Anal. Chem. 2004, 76, 5597-5602
Bonding of Glass Microfluidic Chips at Room Temperatures Zhi-Jian Jia, Qun Fang,* and Zhao-Lun Fang
Institute of Microanalytical Systems, Chemistry Department, Zhejiang University, Hangzhou, 310028, China
A simple, room-temperature bonding process was developed for the fabrication of glass microfluidic chips. Highquality bonding with high yields (>95%) was achieved without the requirement of clean room facilities, programmed high-temperature furnaces, pressurized water sources, adhesives, or pressurizing weights. The plates to be bonded were sequentially prewashed with acetone, detergent, high-flow-rate (10-20 m/s) tap water, and absolute ethyl alcohol and were soaked in concentrated sulfuric acid for 8-12 h. The plates were again washed in high-flow-rate tap water for 5 min and, finally, with demineralized water. The plates were bonded by bringing the cleaned surfaces into close contact under a continuous flow of demineralized water and air-dried at room temperature for more than 3 h. This bonding process features simple operation, good smoothness of the plate surface, and high bonding yield. The procedures can be readily applied in any routine laboratory. The bonding strength of glass chips thus produced, measured using a shear force testing procedure, was higher than 6 kg/cm2. The mechanism for the strong bonding strength is presumably related to the formation of a hydrolyzed layer on the plate surfaces after soaking the substrates in acid or water for extended periods. Microfluidic chips bonded by the above procedure were tested in the CE separation of fluorescein isothiocyanate-labeled amino acids. Chip-based microfluidic systems have attracted broad interest in recent years as a major form for realizing the lab-on-a-chip, or µTAS, concept.1-3 Although microfluidic chips may be produced from glass, quartz, silicon, and various polymeric materials, those fabricated on glass substrates have been employed most frequently owing to their favorable optical properties and support of reproducible electroosmotic flow.1-3 Fabrication of glass microfluidic chips usually involves micro channel/structure fabrication using a photolithographic and wet-etching procedure, followed by the bonding of the etched plate with a flat coverplate. Currently, bonding processes involving high temperatures are employed most frequently. Glass plates are brought into close contact with * To whom correspondence should be addressed. Phone: +86-571-88273496. Fax: +86-571-88273496. E-mail:
[email protected]. (1) Manz, A.; Fettinger, J. C.; Verpoorte, E.; Ludi, H.; Widmer, H. M.; Harrison, D. J. Trends Anal. Chem. 1991, 10, 144. (2) Harrison, D. J.; Manz, A.; Fan, Z.; Lu ¨ di, H.; Widmer, H. M. Anal. Chem. 1992, 64, 1926. (3) Jacobson, S. C.; Hergenro ¨der, R.; Koutny, L. B.; Warmack, R. J.; Ramsey, J. M. Anal. Chem. 1994, 66, 1107. 10.1021/ac0494477 CCC: $27.50 Published on Web 08/11/2004
© 2004 American Chemical Society
each other under clean room conditions and are usually subjected to sophisticated temperature programming in a high-temperature furnace at 500-650 °C.2-4 High-temperature bonding processes usually involve complicated manipulations requiring considerable expertise for achieving high yields and stringent laboratory conditions. Weights made from quartz, graphite, or refractory materials have often been imposed on the plates to increase the bonding pressure. Although such measures may be effective in improving the bonding quality and yield, our experiences have shown that smoothness of the plate surface was often deteriorated, which reduced the optical quality of the chips. Recently, Yin et al.4 reported the high-temperature bonding of glass plates under routine laboratory conditions involving mating the plates under a continuous flow of demineralized water. In contrast to high-temperature bonding processes, lowtemperature bonding of glass microfluidic chips usually involves the use of various adhesives below 100 °C. Such procedures are, therefore, suitable for the fabrication of microfluidic devices containing structural features that are nonresistant to high temperatures, such as electrodes and waveguides. Bonding with adhesives is also suitable for materials with significantly different thermal expansion coefficients. Wang et al. bonded glass plates at 90 °C employing sodium silicate as an adhesive layer.5 Nakanishi et al. reported a room-temperature pressure-assisted procedure for bonding quartz wafers by treating the wafer surface with dilute hydrofluoric acid.6,7 Sayah et al. described two methods for direct pressure-assisted low-temperature bonding of glass substrates,8 involving epoxy gluing at 90 °C in the first method and exposure of the glass stack to high pressure in the 100-200 °C temperature range in the second. Chiem et al. proposed a roomtemperature bonding procedure featuring stringent washing of the plate surfaces under clean-room conditions.9 A UV-curable glue was used by Huang et al.10 for bonding glass chips at room temperatures. The glue was filled into “guide channels” on the microchip architecture, followed by exposure to UV light. (4) Yin, X. F.; Shen, H.; Fang, Z. L. Fenxi Huaxue (Chinese J. Anal. Chem.) 2003, 31, 116. (5) Wang, H. Y.; Foote, R. S.; Jacobson, S. C.; Schneibel, J. H.; Ramsey, J. M. Sens. Actuators, B 1997, 45, 199. (6) Nakanishi, H.; Nishimoto, T.; Nakamura, N.; Nagamachi, S.; Arai, A.; Iwata, Y.; Mito, Y. IEEE. 1997, 299. (7) Nakanishi, H.; Nishimoto, T.; Kanai, M.; Saitoh, T.; Nakamura, K.; Shoji, S. Proc. Transducers ’99, Sendai, Japan, 1999, 1332. (8) Sayah, A.; Solignac, D.; Cueni, T.; Gijs, M. A. M. Sens. Actuators, A 2000, 84, 103. (9) Chiem, N.; Lockyear-Shultz, L.; Andersson, P.; Skinner, C.; Harrison, D. J. Sens. Actuators, B 2000, 63, 147.
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In this work, we report a simple low-temperature bonding process with high bonding quality and yield and without the need for clean-room conditions, adhesives, pressurizing equipment, and temperature programmable furnaces. EXPERIMENTAL SECTION Apparatus and Equipment. UV lithography was performed using a model JKG-2A lithographic device (Photomechanical Co. Shanghai). Thermal bonding for glass chips was performed in a programmable furnace (model SX2-4-10, Shenyang Electrical Furnace Co., Shenyang). An electronic testing machine (model WDS-100, KN, Shijin Group Co., Jinan) was used for measurement of bonding strength of the glass chips. A model 9323-HVPS HV power supply (Institute of Applied New Technology, Beijing), variable in the range 0-30 kV, was used for endurance testing of the bonded chips under high voltage. A homemade high-voltage power unit,11 variable in the range 0-1500 V was used for on-chip sample injection and CE separation. A LIF detection device, as detailed in a previous report,11 with an argon ion laser (488 nm, 4 mW, model 367, Nanjing Electronic Equipment Co. Nanjing) was employed for detection. The signal output from the detector was recorded using a model XWTD-164 chart recorder (Dahua Instruments. Shanghai). Reagents. All reagents used were of analytical grade, and demineralized water was used throughout. Sodium tertraborate buffer (5 mM, pH 9.2) was used as the working electrolyte for capillary electrophoresis (CE) separation. Stock solutions of 10 mM amino acids were prepared from L-arginine, D,L-β-phenylalanine, and glycine purchased from Kangda Amino Acid Works (Shanghai, China). Fluorescein isothiocyanate (36 mM, FITC) labeling reagent was prepared by dissolving 8.5 mg of FITC obtained from Acros Organics (Geel, Belgium) in 0.6 mL of acetone (with 0.03 mL of pyridine added) before use.11 FITC-labeled amino acid solutions containing 2.5 mM of each amino acid were prepared by mixing 0.5 mL of the stock solutions of each of the amino acids with 1.3 mL of 10 mM sodium tetraborate buffer (pH 9.2) and 0.2 mL of FITC reagent. The mixture was allowed to stand overnight in the dark. A working solution containing a mixture of 20 µM of each amino acid was prepared by mixing 0.4 mL of each of the labeled amino acid solutions and diluting to 50 mL with 5 mM borate buffer. Procedures. Fabrication of Glass Chips. Photolithographic and wet chemical etching techniques were used for fabricating microchannels onto a 1.6-mm-thick 20 × 60-mm glass substrate. Type SG2506 glass substrate with chromium and AZ1805 photoresist coating and glass cover plates of the same dimensions were obtained from Shaoguang Microeletronics Corp. (Changsha, China). Production of the photomask and procedures for fabrication of microchannels were previously described.4 Briefly, a design on a photomask with microchannels was transferred onto the glass plate following a UV exposure. The microchannels were etched into the plate in a well-stirred bath containing dilute HF/NH4F. Four 1.2-mm-diameter access holes were drilled on the etched plate at channel terminals using an emery drill-bit, forming four reservoirs. Microchips with a crossed-channel design were used for testing the bonding strength as well as their performance in (10) Huang, Z. L.; Sanders, J. C.; Dunsmor, C.; Ahmadzadeh, H.; Lander, J. P. Electrophoresis 2001, 11, 382. (11) Fang, Q.; Xu, G. M.; Fang, Z. L. Anal. Chem. 2002, 74, 1223.
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Figure 1. Processes of the room-temperature bonding approach: (a) plates washed with tap water at high flow rate (10-20 m/s); (b) plates soaked in concentrated H2SO4 or water; (c) plates brought into close contact under a continuously flowing stream of demineralized water; (d) combined plates stand at room temperature for more than 3 h.
CE separations. Channel dimensions were 30 µm deep and 85 µm wide, with a 10-mm-long sampling channel, and a 40-mm separation channel. The bonding process of the glass microchips was as follows: Etched substrates and cover plates were washed sequentially with acetone, household dishwashing detergent, tap water at high flowrate (10-20 m/s), and ethanol to remove solid particles and organic contaminants from the glass surface as well as the micro features. The plates were dried with a hair drier before being soaked in concentrated sulfuric acid for 8-12 h (as shown in Figure 1a). Subsequently, the plates were aligned vertically; held with a space of 1-2 mm between the surfaces, the channel structures of the etched plate facing inside; and washed again under a low flow of tap water (1.3-2.3 m/s) for 5 min. Finally, the aligned plates were brought into close contact under a continuous stream of demineralized water flowing between the two vertically held plates (Figure 1b). The combined plates were
Figure 2. Schematic diagram of chip structure for endurable shear force testing. The device was composed of two 30 × 14 × 1.2 mm glass plates with 3-mm-i.d. holes, bonded with an overlapping area of 15 × 14 mm.
then allowed to stand at room temperature for more than 3 h (as shown in Figure 1c) after removing the residual water film from the exposed chip surface using filter paper. The bonding was weak during the first few hours, and care was taken not to disturb the position of the aligned plates during this period. This period could be shortened to 15 min by drying the chips with a hair drier (80100 °C). When bonding quality is unsatisfactory owing to insufficient treatment, which could be observed by the appearance of interference fringes, the bonded plates may be separated for recleaning immediately after bonding. This could be achieved by soaking the chip in 50-60 °C water for a few minutes and then carefully forcing a sharp blade into the bonding interface. Testing of Bonding Quality for Chips. Tests were carried out using the bonded chips produced from type SG2506 blank glass substrate with a bonding area of 15 × 15 mm2. The maximum shear force endurable by the chips (before the bonding failed) was measured using an electronic testing machine. A schematic diagram of the testing arrangement is shown in Figure 2. Holes (3-mm) were drilled on the two bonded plates at the center of the nonbonded regions. Nylon strings were inserted through the holes and fixed on the testing machine. The plates were then pulled with the force increasing until a bonding failure occurred. The endurance to high voltage of the room-temperature bonded chips with crossed-channel design was tested by continuously applying a voltage drop of 1 kV at two reservoirs connected by a 4-cm-long channel (field strength, 250 V/cm), filled with 5 mM borate buffer, for 50-100 h. The current was digitally displayed, and recorded at 15-min intervals. Potentials of 6-15 kV were also applied under otherwise identical conditions for identifying an electrical breakdown value. The endurance of the room-temperature-bonded chips to hydrodynamic pressure was tested using two 20 × 60-mm chips on which were fabricated 85 µm (w) × 30 µm (d) × 40 mm (l) straight channels, with 1.5-mm diameter holes drilled at the channel terminals. MicroLine tubings (0.5 mm i.d., 1.5 mm o.d.; Thermoplastics, Sterling, NY) were inserted in the inlet holes of the chips and secured with epoxy. The distal ends of the tubings were push-fitted over the outlet needle of a syringe micropump (model WZS-50F, ZJU Medical Instruments, Hangzhou) and secured by applying epoxy at the connection point. Demineralized water was pumped through the microchannels at varying flow rates while they were monitored for leakages at the bonding surfaces. A further test was conducted by directly connecting a plastic syringe to a conical pipet tip epoxied to the chip inlet and applying pressure to the water-charged syringe using the electronic testing equipment employed for bonding strength testing. The hydrodynamic pressures exerted on the walls of the chip channels were estimated from the meter readings and the plunger cross-sectional area of the syringe.
CE Separation of Amino Acids. Chip-based CE separation with LIF detection employing the room-temperature-bonded chips was performed using a multicontact high-voltage power supply. Before use, the microchannels on chips were treated with 0.1 M NaOH or concentrated H2SO4 for 1 h and rinsed with demineralized water and 5 mM sodium tetraborate buffer (pH 9.2). Buffer solution was filled into the buffer and waste reservoirs, and a sample with FITClabeled amino acids was filled into the sample reservoir. Platinum wire electrodes were inserted into the reservoirs. The sample was injected into the CE separation channel by pinched injection mode. A field strength of 220 V/cm was applied for CE separation, with an effective separation length of 18 mm. Safety Considerations. The soaking of glass plates in concentrated sulfuric acid should be handled with care using PTFE forceps, while wearing protective gloves and goggles. Plates should not be immersed in the acid immediately after washing, but should be first dried as indicated in the procedures. Immersing of wet plates into the acid can cause disastrous results. The vessel containing the acid should be covered and labeled during treatment. RESULTS AND DISCUSSION Cleaning and Bonding of Glass Substrates. One of the most important factors affecting successful bonding of planar glass chips is the cleanliness of the bonding surfaces of glass substrates. This was reported by Chiem et al.9 and has now become a broadly accepted reason for the requirement of a clean-room environment during chip fabrication. Other factors often reported included surface flatness of the glass plates, bonding temperature, and pressure. Our experiences also strongly support the importance of cleanliness, having shown that the most unsuccessful bonding events are often associated with solid particles or organic material remaining on the glass surfaces, for example, dust, residual photoresist or glass particles, and grease, etc., prior to bonding. The washing and cleaning of glass substrates are often performed using detergents, water-soluble organic solvents (e.g., ethanol, acetone), concentrated sulfuric acid, and high-flow-rate tap water. High-flow-rate tap water showed better effects for removing minute solid particles on chip surfaces.9 In this work, most surface contaminants were removed by such treatment, and those remaining in the microstructures were removed using a cotton swab soaked with absolute ethyl alcohol. To remove further residual organic contaminants, the plates were immersed in concentrated sulfuric acid and soaked for 8-12 h. This soaking procedure proved to be most effective for eliminating such contaminants and also significantly contributed to the formation of a hydrolyzed layer on the glass surface, which is important for improving bonding quality and yield at room temperature12 (see the Evaluation of Bonding Quality section for details). We found that glass substrates that are free from organic contamination do not require such rigorous treatment. Instead, soaking in demineralized water or dilute sulfuric acid solution for the same or shorter period was in most cases sufficient for achieving satisfactory bonding. Another important practice before bonding is preventing exposure of the cleaned and hydrolyzed glass surfaces to an unclean environment, such as that in a routine laboratory. This (12) Fang, Q.; Jia, Z. J.; Fang, Z. L. Chinese Patent, Appl. no. 200410016216.3, 2004.
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is the reason most published processes required operations under clean-room conditions. In an earlier report from our group, employing a high-temperature bonding process, glass substrates and cover plates were brought into contact under a continuous stream of demineralized water flow4 to avoid contamination from the environment, immediately before bonding (see Figure 1b). This approach was adopted in the room-temperature bonding process to avoid the need for clean-room facilities. In contrast to our previous approach, which still required a high-temperature treatment, in this method, reliable bonding was achieved at room temperature following a period of standing. This is due to the more stringent prebonding treatment discussed in the preceding paragraph. A more than 95% high bonding yield was achieved in our routine laboratory during the fabrication of 150 pieces of glass microchips with various dimensions. Surface flatness of the glass plates is another factor that affects close contact of surfaces and, therefore, the bonding quality. These effects were studied using planar substrates and cover plates, described in the Experimental Section (0-2 µm flatness), as well as untreated microscope slides (3-5 µm flatness) with identical chip areas (25 × 25 mm). Stronger bondings were obtained for the former compared with the latter, with bonded areas of 100% (20 × 60 mm2 chip area) and 30-60% (15 × 25 mm2 chip area), respectively. The bonding yield for microscope slides was strongly dependent on the chip area, with the yield increasing for smaller chip areas, apparently because extreme flatness is more difficult to ensure over larger areas. Thus, for a 13 × 15 mm2 chip, the bonded area was increased to over 90%. Although the room-temperature bonding procedure is sufficient for most chip-based applications, a stronger permanent bonding may be achieved by a subsequent high-temperature treatment if required. In that case, the present approach may be used as a pretreatment before the furnace heating process, during which no pressurizing weights (which could affect glass surface smoothness) on the chip surfaces are then required, and almost 100% bonding yield could be obtained. Typical high-temperature-treated glass chips produced using pressurized weights during heating and one without weights following room-temperature bonding are shown in Figure 3a and b, respectively. Mechanism of Room-Temperature Bonding for Glass Chips. Our work has shown that considerably strong bonding of glass chips, sufficient for most microfluidic operations, could be obtained at room temperature by employing the approach described in the Experimental section. In a low-temperature bonding approach for glass chips employing sodium silicate as an adhesive, Wang et al.5 proposed a bonding mechanism involving siloxane bond formation between the sodium silicate and glass surfaces. Our observations and results support the assumption that a hydrolyzed gel layer is formed on the glass substrate surface after soaking the plates in acid or water for relatively extended periods of at least a few hours, during which most of the Si-ONa groups near the surface of the chips are transformed into Si-OH groups, as shown in Figure 4a. After intimate contact of the hydrolyzed surface and standing at room temperature, the Si-OH groups gradually dehydrate, forming siloxane bonds, and terminating with a condensation-polymerization reaction, as shown in Figure 4b. The natural dehydration process is slow, apparently being determined by the speed of water evaporation from the bonded 5600 Analytical Chemistry, Vol. 76, No. 18, September 15, 2004
Figure 3. Comparison of surface smoothness of glass chips (a) using pressurizing weights in high-temperature bonding process and (b) with the room-temperature bonding method, both after being subjected to the same high-temperature treatment.
surface, and in this work, usually 30 days was required to reach an equilibrium state, when maximum bonding strength was achieved. Such observations also support the presumed bonding mechanism. Evaluation of Bonding Quality. The bonding quality of glass chips using the present approach was compared with those using a conventional high-temperature bonding method employing both SEM images and bonding-strength testing. The cross sections of a bonded chip using the different bonding approaches are shown in Figure 5. No obvious differences at the bonding interface were observed. In this study, the bonding strengths of room-temperature (1525 °C)-bonded chips following different standing periods were evaluated using a shear force testing technique. A total number of 100 separate chips were subjected to the tests, and successfully bonded chips (98% bonding yield) were used for testing. The effects of postbonding standing time on bonding strength for chips soaked in water or sulfuric acid were studied, and the results are shown in Figure 6a and b, respectively. The first measurements were made 3 h after initial bonding, and tests were conducted until a 1-month standing period. Since each chip was destroyed during the test, the data for varying standing times were collected from different chips. For either mode of pretreatment, bonding strengths increased on standing, and gradually stabilized at a maximum value 20-30 days after bonding. A maximum of 72.2 ( 4.1 N/cm2 was reached for plates treated with acid, as compared to 61.8 ( 2.6 N/cm2 for those soaked in water. It should be noted that ∼80% of maximum bonding strength could be achieved 3-4 days after bonding, when the strength was sufficient for most applications. It should also be noted that although the bonding strengths of chips soaked in water or acid did not differ significantly, the yield for successful bonding of water-treated chips was significantly lower, by ∼20%. This might be explained by the stronger cleaning power of the acid in removing minute particles from the glass surface. Endurances of the room-temperature-bonded chips to high voltage and hydrodynamic pressure were tested as described in the Experimental Section. Bonding quality was monitored by
Figure 4. Presumed mechanism for room-temperature bonding process: (a) the formation of hydrolyzed layer on the substrate surface and (b) dehydration and condensation/polymerization of Si-OH groups.
Figure 5. SEM images of channel cross-sectional profiles of glass chips fabricated using different boding procedures: (a) high-temperature bonding and (b) room-temperature bonding.
observing current fluctuations under different applied voltages. With an applied voltage of 1 kV (250 V/cm field strength), the observed variation in current during a total testing time of 50100 h for 3 chips showed an average reproducibility of 2.4-4.7% RSD. The largest RSD for a single chip was 4.7% (n ) 24). The longest continuously operated period during this study was 12 h, during which the current RSD was 4.3% (n ) 30). Under an applied voltage of 1.5 kV (375 V/cm field strength), results similar to those obtained under 250 V/cm field strength were obtained. Under applied voltages of 6-8 kV (1.5-2.0 kV/cm field strength), constant currents were maintained for at least 3 h. When higher voltages (>12 kV, 3.0 kV/cm field strength) were applied, excessive current fluctuations occurred owing to partial bonding failure. Endurance of the room-temperature-bonded chips to hydrodynamic pressure was tested by increasing the flow rate of demineralized water through the chip channel described in the Experimental Section to a maximum of 100 µL/min (corresponding to a linear flow rate of 65 cm/s), leaving a residence time of only 60 ms for any fluid element flowing through the channel. Above this flow rate, the pump stopped working owing to actuation
Figure 6. Effect of standing time vs bonding strength of glass chips using room-temperature bonding approach: (a) plates soaked in concentrated H2SO4 and (b) plates soaked in water, before bonding.
of the protective circuitry when an upper pressure limit was exceeded. A further test was conducted using an electronic testing device, as described in the Experimental Section. When a pressure of 2.8 MPa (∼30 atm) was reached within the channel, with the flow-rate achieving 700 µL/min, the epoxied connection between the syringe and chip broke away, No leakages or interference fringes were observed at the bonding surfaces during both tests, indicating satisfactory performance of the chip, even under the most demanding hydrodynamic conditions encountered in microfluidic applications. The reliability and performance of glass microfluidic chips bonded at room temperature were tested in the CE separation of a mixture of FITC-labeled amino acids under an applied voltage of 800 V. Electropherograms obtained are shown in Figure 7b, with a comparison to those obtained using a high-temperatureAnalytical Chemistry, Vol. 76, No. 18, September 15, 2004
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Figure 7. Typical LIF electropherograms of repetitively introduced samples of a mixture of 20 µM FITC-labeled arginine, phenylalanine, and glycine employing glass microchips fabricated by (a) conventional high-temperature bonding method and (b) room-temperature bonding method. Working electrolyte, 5 mM borax buffer; applied field strengths for filling of sampling channel and CE separation were 500 and 200 V/cm, respectively; effective separation length, 18 mm.
bonded chip fabricated with conventional procedures (Figure 7a). RSD values of the fluorescence intensity ranged from 2.1% for arginine to 3.0% for glycine (n ) 10) using conventional hightemperature-bonded chips (plate numbers 7000-11000), and ranged from 1.9% for arginine to 1.5% for glycine (n ) 21) using a room-temperature-bonded chip (plate numbers 7600-13000). Precision of the residence times for different amino acids using chips fabricated by the high-temperature and room-temperature bonding approaches were both better than 1% RSD. The results show that the CE performance of room-temperature-bonded chips are comparable to those obtained using chips bonded at high temperatures. CONCLUSION Effective bonding of glass chips has been achieved at room temperature without the requirement of clean-room facilities, adhesives, pressurized water sources, high-temperature furnaces, or pressurizing weights. Bonding the plates within a continuous stream of water avoided the requirement of a clean room facility. The method has been employed in our routine laboratory for more than a year with high yields, during which time glass microfluidic chips with various dimensions and designs were fabricated, demonstrating performance comparable to high-temperature-
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bonded chips. Three to four days is required for stabilizing the bonding quality, which is sufficiently short for most applications. However, for urgent use, the room-temperature bonded chips may be subjected to a high-temperature treatment at 550 °C immediately after forced drying. No weights were required during the latter treatment, and chips may be ready for use on the very day of designing the channel structures. The labor involved in the microfabrication of glass devices is reduced by eliminating the thermal bonding process. Avoiding the use of clean room facilities and programmed furnaces allows the microfabrication process to be easily mastered by students and technicians in almost every analytical lab and with very limited investment. Proliferation of the technique could significantly accelerate research in microfluidic systems, currently often limited by lack of on-site microfabrication capabilities for optimizing chip designs. ACKNOWLEDGMENT This work was supported by the National Natural Science Foundation of China (Grant 20299030). Received for review April 10, 2004. Accepted July 2, 2004. AC0494477
