Anal. Chem. 2004, 76, 238-243
Fabrication of a Spectrophotometric Absorbance Flow Cell Using Injection-Molded Plastic John P. Hulme,* Peter R. Fielden, and Nicholas J. Goddard
Department of Instrumentation and Analytical Science, University of Manchester Institute of Science and Technology, Manchester, U.K. M60 1QD
A microfluidic device with integrated spectrophotometric elements has been injection molded in poly(methyl methacrylate) (PMMA). The strategy for producing the device combined the direct photopatterning, replica molding techniques and a rapid cooled release mechanism in conjunction with material-material transfer to form a multilayer epoxy master on an injection mold insert. An injection-molded flow cell consisting of 1800 diffractive elements patterned on 25-µm-high PMMA pillars was produced. The design incorporated a large cross sectional surround, simplifying the alignment and bonding procedure. The absorbance range of one of the devices was standardized against a commercially available visible spectrophotometer at 605 nm to increasing concentrations of Nile blue perchlorate A dye in aqueous solution. The detection limit for a new integrated device was 1.2 × 10-6 m/Lof the mentioned dye. Of the many optical detection techniques, absorbance is not commonly used in microfluidic systems.1 Miniaturized devices permit only limited detection and often require the use of a multireflective surface to increase the optical path length.2,3 Early improvements in design and absorbance detection limits were achieved in miniaturized silicon flow cells and glass microcapillaries.4,5 In traditional spectrophotometers, the optical element is independent of the microchannels or the detection area, adding to the number of total components used in the instrument’s construction. The integration of the optical element into the microfluidic area would significantly reduce the complexity of the device and reduce the overall cost of its manufacture. Generally, a flow cell has to be rugged, disposable, and inexpensive to produce. These requirements limit the number of fabrication methods and materials from which a flow cell can be constructed. Most modern optical flow cells are fabricated from * To whom correspondence should be addressed. E-mail:
[email protected]. Fax: +44 0161 2004911. (1) Schwarz, A. M.; Hauser, P. C. Lab Chip 2001, 1, 1-6. (2) Salimi-Moosav, H.; Jiang, Y.; Lester, L.; Mckinnon, G.; Harrison, D. J. Electrophoresis 2000, 21, 1291-1299. (3) Salimi-Mossavi, H.; Tang, T.; Harrison, D. J. J. Am. Chem. Soc. 1997, 119, 8716-8717. (4) Manz, A.; Graber, N.; Widmer, H. M. Minaturized total chemical analysis systems: a novel concept for chemical sensing, Sensors Actuators B 1990, 1, 244-248. (5) Zeller, P. N.; Voirin, G.; Kunz, R. E. Biosens. Bioelectron. 2000, 15, 591595.
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injection-molded poly(methyl methacrylate) (PMMA) of a nickel master. However, recent advances in rapid prototyping techniques, which evolved from soft lithography, have allowed a multilayered epoxy master to be fabricated and used for injection molding within 24 hours.6 This allowed a higher level of instrument integration to be achieved at a fraction of the cost compared to similar designs fabricated by conventional silicon micromachining methods.7-18 At that time, the fabrication process suffered from a lack of automation, and the depth of the injection-molded devices was limited. In this paper, we report on a new approach. The emphasis is on the fabrication and elevation of the optical element or waveguide into the surrounding solution to improve the signalto-noise ratio (SNR). A new semiautomated process has been developed (in house) to reliably fabricate the master at a prescribed depth and coupled with a very simple bonding procedure to produce an optically clear chip that does not include any of the refractive index disparities often found in laminated or thermally sealed devices. Of the work published so far, Zeller et al.’s single-pad scheme for integrated optical fluorescence sensing is the closest comparable optical configuration.19-21 The results demonstrated a high (6) Hulme, J. P.; Mohr, S.; Goddard, N. J.; Fielden, P. R. Lab Chip 2002, 2, 203-206. (7) Martynova, L.; Locascio, L. E.; Gaitan, M.; Kramar, G. W.; Christensen, R. G.; MacCrehan, W. A. Anal.Chem. 1997, 69, 4783-4789. (8) McCormick, R. M.; Nelson, R. J.; Alonso-Amigo, M. G.; Benvegnu, D. J.; Hooper, H. H. Anal. Chem. 1997, 69, 2626-2630. (9) Jaszewski, R. W.; Schift, H.; Gobrecht, J.; Smith, P. http://www.snf.ch/ de/fin.asp. (10) Gopel, W. Biosens. Bioelectron. 1998, 13, 723-728. (11) Montamedi, M. E.; Goering, R. SPIE-Int. Soc. Opt. Eng. 1998, 327, 290. (12) Motamedi, M. E. IEEE Trans. Ultrason., Ferroelectr., and Freq. Control 1987, 2, 237. (13) Motamdei, M. E. et al. Opt. Eng. 1997, 36, 1371-1381. (14) Cox, W. R.; Hayes, D. J.; Chen, T.; Ussery, D. W.; McFarlane, D. L.; Wilson, E. SPIE 1995, 2383, 179-183. (15) Duffy, D. C.; McDonald, J. C.; Schueller, O. J. A.; Whiteside, G. M. Anal. Chem. 1998, 70, 4974-4984. (16) Qin, D.; Xia, Y.; Whiteside, G. M. Adv. Mater. 1996, 8, 917. (17) Xia, Y.; Whiteside, G. M. Angew. Chem., Int. Ed. 1998, 37, 551. (18) Renauld, P.; van Lintel, H.; Heuskchel, M.; Guerin, L. In Proceedings of Micro Total Analysis Systems; Kluwer Academic Publishers: Dordrecht, The Netherlands, 1998; pp 17-22. (19) Verpoorte, E.; Manz, A.; Ludi, H.; Bruno, A. E.; Maystre, F.; Krattiger, B.; Widmer, H.; M.; Schoot, B. H.; Rooij. N. F. Sensors Actuators B 1992, 6, 66-70. (20) O′Brien, M. J., II; Brueck, S. R. J.; Perez-Luna, V. H.; Tender, L. M.; Lopez, G. P. Biosens. Bioelectron. 1999, 14, 145-154. 10.1021/ac034755a CCC: $27.50
© 2004 American Chemical Society Published on Web 11/25/2003
efficiency of fluorescence light excitation and collection, as well as a good suppression of the volume background. A single grating device was constructed by Burgess et al. to characterize nanoparticles using grating light reflection spectroscopy and a PDMS microchannel.22 Lu and Collins have detected transition metals electrophoretically separated as their complexes with 4-(2-pyridylazo)resorcinol (PAR) down to about 1 ppm with a green lightemitting diode (LED) as the radiation source.23
Figure 1. Optical apparatus.
EXPERIMENTAL SECTION Materials and Reagents. A UV-vis grating on an aluminized glass substrate with a resolution of 2400 lines/mm and polyester film were supplied by Edmund Scientific (York, U.K.). UV curable epoxy and polyurethane (No. 81, 68) were purchased from Norland Products Inc. (New Brunswick, NJ). These materials were chosen because of their high transmission from 370 to 4000 nm. Poly(dimethylsiloxane) (PDMS) was supplied in the easy-to-use
Figure 2. Fabrication process used in the production of a flexible grating and epoxy master.
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Table 1. Quality and Resolution of Various Gratings Produced by UV Embossing and Injection Molding Compared to the Master grating
period (nm)
depth (nm)
grating master epoxy copy third copy chip 10 chip 25
416 316 300 440 440
35 29 29 36 33
Table 2. Detection Limits of the Injection Flow Cell at 605 nm absorbance (arbitrary units) dye concentration (m/L)
element x
element y
spectrophotometer
0 2.4 × 10-7 1.2 × 10-6 6 × 10-06 1.2 × 10-05
0 0.018 0.746 1.818 3.058
0 0.013 0.664 1.859 3.858
0
R2 background (SDa) SNRb
0.967 0.0246 1447:1
0.993 0.0246 1447:1
0.998
0.013 0.046 1.0
a SD is the standard deviation of the background signal. b SNR is the signal-to-noise ratio of the reflected intensity to the background.
format Sylgard 184 Silicon Elastomer (Dow Corning, Midland, MI). PMMA sheets were purchased from Goodfellow (Huntington, Cambridge, U.K.). Laminar polished glass (25 × 25 × 10 mm) was obtained from Edmund Scientific (Barrington, NJ). The injection molder was a Babyblast 6/6 (Cronoplast S.L., Barcelona, Spain). DQ 501 was purchased from Merck Eurolab (Lutterworth, U.K.). This particular polymer was chosen because of its high stability and good flow rheology. A Dymax 400 UV source was used (Dymax Corp., Torrington, CT). Nile blue A perchlorate, sodium dodecyl sulfate, and sodium phosphate were obtained from Aldrich Chemicals (Gillingham, U.K.). The laminator was a Ledco Digital 42 from Ledco Ltd. (Kitchener, ON, Canada). Instrumentation. Absorbance measurements were measured with 8452A model spectrophotometer (Hewlett-Packard, Menlo Park, CA). The optical arrangement shown in Figure 1 was employed to measure the changes in reflected intensity of the first diffractive order to different aqueous dye solutions. The light source used was 40-W tungsten halogen with a condensing lens, a 350-µm pinhole, and a collimating lens to produce a substantially collimated beam of white light. This beam was used directly for variable-angle and absorbance experiments. Band-pass interference filters of wavelengths 605 and 440 nm (10-nm fwhm bandwidth, Ealing Optics, Watford, U.K.) were used to provide a monochromatic light source providing the optimum angle of reflectivity at a prescribed wavelength, before the light was finally passed through a collimating and cylindrical lens (Edmund Scientific, York, U.K.) onto the flow cell. Changes in the reflected (21) Hulme, J. P.; Malins, C.; Singh, K.; Fielden, P. R.; Goddard, N. J. Analyst 2002, 127, 1233-1236. (22) Smith, S. A.; Brodsky, A. M.; Vahey, P. G.; Burgess, L. W. Anal. Chem. 2000, 72, 4428-4434. (23) Lu, Q.; Collins, G. E. Analyst 2001, 126, 429.
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Figure 3. SEM images of (A) parallel elements, (B) a single pillar and grating, and (C) the grating surface on top of one of the pillars.
intensity were recorded using a CCD color camera (SMZ.2T, 640 × 480 pixels, Nikon, Tokyo, Japan) connected to a standard light microscope (Nikon). Image analysis was performed using a standard software package (Scion Image, Scion Corp., Frederick, MD). Nile blue A solutions were prepared in (0.05 M) sodium phosphate buffer pH (7.4). Solutions were irrigated through a chip at a flow rate of 4 mL min-1. Changes in absorbance were measured at 5-min intervals against a sodium phosphate blank. All designs were drawn using AutoCAD R14 (AutoDesk Inc., San Rafael, CA) and printed onto transparent acetate (J. D. Phototools, Oldham, U.K.) at 8000 dpi resolution; the transparency images were used as the masks for photolithography. Fabrication of a Flexible Grating Copy. A layer of PDMS was formed on a 10-cm2 square sheet of PMMA by pouring 10 mL of a stirred solution of base and catalyzer at a ratio of 1:10 onto the PMMA and placing the coated sheet in a vacuum oven at 70 °C for 15 min. The aluminized grating was then centered
Figure 4. Measurement of the optical path lengths at 440 and 605 nm. A/B ) cos θi. The total path length in which the light interacts with the solution is A + B. CR is the critical angle at the water-PMMA interface.
on the partially cured layer of PDMS, and the sheet was returned to the oven for an additional 2 h. Once cured, the combined grating, PDMS, and PMMA layers were left to cool at room temperature for 30 min before use. One milliliter of adhesive (Norland 81) was then dispensed at the front of the grating block, and a sheet of thin-film polyester was placed on top. The whole device was positioned and rolled (Western Laminator) at a pressure of 20 psi for 10 s. The adhesive layer was then exposed to a UV (365 nm) source for 35 s and left to cool for 10 min at room temperature. The polyester sheet was carefully removed, carrying a copy of the aluminized master. The replica was then post-cured for an additional minute before use. Multiple copies of the master were made in this way. Fabrication of a Single Grating Copy on a Glass Substrate. A flexible grating copy was pretreated in a 0.0005% 40 °C solution of sodium dodecyl sulfate and dried in nitrogen. A line of Norland 81 was dispensed at the front of a polished glass substrate set on a supporting layer of PMMA and PDMS. The grating replica was placed on top of the adhesive, rolled, and exposed to a UV source for 35 s. The whole device was then rested on a bed of dry ice for 2 min to induce separation between the respective layers. The polyester film containing the epoxy daughter was peeled away, leaving an inverse copy on the glass block. This was brought to room temperature in a stream of nitrogen before being post-cured under UV light for 1 min. A summary of the process can be found in Figure 2. Design and Fabrication of the Array. The fabrication of the array master was a two-step procedure. A grating copy was fabricated on a glass block as previously described. The acetate design was dipped in 0.05% SDS solution for 10 s and then dried in nitrogen. One milliliter of Norland 81 was dispensed at the front
of the grating block, and the mask was carefully placed on top. Steps 1-5 in Figure 2 were repeated, and the block was cut from the PDMS cushion with a sharp blade and then developed in acetone (-20 °C) and a stream of CO2 gas. Finally, the master was dried in nitrogen and post-cured for an additional 2 min before use. The master mold was an elongated hexagonal shape measuring 2 cm long by 1 cm in width with a 500-µm-broad line seal around the chip. The detection area consisted of approximately 1800 100-µm-wide sensing wells with a 500-nm diffractive pattern at the base. Injection Molding. Injection molding for all devices was performed at an injection pressure of 70 bar and a nozzle temperature of 210 °C. The cycle time for all devices was set at 30 s. Two inlets were then milled at either end of a device and cleaned in a stream of nitrogen. Finally, 2 nm of chromium followed by 35 nm of gold were deposited onto the chip before it was sealed. Sealing of a Device. The bonding of a device was a long process because of the optical requirements of the experiment. A second design was then fabricated on a borosilicate substrate containing the inner dimensions of an injection-molded chip. A layer of adhesive was again rolled (2 psi) on a polished glass block set on a PDMS cushion for 10 s with a thin sheet of polyester film as the separating layer. The mask was then placed on the top of the polyester, and the whole device was exposed for 26 s. The design was developed in acetone and post-cured as previously described, leaving a mold cavity. Ten milliliters of a solution of PDMS was prepared, and 0.5 mL was then injected into the cavity and cured for 15 min at 70 °C in a vacuum oven. Once cured, the PDMS mold was removed and placed on a flat 9-cm2 square sheet of PMMA. Around the edge of the PDMS, 0.5 mL of Norland 68 Analytical Chemistry, Vol. 76, No. 1, January 1, 2004
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Figure 5. (A) Video photograph of the optical elements x and y in buffered solution. (B) Change in reflected intensity with different dye concentrations.
was dispensed and spin coated at 1500 rpm for 10 s. The surrounding layer was then partially cured by exposure to a UV source for 15 s. The PDMS copy was carefully removed, leaving an adhesive layer patterned to the inner wall dimensions of the injection-molded chip. The PMMA sheet was slowly positioned on the chip and aligned with a light microscope and then postcured for an additional minute. Once the assembly was annealed, an additional layer of adhesive was injected into the cavity between the top and bottom of the device and finally cured for another minute. Tubing (2 mm) was then connected and adhered to the underside of the chip. The depth of the bonding layer was obtained by taking random measurements using a Talistep machine (Rank Instruments, Leicester, U.K.). Characterization of Single Grating. Daughter, third-generation, and injection-molded grating copies were determined by atomic force microscopy (AFM). The data were used to assess grating reproducibility, as well as to provide an indication of the stability and expansion of the epoxy when heated in the mold. 242 Analytical Chemistry, Vol. 76, No. 1, January 1, 2004
Injection-molded copies of the integrated flow cell (unsealed) were assessed using scanning electron microscopy (SEM) and standard light microscopy (sealed). RESULTS AND DISCUSSION Twenty-five copies of the epoxy grating were injection molded, and the period and depth of the 10th and 25th copies were compared to the corresponding dimensions of the aluminized master. The results are shown in Table 1. AFM measurements demonstrate a loss in grating resolution as the AFM tip transferred from one substrate to another. The injection-molded copies showed an increase in period when compared to the aluminized master epoxy daughter and the second copy (Table 1). The transfer of the epoxy daughter into exactly the same material (i.e., the second epoxy copy) is the first of its kind and is known as material-to-material transfer (MMT). The epoxy daughter exhibits a significant reduction in its flexibility after being post-cured. It is this difference in the cross-linked state
Figure 6. Generation of a complete spectrum using a patterned 1-µm grating on a 50-µm-high pillar.
of the epoxy daughter compared with the cross-linked state of the second copy that allows the successful transfer of the nanostructures into the same material (MMT). Although the presence of the anionic surfactant (SDS) aids the separation and inhibits the adhesion of the two layers, fundamentally, it is the rapid cooling of the exposed layer that prevents the reorganization of the hydrogen network at the interface of the two films. Although not shown, the same phenomenon occurs when the surfactant is not present. However, the optimum exposure time relative to the difference in the cross-linked density of the materials has to be controlled to within 0.5 s for the nanostructure to be transferred or imprinted. Finally, the increase in the grating period in the injectionmolded copies is due to the expansion of the glass block when it was heated in the injection molder prior to fabrication. SEM examination of one of the injection-molded pillars in Figure 3 reveals corners that are slightly rounded because of the lack of collimation of the UV source and the resolution of the photo plotter masks purchased. The dimensions of the pillars were 110 (width) × 110 (length) × 25 (height) µm. Further SEM measurements at higher magnification clearly demonstrate (Figure 3C) the 440-nm grating on top of the injection-molded pillar. However, there is uniform shrinkage around the edge of each pillar; this is due to the expansion of the underlying glass substrate on which the master was fabricated. Ideally, each pillar should be 100 µm by 100 µm in length and width. Measurement of the Optical Path Length. Once the chip was sealed and filled with sodium phosphate buffer (pH 7.4), the optimum reflectivity angles of the first-order mode at 605 and 440 nm were measured. These were 80θ and 65θ, respectively. From these values, the total path lengths at 605 and 440 nm were calculated to be 214 and 200 µm, respectively. The average depth of the adhesive layer around the seal of the flow cell was 100 µm
with a standard deviation of 5%. At both wavelengths, the integrating light was assumed to pass through the chip only once. A summary of the calculations and the interactions of the light with the chip is presented in Figure 4. Absorbance Measurements. A video photograph showing a section of the flow cell reflecting at 605 nm through a sodium phosphate buffer layer is presented in Figure 5A. The changes in the reflected intensity in reponse to different dye concentrations compared with those observed for a standard visible spectrophotometer are shown in Table 2 and Figure 5B. Elements x and y both produce a linear response to increasing dye concentrations, although the absorbance of only a single wavelength could be examined at any one time using the setup described in Figure 1. Second, concentrations above 50 ppm of dye were not employed in this assay, as they induced a detectable change in the peak position of the reflected wavelength. The molar absorbitivity of Nile blue A was calculated to be 83 333 L mol-1 cm-1 using the results obtained from the standard spectrometer. Ideally, the whole spectrum should be generated on each element, thereby removing the need for moving parts. The fabrication procedures for such devices are currently under review and cannot be discussed here, but the photograph in Figure 6 demonstrates that it is possible to produce such elements. Finally, the signal-to-noise ratio in Table 2 shows that the signal from the raised platform is more than 3 orders of magnitude greater than the background signal. Conclusions. This report has demonstrated the evolution and automation of a rapid prototyping fabrication process that can be used to produce injection-molded devices with integrated elevated optical elements. Until recently, such devices could not be conceived because of the overall costs in producing a machined master. The problems of sealing injection-molded devices were taken into account in the design of the flow cell, resulting in a simple but reliable bonding procedure that preserves the internal nanostructures. The sensing elements are raised into solution, demonstrating a good signal-to-noise ratio. A device can be easily converted into a surface plasmon resonance (SPR) sensor or waveguide cell for biosensing purposes. The assembly of the device is also compatible with simple microcontact printing procedures.14 Research into such instrumentation is currently being conducted. ACKNOWLEDGMENT J.H. acknowledges the financial support provided by the Bio4sure project (QLK3CT200000607). Mr. J. Wilson of the Corrosion and Protection Center, UMIST, is thanked for his expertise with the AFM analysis. Mr. M. Faulkner of the Department of Materials Science, University of Manchester, is thanked for his help with the SEM analysis and vacuum deposition. Mr. McGowan, Department of Electronics, UMIST, is thanked for his assistance in working the Talistep machine. Received for review July 7, 2003. Accepted September 22, 2003. AC034755A
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