the substrate solution was added to the back surface. This slow color change in the absence of HRP is due to the much slower catalytic reaction of 4-CN with DEPDA in the presence of H2O2. The light blue color of the SU-8 film in Figure 3 is a result of the noncatalytic reaction. The overall rate of reaction in the microreactors is controlled by the mixing of the conjugate with the substrate
and the reaction kinetics of HRP. HRP is known to have a high turnover number of ∼1000 s-1.28 Thus, the substrate molecules that reach the enzyme are almost immediately converted into the insoluble product. This suggests that the overall rate of reaction is determined by the diffusion of the hydrogen peroxide and dye molecules to the HRPstreptavidin conjugate. These molecules are free to move through the membrane, as the nominal pore size of the membranes used in this study was 200 nm. Although the HPR-streptavidin conjugate is free to diffuse in the reactor, its diffusion coefficient is ∼0.05 × 10-5 cm2/s. This is at least 1 order of magnitude slower than that of the dyes and hydrogen peroxide. This suggests that the substrate molecules react with the HPR-streptavidin conjugate in the immediate vicinity of the interface of the membrane and that the overall rate of reaction is set by the diffusion of the substrate to the surface of the membrane through the pores. The fact that the blue color is localized on the membrane surface of the reactor is consistent with this model. The HRP reaction can also be run in a configuration in which the conjugate is first absorbed on the reactor surface of the microreactor and the dye mixture is then added to the opposite side of the membrane. In this case, the rate of reaction was at least 10 times slower. The slower overall reaction time appears to result from the fact that the dye mixture must diffuse through the 60 µm thick mesoporous membrane to the HRP absorbed on the surface of the membrane. In many sensors, it is advantageous to use immobilized receptors, but it is unacceptable to wait for 10 min for the initiation of a reaction. The speed of surface bound asymmetric reactions could be increased by using pressure driven flow to drive a substrate or analyte through the membrane. The design of our microreactor mesoporous membrane array could be integrated with a second or third microfluidic layer to control the delivery of either analyte or substrates. We believe that one of the most important applications of asymmetric biochemical reactors will be for membrane proteins that are asymmetric. Membrane proteins are often asymmetric, as their function is to transduce signals, molecules, or energy across a lipid bilayer. These proteins have reaction centers designed to operate either in the environment of the cytoplasm and periplasm or across the organelle in which they are localized. Membrane protein sensors typically have not been able to fully utilize this asymmetry and thus have not taken full advantage of the protein function. Rather, these sensors have used proteins randomly oriented in lipid vesicles or bilayers supported on a surface.6,7 The micropatterned mesoporous membranes provide at least two advantages for membrane protein sensing. First, the mesoporous membrane structure provides a permeable barrier that protects the delicate vesicle and bilayers from hydrodynamic shear and species that can disrupt their structure. Second, the micropatterned mesoporous membranes allow the environment of a specific side of the bilayer membrane to be addressed. If the membrane proteins are oriented in the bilayers, the pH or concentration of a reactant can be regulated on a specific side of the protein. It will also provide the means to analyze the reaction products at a specific surface of the membrane. To conclude, the development of a low cost means for incorporating mesoporous membranes in polymeric LOC devices has made it possible to employ asymmetric
(26) Nozaki, O.; Ji, X. Y.; Kricka, L. J. J. Biolumin. Chemilumin. 1995, 10, 151-156. (27) Conyers, S. M.; Kidwell, D. A. Anal. Biochem. 1991, 192, 207211
(28) Saunders, B. C.; Holmes-Siedle, A. G.; Stark, B. P. Peroxidase: The Properties and Uses of a Versatile Enzyme and of Some Related Catalysts; Buttersworths: London, 1964.
Figure 3. Image of HRP catalyzed colorimetric reaction in a region of 400 µm microreactors.
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biochemical reactions in affinity, catalytic, membrane, and cell based sensing. The permeability of the membrane was maintained at 80% of its original level through optimization of the development and spin-coating steps and the addition of an Ar/O2 etching step. This device was used to demonstrate an asymmetric reaction, in which an enzyme and its reaction substrate were fed to a membrane surface from opposite sides of the membrane. These devices promise to improve the sensitivity, response time, and stability of several existing sensors and provide new modes of membrane protein sensing.
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Acknowledgment. This research was supported by the Center for Membrane Protein Biotechnology at Purdue University. The authors would like to thank Jinwon Park and Hao Shang for valuable discussions and Bill Crabill and Tim Miller for technical assistance in the Microelectronics & Nanotechnology Research Laboratory. XPS was carried out in the Center for Microanalysis of Materials, University of Illinois, and was partially supported by the U.S. Department of Energy under grant DEFG02-91ER45439. LA0477340
