- Email: [email protected]
glass substrate for immuno-diagnosis
Current Applied Physics 9 (2009) e60–e65
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Current Applied Physics journal homepage: www.elsevier.com/locate/cap
Microfluidic chip with porous anodic alumina integrated with PDMS/glass substrate for immuno-diagnosis Kwang Suk Yang a, Hae Jin Kim b, Jeong Keun Ahn b, Do Hyun Kim a,* a
Department of Chemical and Biomolecular Engineering and Center for Ultramicrochemical Process Systems, KAIST, 373-1, Guseong-dong, Yuseong-gu, Daejeon 305-701, Republic of Korea b Department of Microbiology, School of Bioscience and Biotechnology, Chungnam National University, Daejeon 305-764, Republic of Korea
a r t i c l e
i n f o
Article history: Received 1 July 2008 Received in revised form 1 November 2008 Accepted 1 December 2008 Available online 13 March 2009 PACS: 78.55.Mb 87.85.fk 87.85.M 85.85.+j 81.20.Fw 42.82.Cr Keywords: Anodic aluminum oxide Hepatitis B virus Immuno-sensor ELISA
a b s t r a c t An efficient system for diagnosis of disease marker molecules in microfluidic devices was developed by employing anodic aluminum oxide (AAO) which has highly ordered, uniform, and straight nanopore arrays by a two-step anodization process. AAO on glass substrate was integrated within poly(dimethylsiloxane) (PDMS) microchannel structure. Vacuum-deposited aluminum thin film was anodized by variation of electrolyte composition, applied voltage and anodizing time, for specific pore sizes and depth. The pore was tunable to achieve a size corresponding to target proteins. For enhancement of antibody immobilization and adhesiveness with a PDMS micro-pattern, surface activation of AAO was performed by TMOS–sol spin-coating and calcinations to form a SiO2 layer. The demonstration of diagnosis of biomarker protein was performed by employing conventional sandwich-type immuno-assay for hepatitis B virus (HBV). The anti-hepatitis B surface antigen (anti-HBsAg) was immobilized by bridges using c-aminopropyltriethoxysilane and glutaraldehyde. The hepatitis B surface antigen (HBsAg) was coupled with anti-HBsAg and sheep anti-HBs/horseradish peroxidase conjugate. The result was analyzed by colorimetric assay for comparison with the result using conventional immuno-assay and it showed higher efficiency using microfluidic channels. The AAO inside the PDMS microfluidic channel allows specific immobilization of proteins by controlling the size for access. This study can be extended for a highthroughput system for bio-marker proteins. Ó 2009 Elsevier B.V. All rights reserved.
1. Introduction Microfluidic systems have become widely used for various chemical and biochemical applications, which include sample pre-handling, reagent mixing, separation, enzyme reaction, and detection processes, due to short mass transfer length and transport time [1], very small volume of analytes [2], almost isothermal conditions, and easy parallelization [3]. In the particular case of clinical diagnostics, these inherent properties of microfluidic devices are related to high selectivity and sensitivity in biorecognition. Additionally, the use of high-surface-area micro/nano structure, such as mesoporous materials, in a heterogeneous diagnosis system can increase sensitivity between probe and target molecules by increasing probe loading capacity, a higher target binding specificity and accessibility of targets to the probes, and reducing reaction/hybridization time [4]. Many approaches to increasing the surface-area-to-volume ratio of microchannels have been proposed, including microfabrica* Corresponding author. Tel.: +82 42 350 3929; fax: +82 42 350 3910. E-mail address: [email protected] (D.H. Kim). 1567-1739/$ - see front matter Ó 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.cap.2008.12.031
tion by photolithography, packing microbeads, or patterning of a porous membrane inside the microfluidic channel. However, these approaches require high-end lithographic techniques, expensive equipment and significant amounts of precious materials and labor, due to the large number of manipulations needed for fabrication, because the uniformities of sizes, shapes and volumes of the void spaces in porous materials are directly related to their ability to perform the desired function in a particular application. Anodic aluminum oxide (AAO) membrane, which has a hexagonal pore array of nanometer scale, has been studied for size-selective separation (chemicals or bio-molecules) and template material for metal nanowires (electroplating) or polymer nanotubes. Masuda and co-workers proposed a two-step-anodization method for self-organized formation of a hexagonal pore array [5,6]. The two-step-anodization method includes removal of the amorphous porous layer to expose well-oriented pores. Martin and co-workers [7] devised an elegant method to separate drug enantiomer from racemic mixture by using molecular recognition protein and functionalized AAO, which is prepared by immobilization of antibody on the inner surface of AAO activated by a sol–gel method.
K.S. Yang et al. / Current Applied Physics 9 (2009) e60–e65
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Here, a heterogeneous immuno-assay is developed using an AAO-incorporated PDMS (polydimethylsiloxane) microfluidic system, which has the advantages of rapid analysis time and smaller volumes of samples such as antibodies and antigens. Enzymelinked immunosorbent assay (ELISA), typically carried out in a 96-well microtitre plate, is the most common method of heterogeneous immuno-assay. However, conventional ELISA involves a tedious and labor-intensive protocol that often results in large errors and inconsistent results due to personally operated injection, mixing, and washing steps. Also, insufficient heat and mass transfer during the incubation step contribute to errors and inconsistent results [8]. A porous AAO-incorporated PDMS microfluidic system can provide a wide surface area with chemical modification for the effective immobilization of target antibodies [9,10], highly improved mass transfer, precise temperature control, and semiautomation. In this work, the surface of the size-controlled AAO nanopore array is modified by SiO2. Then, anchor and linker molecules are introduced into the SiO2 pores. Finally, hepatitis B surface antibody (HBsAb) is immobilized in the glutaraldehyde-activated SiO2–AAO pores. The ELISA test was carried out with a naked-eye method.
2. Experimental 2.1. Chemicals
c-Aminopropyltriethoxysilane (APTES), tetramethylorthosilicate (TMOS, 98%), ethanol, HCl (38%), oxalic acid, phosphoric acid, hydrogen peroxide, and sulfuric acid were purchased from Sigma– Aldrich (St. Louis, MO, USA). Hepatitis B virus surface antibodies (mono- and polyclonal: anti-HBs) were purchased from Chemicon (Temecula, CA, USA). 96-well ELISA kit for hepatitis B surface antigen (HBsAg) and sheep Anti-HBs/horseradish peroxidase conjugate diluted with bovine serum albumin–phosphate buffer, normal human serum, tetramethylbenzidine (TMB) solution, and dimethylsulfoxide (DMSO) were purchased from Green Cross Medical Science Corp. (GyeongGi-Do, Korea). All solutions were made in Milli-Q-grade deionized water from a Human UP 900 (Human Corp., Korea). Other chemicals and reagents used were of analytical grade. 2.2. Fabrication of AAO nanopore array As shown in Fig. 1A, a two-step-anodization method was used for self-organized formation of a hexagonal pore array [5]. A microscope glass slide was cleaned following the standard cleaning process. As a source of anodic alumina layer, 3-lm thick Al layer was deposited on the glass slide at 10-6 torr. Nanoporous anodic alumina layer was grown in 0.3 M oxalic acid solution as an electrolyte. 40–80 V DC anodic voltage was applied for 60–120 s using an EDP-1501 DC power supply (PNCYS Co., Ltd., Korea). Inter-pore distance was 100 nm at 40 V and 15 °C. Prepared Al2O3 surface was removed by 0.6 M H3PO4 and 0.2 M CrO3 for 180 s at 60 °C to give substructure. Then, second anodizing was carried out to grow AAO with thickness of 1.45 lm under the same condition of 0.3 M oxalic acid under 40–80 V DC voltage for 90–180 s at 15 °C. Here, the thickness of AAO layer is considered as the pore depth. Finally, the pore widening process was performed with 0.1 M H3PO4 solution for 300 s at 30 °C to give an opening diameter of 80 nm, which is related to the surface area. 2.3. Fabrication of PDMS microchannel PDMS (Dow Chemical, Midland, MI, USA) microchannel was fabricated using standard soft lithography and PDMS replica mold-
Fig. 1. (A) Patterning procedure of AAO on a glass slide substrate: (a) first anodizing process to obtain hexagonally oriented pores; (b) removal of first Al2O3 layer for pore arrangement; (c) second anodization for depth of pores; (d) pore widening for pore diameter. Pre-widening condition: 0.3 M oxalic acid, 80 V, 15 °C; etching condition: 0.6 M H3PO4, 60 °C. (B) Scanning electron microscopic images of each step.
ing techniques. The general procedure is described in Fig. 2. AZÒ 9260 (Clariant Corporation AZ Electronic Materials, Wilmington, DE, USA) photoresist was spin-coated onto Si wafer at 2000 rpm. This wafer was baked at 110 °C on a contact hotplate for 120 s. Then, it was cooled and exposed to 1300 mJ/cm2 365-nm i-line UV using a CA-4/6 M mask aligner system (Shinumst Co., Ltd., Korea). The photoresist was developed with AZ 400 K developer (AZ Electronic Materials, Wilmington, DE, USA) for 10 min. Then, the wafer was washed with deionized water and dried with N2 gas. The PDMS replica molding process was carried out by casting PDMS mixture composed of a 10:1 ratio of base and curing agent on the AZÒ 9260 master pattern. After evacuation, PDMS mixture was cured at 70 °C for 4 h. Then, PDMS molding was removed carefully after cooling to room temperature. The resulting PDMS mold was cut and punched for use.
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2.4. Preparation of TMOS–sol solution
2.5. Surface modification of AAO with polyclonal anti-HBsAg
Sol–gel solution for coating was typically prepared with a mole ratio of 1:4:0.003:4 (TMOS:ethanol:HCl:H2O) [11]. The mixture was stirred for 2 h at 60 °C. To hydrolyze the TMOS, hydrochloric acid (38%) was added dropwise. Then, the flask was sealed and the temperature was raised to 80 °C for the reaction to give TMOS–sol. Prepared TMOS–sol was coated on the top of porous AAO substrate by spin-coating at 1500 rpm as shown in Fig. 3.
2.5.1. Silanization of SiO2–AAO A freshly prepared solution containing 10 mL ATPES in 90 mL water, was recirculated at a feeding speed of 2 lL/min using a syringe pump (KDS-100; KD Scientific Inc., PA, USA). The reaction was carried out at room temperature for 2 h. After reaction, the device was washed with distilled water until neutral.
Fig. 2. Photolithography and PDMS replica molding process: (a) photoresist spin-coating; (b) UV exposure and developing; (c) PDMS casting on photoresist mold; (d) curing of PDMS; (e) detaching of PDMS from mold and punching of fluidic ports; (f) oxygen plasma treatment; (g) alignment and bonding of PDMS mold with AAO on a glass slide.
Fig. 3. SiO2 coating and anti-HBs immobilization in AAO nanopores: (a) TMOS–sol coating; (b) formation of amine group using ATPS; (c) surface modification with glutaraldehyde; (d) immobilization of anti-HBs (primary antibody).
K.S. Yang et al. / Current Applied Physics 9 (2009) e60–e65
2.5.2. Coupling of anti-HBsAg on SiO2–AAO pore The ATPES-modified SiO2–AAO membrane was first equilibrated with 0.2 M NaAc–HAc buffer (pH 7.50) for 10 min at room temperature. After removal of buffer, 100 mL of 25 wt% glutaraldehyde aqueous solution was circulated at 1 mL/min for 3 h at 40 °C. After reaction, the device was washed with distilled water. The device activated with glutaraldehyde was then subjected to a reacting solution containing 10 mg HBV monoclonal antibody in 10 mL of 0.01 M CaCl2 and 0.05 M Tris–HCl buffer (pH 8.5). The immobilization was carried out by circulating the antibody solution through the microfluidic device for 2 h at a flow rate of 1 mL/min. The activity of the immobilized enzyme was monitored by taking control samples. 2.6. Enzyme-linked iummunosorbent assay (ELISA) In the ELISA, HBsAg was used as antigen, Anti-HBsAg was used as the primary antibody, and horseradish peroxidase-conjugated monoclonal goat Anti-HBs was used as secondary antibody conjugate. HBsAg was used as positive control, and normal human serum was used as negative control. Positive control diluted to 1/4 of original concentration with negative control was used as a test sample. Washing buffer was prepared from pH 7 PBS. The substrate solution was prepared by diluting TMB (5.25 g/L in DMSO) with citrate–phosphate buffer as prepared from the company. In this experiment, the determination was carried out by colorimetric analysis (naked-eye method). The mean absorbance of the positive control should be greater than or equal to that of the sample. If the absorbance of the test sample does not reach that of the negative control, the test sample is considered negative for the presence of hepatitis B antigen. The opposite result can be considered positive. 2.6.1. ELISA in 96-well plates One hundred micro litres of negative control and positive control, and the test sample were added to each well. Then, prepared enzyme-conjugate was added into each well. The well-mixed wells were incubated at 37 ± 1 °C for 90 min. Individual wells were evacuated and washed five times with 300 lL washing buffer. Washing buffer was completely removed, and 100 lL of enzyme–substrate solution was added into each well. Then, it was incubated for 30 min at room temperature. For colorimetric analysis, an image of the color change was taken by Olympus CAMEDA C-5060 digital camera when the reaction time had reached 30 min. 2.6.2. ELISA in AAO-incorporated PDMS microfluidic channels An AAO-incorporated PDMS microfluidic chip was placed on a warm (37 °C) hot plate with feeding of positive control, negative control and sample by syringe pumps at a rate of 2.5 lL/min to microfluidic channels for 20 min. HRP conjugate was loaded with the same speed without the washing step. Unthreaded HRP conjugate was removed by using washing buffer for 5 min. Enzyme–substrate solution was loaded with the same feeding speed for 15 min. The eluant was collected from each drain port. The colors of the collected substrate solutions were not changed, due to the isolation with enzyme bound into the porous AAO substrate.
3. Results and discussion 3.1. Fabrication of AAO on SiO2 glass slide Electrolyte composition, temperature and anodizing voltage may be varied depending on the desired parameters of the anodic alumina substrate, such as pore density, diameter, and thickness. The inter-pore distance was observed to decrease with decreasing
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anodizing voltage, while the layer growth rate was observed to depend on the desired pore diameter and electrolyte composition, being proportional to the current density. The anodizing conditions should be optimized for the dimensions of the target protein. A two-step-anodizing procedure is adopted to achieve regular opening and positioning of the pores. A large hexagonal pore array of anodic aluminum oxide (AAO) using the two-step-anodizing technique serves as a nano-well bioreactor for the immuno-diagnosis. The opening size of the AAO nano-pore as prepared is about 100 nm, which is wide enough for sandwich-type heterogeneous immuno-assay. Fig. 1B shows SEM images of AAO on the slide after anodizing at 40 V applied voltage in 0.3 M oxalic acid. The anodizing process turns the Al layer into mirror-like AAO as transparent alumina, as shown in Fig. 4B. Transparency is a desired property for the application of an optical detection technique, especially naked-eye or fluorescence determination, in diagnosis. 3.2. Design and fabrication of PDMS/AAO microfluidic device The device shown in Fig. 4A is composed of several parts, comprising five inlet ports with a main channel for washing, a reaction chamber for enzyme reaction and outlet ports. The width of the washing channel is 200 lm, but the ports for controls and the test sample ports are 100 lm. This will prevent reverse flow of the main stream to the sample inlets. The overall channel wall thickness is 25 lm. AAO was modified by the sol–gel method to improve the bonding with the PDMS microchannel and then treated with piranha solution to obtain immobilization efficiency of primary antibody. TMOS–sol prepared by a typical method was coated on AAO and the organic part was removed by calcination at 250 °C. A photograph of the device is shown in Fig. 4B with an illustration of the cross-sectional view. 3.3. Immobilization of HBV antibody TMOS–sol serves as a bi-functional material. Firstly, to improve the adhesion of PDMS to AAO, TMOS–sol was applied to the AAO surface. Secondly, hydroxyl groups on the SiO2 layer induced by piranha treatment act as anchoring groups for chemical modification. As shown in Fig. 3, ATPES reacted with –OH groups on SiO2– AAO structure by circulation. The terminal amino group of the silane molecule was coupled with glutaraldehyde as a linker molecule. HBV antibody was linked with the unreacted aldehyde group of glutaraldehyde. 3.4. ELISA in the microfluidic device Typical ELISA tests were carried out in a commercial 96-well plate coated with primary antibody corresponding to the target protein. Here, HBsAg was used as a model system. The test sample was prepared by 5 dilution of the concentration of HBsAg positive control. Positive and negative controls and model samples were introduced into the AAO/PDMS device by using syringe pumps. While maintaining 35 °C constantly using a hotplate, sample solutions were circulated at a rate of 15 lL/min for 2 min. Then, the remaining sample solution inside the device was washed with washing buffer at a rate of 30 lL/min. Sheep anti-HBs/HRP conjugate solution was injected and washed in the same manner and for the same time as above. To determine the assay, TMB (substrate for horseradish peroxidase) solution was introduced slowly and reacted for 10 min. Conversion of TMB resulted in color changes from transparent to blue. The enzyme reaction was quenched by sulfuric acid solution. The results were analyzed by naked-eye determination based on colorimetric assay [12] and compared with commercial 96-well
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ELISA and AAO nano-well ELISA as shown in Fig. 5. The colorimetric assay shows that the absorbance (f) corresponding to 60% of (e) is
similar to (c) corresponding to 80% of (b). Total analysis time, including test sample loading, reaction and washing, was around
Fig. 4. (A) Layouts and (B) photograph of microfluidic device with the illustration of PDMS/AAO.
Fig. 5. (A) Commercial 96-well ELISA, and (B) AAO nano-well immuno-assay in a microfluidic device. (a), (b) and (c) are obtained from commercial ELISA kit. (d), (e) and (f) are obtained from the AAO nano-well system. (a) and (d) are negative controls with no antigen. (b) and (e) are positive controls with antigen (10 mg/mL). (c) and (f) are test samples. The test sample was prepared by 5 times dilution of the positive sample. Sample volume was 100 lL for (a), (b) and (c), and 25 lL for (d), (e) and (f). (a0 )–(f0 ) are the absorbances corresponding to (a)–(f), respectively.
K.S. Yang et al. / Current Applied Physics 9 (2009) e60–e65
15 min, based on the overall feeding rate (5 lL/min). However, the conventional ELISA test needed over 130 min. Due to the short vertical dimension (25 lm) and wide surface area, this device provides rapid immune interaction with the capability to hold more antibody molecules and short mass/heat transport distances between the porous alumina surface and the reaction chambers.
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Acknowledgements This work was supported by the Basic Research Program (Grant No. R01-2004-000-10681-0) of KOSEF and the Center for Ultramicrochemical Process Systems sponsored by KOSEF. References
4. Conclusion We demonstrated the possibility of sandwich-type enzymelinked immunosorbent assay (ELISA) in a PDMS microfluidic chip integrated with porous anodic aluminum oxide (AAO). The performance of our microfluidic chip was compared with that of a conventional ELISA system. Anodizing of the aluminum layer on glass substrate yielded a regularly ordered hexagonal pore array. Variation of the opening size of the nanopores was also investigated by changing the anodizing conditions such as voltage, current density, and electrolyte composition and selection. The control of opening size of pores may offer versatility for a variety of target molecules and concentrations. Hepatitis B surface antigen is used as a model target protein. To accommodate the primary antigen, antibody, and second antibody conjugated HRP, the pore opening used is about 80 nm. For the ELISA test, a sol–gel method was employed for the adhesion of PDMS with AAO as well as the surface activation of AAO for the immobilization of the antibody. The sol–gel method was successful for further biochemical modification. One of the intrinsic properties of porous AAO is the lack of actual contact area with PDMS microstructure. Therefore, the bonding property should be improved by the local anodizing technique by adopting a masking layer such as SiO2 during the anodization process. This platform technology using a microfluidic device with porous alumina is useful in the application of a heterogeneous immuno-assay system and other fields of biosensors and diagnostics with reduced analysis time and analyte volumes.
[1] P. van Male, M.H.J.M. de Croon, R.M. Tiggelaar, A. van den Berg, J.C. Schouten, Heat and mass transfer in a square microchannel with asymmetric heating, Int. J. Heat Mass Trans. 47 (1) (2004) 87–99. [2] M.J. Heller, A. Guttman, Integrated Microfabricated Biodevices, Marcel Dekker, New York, 2002. [3] F. Vint, P. Chaton, Y. Fouillet, Microarrays and microfluidic devices: miniaturized systems for biological analysis, Microelectron. Eng. 6162 (2002) 41–47. [4] A. Bange, H.B. Halsall, W.R. Heineman, Microfluidic immunosensor systems, Biosens. Bioelectron. 20 (12) (2005) 2488–2503. [5] H. Masuda, K. Fukuda, Ordered metal nanohole arrays made by a two-step replication of honeycomb structures of anodic alumina, Science 268 (1995) 1466–1468. [6] H. Masuda, M. Satoh, Fabrication of gold nanodot array using anodic porous alumina as an evaporation mask, Jpn. J. Appl. Phys. 35 (1996) L126–L129. [7] S.B. Lee, D.T. Mitchell, L. Trofin, T.K. Nevanen, H. Soederlund, C.R. Martin, Antibody-based bio-nanotube membranes for enantiomeric drug separations, Science 296 (2002) 2198–2200. [8] M. Kakuda, H. Takahashi, S. Kazuno, K. Murayama, T. Ueno, M. Tokeshi, Development of the microchip-based repeatable immunoassay system for clinical diagnosis, Meas. Sci. Technol. 17 (2006) 3189–3194. [9] W. Guo, E. Ruckenstein, Modified glass fiber membrane and its application to membrane affinity chromatography, J. Membr. Sci. 215 (1–2) (2003) 141–155. [10] M.-Y. Lee, A. Srinivasan, B. Ku, J.S. Dordick, Multienzyme catalysis in microfluidic biochips, Biotechnol. Bioeng. 83 (1) (2003) 20–28. [11] K. Kusakabe, S. Sakamoto, S. Morooka, Pore structure of silica membranes formed by a sol–gel technique using tetraethoxysilane and alkyltriethoxysilanes, Sep. Purif. Technol. 6 (2) (1999) 139–146. [12] T. Mosmann, Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays, J. Immunol. Methods 65 (1–2) (1983) 55–63.
Contents lists available at ScienceDirect
Current Applied Physics journal homepage: www.elsevier.com/locate/cap
Microfluidic chip with porous anodic alumina integrated with PDMS/glass substrate for immuno-diagnosis Kwang Suk Yang a, Hae Jin Kim b, Jeong Keun Ahn b, Do Hyun Kim a,* a
Department of Chemical and Biomolecular Engineering and Center for Ultramicrochemical Process Systems, KAIST, 373-1, Guseong-dong, Yuseong-gu, Daejeon 305-701, Republic of Korea b Department of Microbiology, School of Bioscience and Biotechnology, Chungnam National University, Daejeon 305-764, Republic of Korea
a r t i c l e
i n f o
Article history: Received 1 July 2008 Received in revised form 1 November 2008 Accepted 1 December 2008 Available online 13 March 2009 PACS: 78.55.Mb 87.85.fk 87.85.M 85.85.+j 81.20.Fw 42.82.Cr Keywords: Anodic aluminum oxide Hepatitis B virus Immuno-sensor ELISA
a b s t r a c t An efficient system for diagnosis of disease marker molecules in microfluidic devices was developed by employing anodic aluminum oxide (AAO) which has highly ordered, uniform, and straight nanopore arrays by a two-step anodization process. AAO on glass substrate was integrated within poly(dimethylsiloxane) (PDMS) microchannel structure. Vacuum-deposited aluminum thin film was anodized by variation of electrolyte composition, applied voltage and anodizing time, for specific pore sizes and depth. The pore was tunable to achieve a size corresponding to target proteins. For enhancement of antibody immobilization and adhesiveness with a PDMS micro-pattern, surface activation of AAO was performed by TMOS–sol spin-coating and calcinations to form a SiO2 layer. The demonstration of diagnosis of biomarker protein was performed by employing conventional sandwich-type immuno-assay for hepatitis B virus (HBV). The anti-hepatitis B surface antigen (anti-HBsAg) was immobilized by bridges using c-aminopropyltriethoxysilane and glutaraldehyde. The hepatitis B surface antigen (HBsAg) was coupled with anti-HBsAg and sheep anti-HBs/horseradish peroxidase conjugate. The result was analyzed by colorimetric assay for comparison with the result using conventional immuno-assay and it showed higher efficiency using microfluidic channels. The AAO inside the PDMS microfluidic channel allows specific immobilization of proteins by controlling the size for access. This study can be extended for a highthroughput system for bio-marker proteins. Ó 2009 Elsevier B.V. All rights reserved.
1. Introduction Microfluidic systems have become widely used for various chemical and biochemical applications, which include sample pre-handling, reagent mixing, separation, enzyme reaction, and detection processes, due to short mass transfer length and transport time [1], very small volume of analytes [2], almost isothermal conditions, and easy parallelization [3]. In the particular case of clinical diagnostics, these inherent properties of microfluidic devices are related to high selectivity and sensitivity in biorecognition. Additionally, the use of high-surface-area micro/nano structure, such as mesoporous materials, in a heterogeneous diagnosis system can increase sensitivity between probe and target molecules by increasing probe loading capacity, a higher target binding specificity and accessibility of targets to the probes, and reducing reaction/hybridization time [4]. Many approaches to increasing the surface-area-to-volume ratio of microchannels have been proposed, including microfabrica* Corresponding author. Tel.: +82 42 350 3929; fax: +82 42 350 3910. E-mail address: [email protected] (D.H. Kim). 1567-1739/$ - see front matter Ó 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.cap.2008.12.031
tion by photolithography, packing microbeads, or patterning of a porous membrane inside the microfluidic channel. However, these approaches require high-end lithographic techniques, expensive equipment and significant amounts of precious materials and labor, due to the large number of manipulations needed for fabrication, because the uniformities of sizes, shapes and volumes of the void spaces in porous materials are directly related to their ability to perform the desired function in a particular application. Anodic aluminum oxide (AAO) membrane, which has a hexagonal pore array of nanometer scale, has been studied for size-selective separation (chemicals or bio-molecules) and template material for metal nanowires (electroplating) or polymer nanotubes. Masuda and co-workers proposed a two-step-anodization method for self-organized formation of a hexagonal pore array [5,6]. The two-step-anodization method includes removal of the amorphous porous layer to expose well-oriented pores. Martin and co-workers [7] devised an elegant method to separate drug enantiomer from racemic mixture by using molecular recognition protein and functionalized AAO, which is prepared by immobilization of antibody on the inner surface of AAO activated by a sol–gel method.
K.S. Yang et al. / Current Applied Physics 9 (2009) e60–e65
e61
Here, a heterogeneous immuno-assay is developed using an AAO-incorporated PDMS (polydimethylsiloxane) microfluidic system, which has the advantages of rapid analysis time and smaller volumes of samples such as antibodies and antigens. Enzymelinked immunosorbent assay (ELISA), typically carried out in a 96-well microtitre plate, is the most common method of heterogeneous immuno-assay. However, conventional ELISA involves a tedious and labor-intensive protocol that often results in large errors and inconsistent results due to personally operated injection, mixing, and washing steps. Also, insufficient heat and mass transfer during the incubation step contribute to errors and inconsistent results [8]. A porous AAO-incorporated PDMS microfluidic system can provide a wide surface area with chemical modification for the effective immobilization of target antibodies [9,10], highly improved mass transfer, precise temperature control, and semiautomation. In this work, the surface of the size-controlled AAO nanopore array is modified by SiO2. Then, anchor and linker molecules are introduced into the SiO2 pores. Finally, hepatitis B surface antibody (HBsAb) is immobilized in the glutaraldehyde-activated SiO2–AAO pores. The ELISA test was carried out with a naked-eye method.
2. Experimental 2.1. Chemicals
c-Aminopropyltriethoxysilane (APTES), tetramethylorthosilicate (TMOS, 98%), ethanol, HCl (38%), oxalic acid, phosphoric acid, hydrogen peroxide, and sulfuric acid were purchased from Sigma– Aldrich (St. Louis, MO, USA). Hepatitis B virus surface antibodies (mono- and polyclonal: anti-HBs) were purchased from Chemicon (Temecula, CA, USA). 96-well ELISA kit for hepatitis B surface antigen (HBsAg) and sheep Anti-HBs/horseradish peroxidase conjugate diluted with bovine serum albumin–phosphate buffer, normal human serum, tetramethylbenzidine (TMB) solution, and dimethylsulfoxide (DMSO) were purchased from Green Cross Medical Science Corp. (GyeongGi-Do, Korea). All solutions were made in Milli-Q-grade deionized water from a Human UP 900 (Human Corp., Korea). Other chemicals and reagents used were of analytical grade. 2.2. Fabrication of AAO nanopore array As shown in Fig. 1A, a two-step-anodization method was used for self-organized formation of a hexagonal pore array [5]. A microscope glass slide was cleaned following the standard cleaning process. As a source of anodic alumina layer, 3-lm thick Al layer was deposited on the glass slide at 10-6 torr. Nanoporous anodic alumina layer was grown in 0.3 M oxalic acid solution as an electrolyte. 40–80 V DC anodic voltage was applied for 60–120 s using an EDP-1501 DC power supply (PNCYS Co., Ltd., Korea). Inter-pore distance was 100 nm at 40 V and 15 °C. Prepared Al2O3 surface was removed by 0.6 M H3PO4 and 0.2 M CrO3 for 180 s at 60 °C to give substructure. Then, second anodizing was carried out to grow AAO with thickness of 1.45 lm under the same condition of 0.3 M oxalic acid under 40–80 V DC voltage for 90–180 s at 15 °C. Here, the thickness of AAO layer is considered as the pore depth. Finally, the pore widening process was performed with 0.1 M H3PO4 solution for 300 s at 30 °C to give an opening diameter of 80 nm, which is related to the surface area. 2.3. Fabrication of PDMS microchannel PDMS (Dow Chemical, Midland, MI, USA) microchannel was fabricated using standard soft lithography and PDMS replica mold-
Fig. 1. (A) Patterning procedure of AAO on a glass slide substrate: (a) first anodizing process to obtain hexagonally oriented pores; (b) removal of first Al2O3 layer for pore arrangement; (c) second anodization for depth of pores; (d) pore widening for pore diameter. Pre-widening condition: 0.3 M oxalic acid, 80 V, 15 °C; etching condition: 0.6 M H3PO4, 60 °C. (B) Scanning electron microscopic images of each step.
ing techniques. The general procedure is described in Fig. 2. AZÒ 9260 (Clariant Corporation AZ Electronic Materials, Wilmington, DE, USA) photoresist was spin-coated onto Si wafer at 2000 rpm. This wafer was baked at 110 °C on a contact hotplate for 120 s. Then, it was cooled and exposed to 1300 mJ/cm2 365-nm i-line UV using a CA-4/6 M mask aligner system (Shinumst Co., Ltd., Korea). The photoresist was developed with AZ 400 K developer (AZ Electronic Materials, Wilmington, DE, USA) for 10 min. Then, the wafer was washed with deionized water and dried with N2 gas. The PDMS replica molding process was carried out by casting PDMS mixture composed of a 10:1 ratio of base and curing agent on the AZÒ 9260 master pattern. After evacuation, PDMS mixture was cured at 70 °C for 4 h. Then, PDMS molding was removed carefully after cooling to room temperature. The resulting PDMS mold was cut and punched for use.
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2.4. Preparation of TMOS–sol solution
2.5. Surface modification of AAO with polyclonal anti-HBsAg
Sol–gel solution for coating was typically prepared with a mole ratio of 1:4:0.003:4 (TMOS:ethanol:HCl:H2O) [11]. The mixture was stirred for 2 h at 60 °C. To hydrolyze the TMOS, hydrochloric acid (38%) was added dropwise. Then, the flask was sealed and the temperature was raised to 80 °C for the reaction to give TMOS–sol. Prepared TMOS–sol was coated on the top of porous AAO substrate by spin-coating at 1500 rpm as shown in Fig. 3.
2.5.1. Silanization of SiO2–AAO A freshly prepared solution containing 10 mL ATPES in 90 mL water, was recirculated at a feeding speed of 2 lL/min using a syringe pump (KDS-100; KD Scientific Inc., PA, USA). The reaction was carried out at room temperature for 2 h. After reaction, the device was washed with distilled water until neutral.
Fig. 2. Photolithography and PDMS replica molding process: (a) photoresist spin-coating; (b) UV exposure and developing; (c) PDMS casting on photoresist mold; (d) curing of PDMS; (e) detaching of PDMS from mold and punching of fluidic ports; (f) oxygen plasma treatment; (g) alignment and bonding of PDMS mold with AAO on a glass slide.
Fig. 3. SiO2 coating and anti-HBs immobilization in AAO nanopores: (a) TMOS–sol coating; (b) formation of amine group using ATPS; (c) surface modification with glutaraldehyde; (d) immobilization of anti-HBs (primary antibody).
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2.5.2. Coupling of anti-HBsAg on SiO2–AAO pore The ATPES-modified SiO2–AAO membrane was first equilibrated with 0.2 M NaAc–HAc buffer (pH 7.50) for 10 min at room temperature. After removal of buffer, 100 mL of 25 wt% glutaraldehyde aqueous solution was circulated at 1 mL/min for 3 h at 40 °C. After reaction, the device was washed with distilled water. The device activated with glutaraldehyde was then subjected to a reacting solution containing 10 mg HBV monoclonal antibody in 10 mL of 0.01 M CaCl2 and 0.05 M Tris–HCl buffer (pH 8.5). The immobilization was carried out by circulating the antibody solution through the microfluidic device for 2 h at a flow rate of 1 mL/min. The activity of the immobilized enzyme was monitored by taking control samples. 2.6. Enzyme-linked iummunosorbent assay (ELISA) In the ELISA, HBsAg was used as antigen, Anti-HBsAg was used as the primary antibody, and horseradish peroxidase-conjugated monoclonal goat Anti-HBs was used as secondary antibody conjugate. HBsAg was used as positive control, and normal human serum was used as negative control. Positive control diluted to 1/4 of original concentration with negative control was used as a test sample. Washing buffer was prepared from pH 7 PBS. The substrate solution was prepared by diluting TMB (5.25 g/L in DMSO) with citrate–phosphate buffer as prepared from the company. In this experiment, the determination was carried out by colorimetric analysis (naked-eye method). The mean absorbance of the positive control should be greater than or equal to that of the sample. If the absorbance of the test sample does not reach that of the negative control, the test sample is considered negative for the presence of hepatitis B antigen. The opposite result can be considered positive. 2.6.1. ELISA in 96-well plates One hundred micro litres of negative control and positive control, and the test sample were added to each well. Then, prepared enzyme-conjugate was added into each well. The well-mixed wells were incubated at 37 ± 1 °C for 90 min. Individual wells were evacuated and washed five times with 300 lL washing buffer. Washing buffer was completely removed, and 100 lL of enzyme–substrate solution was added into each well. Then, it was incubated for 30 min at room temperature. For colorimetric analysis, an image of the color change was taken by Olympus CAMEDA C-5060 digital camera when the reaction time had reached 30 min. 2.6.2. ELISA in AAO-incorporated PDMS microfluidic channels An AAO-incorporated PDMS microfluidic chip was placed on a warm (37 °C) hot plate with feeding of positive control, negative control and sample by syringe pumps at a rate of 2.5 lL/min to microfluidic channels for 20 min. HRP conjugate was loaded with the same speed without the washing step. Unthreaded HRP conjugate was removed by using washing buffer for 5 min. Enzyme–substrate solution was loaded with the same feeding speed for 15 min. The eluant was collected from each drain port. The colors of the collected substrate solutions were not changed, due to the isolation with enzyme bound into the porous AAO substrate.
3. Results and discussion 3.1. Fabrication of AAO on SiO2 glass slide Electrolyte composition, temperature and anodizing voltage may be varied depending on the desired parameters of the anodic alumina substrate, such as pore density, diameter, and thickness. The inter-pore distance was observed to decrease with decreasing
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anodizing voltage, while the layer growth rate was observed to depend on the desired pore diameter and electrolyte composition, being proportional to the current density. The anodizing conditions should be optimized for the dimensions of the target protein. A two-step-anodizing procedure is adopted to achieve regular opening and positioning of the pores. A large hexagonal pore array of anodic aluminum oxide (AAO) using the two-step-anodizing technique serves as a nano-well bioreactor for the immuno-diagnosis. The opening size of the AAO nano-pore as prepared is about 100 nm, which is wide enough for sandwich-type heterogeneous immuno-assay. Fig. 1B shows SEM images of AAO on the slide after anodizing at 40 V applied voltage in 0.3 M oxalic acid. The anodizing process turns the Al layer into mirror-like AAO as transparent alumina, as shown in Fig. 4B. Transparency is a desired property for the application of an optical detection technique, especially naked-eye or fluorescence determination, in diagnosis. 3.2. Design and fabrication of PDMS/AAO microfluidic device The device shown in Fig. 4A is composed of several parts, comprising five inlet ports with a main channel for washing, a reaction chamber for enzyme reaction and outlet ports. The width of the washing channel is 200 lm, but the ports for controls and the test sample ports are 100 lm. This will prevent reverse flow of the main stream to the sample inlets. The overall channel wall thickness is 25 lm. AAO was modified by the sol–gel method to improve the bonding with the PDMS microchannel and then treated with piranha solution to obtain immobilization efficiency of primary antibody. TMOS–sol prepared by a typical method was coated on AAO and the organic part was removed by calcination at 250 °C. A photograph of the device is shown in Fig. 4B with an illustration of the cross-sectional view. 3.3. Immobilization of HBV antibody TMOS–sol serves as a bi-functional material. Firstly, to improve the adhesion of PDMS to AAO, TMOS–sol was applied to the AAO surface. Secondly, hydroxyl groups on the SiO2 layer induced by piranha treatment act as anchoring groups for chemical modification. As shown in Fig. 3, ATPES reacted with –OH groups on SiO2– AAO structure by circulation. The terminal amino group of the silane molecule was coupled with glutaraldehyde as a linker molecule. HBV antibody was linked with the unreacted aldehyde group of glutaraldehyde. 3.4. ELISA in the microfluidic device Typical ELISA tests were carried out in a commercial 96-well plate coated with primary antibody corresponding to the target protein. Here, HBsAg was used as a model system. The test sample was prepared by 5 dilution of the concentration of HBsAg positive control. Positive and negative controls and model samples were introduced into the AAO/PDMS device by using syringe pumps. While maintaining 35 °C constantly using a hotplate, sample solutions were circulated at a rate of 15 lL/min for 2 min. Then, the remaining sample solution inside the device was washed with washing buffer at a rate of 30 lL/min. Sheep anti-HBs/HRP conjugate solution was injected and washed in the same manner and for the same time as above. To determine the assay, TMB (substrate for horseradish peroxidase) solution was introduced slowly and reacted for 10 min. Conversion of TMB resulted in color changes from transparent to blue. The enzyme reaction was quenched by sulfuric acid solution. The results were analyzed by naked-eye determination based on colorimetric assay [12] and compared with commercial 96-well
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ELISA and AAO nano-well ELISA as shown in Fig. 5. The colorimetric assay shows that the absorbance (f) corresponding to 60% of (e) is
similar to (c) corresponding to 80% of (b). Total analysis time, including test sample loading, reaction and washing, was around
Fig. 4. (A) Layouts and (B) photograph of microfluidic device with the illustration of PDMS/AAO.
Fig. 5. (A) Commercial 96-well ELISA, and (B) AAO nano-well immuno-assay in a microfluidic device. (a), (b) and (c) are obtained from commercial ELISA kit. (d), (e) and (f) are obtained from the AAO nano-well system. (a) and (d) are negative controls with no antigen. (b) and (e) are positive controls with antigen (10 mg/mL). (c) and (f) are test samples. The test sample was prepared by 5 times dilution of the positive sample. Sample volume was 100 lL for (a), (b) and (c), and 25 lL for (d), (e) and (f). (a0 )–(f0 ) are the absorbances corresponding to (a)–(f), respectively.
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15 min, based on the overall feeding rate (5 lL/min). However, the conventional ELISA test needed over 130 min. Due to the short vertical dimension (25 lm) and wide surface area, this device provides rapid immune interaction with the capability to hold more antibody molecules and short mass/heat transport distances between the porous alumina surface and the reaction chambers.
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Acknowledgements This work was supported by the Basic Research Program (Grant No. R01-2004-000-10681-0) of KOSEF and the Center for Ultramicrochemical Process Systems sponsored by KOSEF. References
4. Conclusion We demonstrated the possibility of sandwich-type enzymelinked immunosorbent assay (ELISA) in a PDMS microfluidic chip integrated with porous anodic aluminum oxide (AAO). The performance of our microfluidic chip was compared with that of a conventional ELISA system. Anodizing of the aluminum layer on glass substrate yielded a regularly ordered hexagonal pore array. Variation of the opening size of the nanopores was also investigated by changing the anodizing conditions such as voltage, current density, and electrolyte composition and selection. The control of opening size of pores may offer versatility for a variety of target molecules and concentrations. Hepatitis B surface antigen is used as a model target protein. To accommodate the primary antigen, antibody, and second antibody conjugated HRP, the pore opening used is about 80 nm. For the ELISA test, a sol–gel method was employed for the adhesion of PDMS with AAO as well as the surface activation of AAO for the immobilization of the antibody. The sol–gel method was successful for further biochemical modification. One of the intrinsic properties of porous AAO is the lack of actual contact area with PDMS microstructure. Therefore, the bonding property should be improved by the local anodizing technique by adopting a masking layer such as SiO2 during the anodization process. This platform technology using a microfluidic device with porous alumina is useful in the application of a heterogeneous immuno-assay system and other fields of biosensors and diagnostics with reduced analysis time and analyte volumes.
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