Description:

The resource has witnessed an increasing demand for applications targeting metabolism. To facilitate this we have developed several new technologies or integrated others to allow the study of cellular metabolism through several approaches. A number of these are reported on elsewhere. A key technique has been the application of self-referencing microsensors which to date have been constructed by hand. Although our efficiency here has greatly improved, manual production of amperometric sensors remains a bottleneck. To circumvent this some form of automated or semi automated production needs to be developed providing a supply of microelectrodes meeting the exacting requirements of a non-invasive modulation technique for signal detection.

Given the importance of metabolism to the pathology of diseases the availability of the new oxygen sensor will facilitate numerous biomedical investigations and potentially provide a novel methodology for targeting drug development at the level of single cell assays.

Progress:

Glass housing for the oxygen sensors are now pulled on a laser puller (Sutter P-2000), rather than the filament puller. This improves repeatability and simplifies maintenance.

Platinum Wire and Tip Etching:
Previously, a Potassium Cyanide (KCN) and Sodium Hydroxide (NaOH) mix was used to electrochemically etch a 1-2um tip from a 25um platinum wire. It was found that NaOH could be used by itself, eliminating the need for the use of Cyanide. This means that etching does not have to occur in a chemical hood. Etching with NaOH takes longer than with KCN, however, in an automated system, multiple electrodes could be etched simultaneously, in batches throughout a 24 hour period in open air.

Platinum Wire Glass Insertion:
Breakage can occur when inserting the very delicate 1-2um tipped 25um platinum wire down the pulled glass capillary. The fine tip can bend or break on the side of the glass. Inserting the tip too far out of the end of the pulled glass capillary can break the glass tip. To avoid these problems a 24 gauge platinum wire is now being used. As 24 gauge wire is close to the inner diameter of the glass capillary the 2-3um tip never touches the side of the glass. As the 24 gauge platinum wire and the fine tip are one piece breakage does not occur when the resin is drying and the final product exhibits reduced noise. A semi automated method has been developed for the wire insertion.

Recessed Etch:
The new automation of the oxygen sensor seeks to eliminate the recessed etch altogether. Instead of inserting the platinum wire passed the opening of the glass capillary, it will be inserted just shy (~4um) from the opening and glued in the back. The sensor is then front-filled with UV curable optical resin, and a drop of glycerol is put on the tip. The glycerol pushes the resin back to the already recessed platinum wire. The resin is then cured, and the glycerol is flushed out by dipping into water. As a recess has already been created, there is no need to perform a recessed etch.

Cellulose Acetate:
Getting cellulose acetate on the tip of the oxygen sensor, and then confirming that it is there, has been a problem. This step is currently done manually.

The technique for electrochemically etching the 24 gauge, pre-beveled wire, to 1-2um is currently being perfected. A complete oxygen sensor prototype, constructed from the steps above, has yet to be tested for sensitivity and repeatability of construction and performance.