2.2. Construction of a Steam-Sterilizable Dissolved Oxygen Electrode
2.2.1. Fabrication of DO Sensor
DO Measurement. Oxygen must be supplied to growing microorganisms just like we need it for maintaining our lives. Oxygen dissolves very sparingly in water - about 8 ppm (parts per million by weight) at 20oC. Therefore, one of the major design variables for a bioreactor is the oxygen transfer rate (OTR) because the maximum growth rate of an aerobic microorganism depends on it. The dissolved oxygen in bioreactors are usually measured by a dissolved oxygen (DO) sensor. In this Section, we will construct a steam-sterilizable DO sensor suitable for bioreactor applications. The idea is to appreciate the relationship between the sensor design/fabrication methods and the sensor performance.
Materials of Construction. The following materials are needed:
Chemicals for cleaning and plating
Step 1. Cut a short length of copper wire (20 cm) and attach 75 µm Pt wire by soldering.
Step 2. Clean the Pt wire (just the Pt wire portion, not the Cu wire) by dipping in a 1:1 concentrated nitric and sulfuric acid solution. Wash with DI water.
Step 3. Place it in the glass capillary tube (cleaned) as shown. Flame the end until the glass melts and fuses around the Pt wire. A good wetting of the Pt wire with molten glass is necessary. Lead glass is best for this purpose because, it wets the metal well and the thermal expansion coefficient is closest to that of Pt.
Step 4. Grind the flamed end flat (use a sand paper: start with a coarse one and then gradually use finer grit sand paper). The end should looks like:
It is important that the glass seals the metal tightly around the metal wire. The goodness of the seal may be observed under a microscope but a better way is to measure the conductivity in an electrolyte solution (by using Ag/AgCl as the counter electrode). The conductivity should remain more or less constant. If it increases with time, the seal is not good and a DO sensor made with such a leaky seal tend to be unstable.
Step 5. Grind flat both ends of the 10 mm OD glass tube. Grind the edge of one end round.
Step 6. Make a Ag/AgCl reference electrode by electrolytic chlorination of Ag wire.
Clean Ag wire by dipping in 1 M nitric acid for 10 s.
Anodize the wire in 0.1M HCl at current density of 0.4 mA/cm2 for 30 min.
Store in 1M KCl solution overnight before use.
When the chlorination is done properly, the wire will have a brownish coat on it.
Step 7. Put the glass capillary tube in the large glass tube, and insert the chlorinated silver wire and the vent tube.
Step 8. Connect the copper wire to the center pin of BNC connector; connect the Ag/AgCl wire to the outer body of BNC by soldering.
Step 9. Fix the BNC connector and the vent tube by molding with epoxy.
Step 10. Cut a 3/4" length of silicone tubing and place it in methyl chloroform for 2 min. The tubing swells. Cover the free end of the sensor assembly with a Teflon membrane and fix it tightly in place with the swelled silicone tubing. Make sure the membrane is tightly fit. Let the solvent evaporate.
Step 11. Fill the electrolyte solution with a syringe via the vent hole. After the filling, clamp the vent tube.