- Material Specifications
Glass is a popular substrate for microfluidic (mF) chips, although plastics are becoming more common due to the ease in fabrication. Several types of glass are used in mF devices, such as soda lime, quartz, and borosilicate. Soda lime glass is easy to work with as it etches quickly, bonds at relatively low temperatures, and is inexpensive; however, the etching quality is suspect and autofluorescence of the glass can be problematic. Therefore, this type of glass is typically used in preliminary studies. The most optically useful substrate is quartz as it is transparent from ultraviolet through infrared. Unfortunately, quartz is difficult to work with as the etching rate is slow and the annealing temperature is approximately 1000 °C, too high for some furnaces. Additionally, the cost of quartz substrates can be almost 2-3-fold higher than soda lime glass; however, quartz is one of the few substrates suitable for low noise optical detection with UV-excitable dyes. The most common glass substrate used in the production of mF chips is borosilicate glass because of its optical characteristics (transparent from approximately 350 nm through 700 nm) and its physical properties (annealing temperature of 640 °C, resistant to most chemicals).
Borosilicate glass incorporates a variety of subtypes, with all borosilicate glasses containing a minimum of 5% B2O3 [Corning Glass website, 2003]. Most borosilicate glasses used for mF chips are produced by the float method, which is a technique used to produce optically flat glass. In the float method, molten borosilicate glass is floated on molten tin and the glass is drawn from the tin producing an extremely flat substrate. Flat glass is necessary for the manufacture of mF chips as uneven glass is difficult to bond to other pieces of glass.
Fabrication of mF chips using borosilicate glass produced by the float method (borofloat glass) involves photolithography, wet chemical etching, and bonding. Each of these steps will be explained in detail in the following sections.
The intended channel design is drawn in AutoCAD 2000 (San Rafael, CA) and submitted to Digidat (Pasadena, CA) for the production of a right reading, anti-reflective chrome down, 4” x 4” x 0.90” soda lime or white crown photomask. Photomasks should be handled with care as any scratches in the chrome can ruin the design and these types of masks are ~$300-$500. For cheaper masks, the channel design can be drawn with Adobe Illustrator and printed onto a transparent film (such as an overhead film) with a high resolution printer. These masks are ~ $15, but the linewidth resolution is only ~20 um as opposed to 5 um for the chrome masks.
The glass used to produce mF chips are intended for the production of photomasks, and are known as photomask blanks. Schott borofloat photomask blanks are acquired from Telic Company (Santa Monica, CA) with a 520 nm layer of AZ1518 positive photoresist on a 120 nm layer of chrome. Although other companies claim to sell Schott borofloat glass, the material specifications do not always correspond to the specifications given by Schott. Acquisition of Schott borofloat glass is essential since the rest of this fabrication procedure has been optimized for this type of glass. Physical characteristics of Schott borofloat glass are: 560 °C annealing temperature, 32.5 x 10-7 K-1 thermal expansion, 2.23 g/cm3 density, and 1.472 refractive index. Therefore, if photomask blanks are purchased from a different company, the physical characteristics of the new glass should be compared to the above to ensure the procedure outlined below is relevant.
- UV Exposure
The following steps are performed in a class 10000 cleanroom unless stated otherwise. Filtered N2 is used to remove surface particles from the photomask and a photomask blank. The UV exposure unit currently used in the lab (Optical Associates, Inc., Milpitas, CA) is intended for contact exposure, meaning the photomask and photomask blank must touch during exposure to ensure even linewidths in the photoresist. The photomasks presently used in the lab are fabricated such that the chrome surface (brownish-gold in color) should be in contact with the photomask blank. To secure the photomask to the photomask blank, two methods are used. The first method entails a cassette that the photomask blank is placed in and the mask is laid over the top. This method is the most reproducible in chip-to-chip production. The second method uses two pieces of scotch tape (one at either end of the blank) to secure the mask to the blank. The only precaution in the UV exposure step is to ensure that the mask is centered on the blank. Centering the mask prevents overhanging reservoirs in the finished mF chip. The photomask/photomask blank assembly is placed under the UV exposure unit, which has been turned on for at least 5 min to warm up the lamp, and the chip is exposed for a minimum of 1 s at 26 mW/cm2. After exposure, the mask is immediately placed in its protective holder to protect from solutions in the remaining steps.
- Development and Chrome Etching
To aid in bonding, excess photoresist is removed from the photomask blank. Removal of excess photoresist allows the glass non-adjacent to the channels to be etched in a future step leaving only a small area of the glass to bond. Using a cotton swab saturated with acetone, photoresist 2-3 mm away from all channels is removed. The chip must be held at a certain angle in the light to reveal the channels, although a cursory movement of the hand enables visualization. Enough photoresist must remain at the end of each channel so that fluidic access holes can be drilled. Removing excess photoresist prior at this point is a precautionary measure so that if a UV-exposed region is accidentally removed, little time has been invested in the fabrication process.
After removing excess photoresist, the exposed blank is placed in ~15 mL of AZ915MIF (Clariant Corporation, Summerville, NJ) developer. The solution is swirled over the top of the blank for 15-20 s then rinsed with deionized water. After development, the photomask blank is placed in 25 mL CEP-200 chrome etchant (Microchrome Technologies, Inc., San Jose, CA). The solution is swirled over the top of the chip until no chrome remains, typically 1-2 min. Chrome etchant is a hazardous material and is only shipped by truck. Shipping takes approximately a month for delivery; therefore, it is best to order the chrome etch solution well in advance.
- Wet Etching of Glass
The next step in the fabrication procedure is to etch the photomask blank. Etching solution contains a mixture of 14:20:66 (v:v:v) HNO3:HF:H2O. Extreme caution needs to be taken when working with HF as the acid is a highly dangerous substance. Plastic dishes should be used for all steps and butyl gloves and eye goggles should be worn. In a clean plastic dish, 17 mL HNO3 is added to a stirring solution of 79 mL H2O; afterwards, 24 mL of 40% HF is added. The solution is allowed to stir for ~1 min to ensure homogeneity of the solution. During this time, the developed photomask blank is rinsed with water to remove any particles and then placed in the etching solution for an appropriate amount of time. The etching rate has previously been found to be 0.3 mm/min, although the etching rate should be periodically tested with a profilometer. After the etched blank is removed with plastic forceps and placed in a 1 L beaker of water, etching solution is discarded into the proper waste receptacle. The etched glass is removed from the 1 L beaker of water, rinsed extensively with deionized water, dried, and brought outside the cleanroom to drill fluidic access holes.
- Drilling of Fluidic Access Holes
Using a drill press and an appropriate sized diamond-tipped drill bit (Tarton Tool Co., Troy, MI), fluidic access holes at the end of each channel are drilled. It is best to drill with the etched channels toward the drill bit as the glass will “pop” out the backside when drilling. Care must be taken so that the glass is not broken during drilling or the bit does not come into contact with more than one channel. After drilling, the etched chip is aggressively rinsed with water to remove all glass fragments. A good method to aggressively rinse the etched chip is to pinch a hose attached to a water spigot, increasing the velocity of the water. To further facilitate removal of glass particles, the etched glass is placed (channels down) in a clean beaker and sonicated for 10-15 min in Milli-Q (Millipore, Bedford, MA) water.
- Cleaning of Drilled Chips
There are multiple steps in the cleaning procedure and it is vital that the glass be cleaned properly prior to bonding to have a functional chip. Because of this reliance on cleaning, individual steps in the following procedure are numbered. (1) In the cleanroom, acetone and chrome etchant are used to remove the remaining photoresist and chrome from the etched blank and another piece of photomask blank (used for the top plate). It is imperative to use matching types of glasses for the etched chip and top plate because different thermal expansions will cause the glass to shatter during the bonding process. In addition to having the same type of glass, all chrome must be removed from both the etched blank and top plate as a small amount of chrome left on either piece of glass will ruin bonding; therefore, both pieces of glass are left in a stirring chrome etch solution for ~ 10 min. (2) While etching the chrome, 150 mL deionized water is poured into a clean, 250 mL beaker and placed on a hotplate in the hood. The temperature of the hot plate is set to 60 °C. (3) To this beaker, 30 mL NH4OH is added and the hot plate with the beaker are moved to the back of the hood to allow the temperature to reach 60 °C. (4) Another clean, 250 mL beaker is placed in the hood and filled with 180 mL H2SO4. The next step is very exothermic and should be performed with great care. (5) The sulfuric acid is returned to its cabinet and 60 mL of 30% H2O2 is added to the beaker with H2SO4. This mixture is called piranha solution. (6) The piranha solution is stirred with a metal spatula and the etched blank and top plate are rinsed of chrome etch solution and placed in piranha for 20 min. Piranha solution is used to aggressively clean the glass of any organic material; therefore, all organic solvents should be removed from the hood as they are highly reactive with piranha. (7) During the cleaning with piranha, 30 mL of 30% H2O2 is added to the beaker on the hot plate, resulting in a mixture known as RCA solution. (8) After 20 min in piranha, the chip and top plate are removed and rinsed for 2-3 min under the deionized water spigot tracing the channels with the water stream. (9) After rinsing, the chip and top plate are placed in the RCA solution for 20-40 min. The chip and top plate must be completely covered with RCA solution during the cleaning process. If the solution evaporates, more water is added.
After cleaning with RCA, the chip and top plate are removed with forceps and each piece is briefly rinsed with deionized water. After this initial rinsing, both pieces of glass are held in one hand and vigorously rinsed with deionized water from the spigot for 4-5 min. It is best to trace the etched regions with the water stream, ensuring that no debris is left in the channels. This rinsing step is crucial to obtaining clean chips prior to bonding. Immediately after cleaning, the plates are brought into contact while still wet. Filtered N2 or a crew wipe is used to dry the outside of the chip. The chip is squeezed from the middle towards the outside to removed trapped air bubbles. If this process is followed, the assembled chip should be fairly stable with capillary action holding the two pieces of glass together.
- Bonding Chips
The assembled chip is placed inside a Neytech Centurion Qex furnace (Pacific Combustion, Los Angeles, CA) (or a normal muffle furnace) between two pieces of ¼” thick Macor ceramic plates. A 400 g stainless steel weight is placed on top of the Macor plates and the temperature is ramped, under vacuum (~20 mm Hg) at 10 °C/min to 640 °C, held for 6-8 hours, and ramped down to room temperature at 10°C/min. After bonding, a microscope is used to examine all channels in the chip. If any particulates are in the channels, it is best to discard the chip and make another. Since the electric fields and therefore flow rates are dependent on the resistances of the channels, a small particle can have a large impact on the performance of the device. If there are no particulates in the channels and the chip has unbonded regions that intersect a channel, a second bonding cycle is performed by placing the chip in the furnace (with the opposite side facing up as compared to the first bonding) for another 6-8 hours using the same temperature ramp as before. When bonding is complete, chips are stored in capped 50 mL plastic centrifuge tubes to ensure no dust or particulates come into contact with the chip. No solutions should be introduced to the chip until reservoirs have been applied. Microfluidic reservoirs are bought from Upchurch Scientific (Oak Harbor, WA) and applied according to the manufacturer’s instructions.
This fabrication protocol is from Roper Laboratory at Florida State University
Baker, C. A.; Roper, M. G. “Continuous-flow, microchip electrophoresis fraction collector” J. Chromatogr. A 2010, 1217, 4743-4748.