Fabrication of Glass Microfluidic Chips

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.

Photolithography
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 mm as opposed to 5 mm 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
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) 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 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.