Description:

For the flux measurements of molecules that are not electrochemically active, we have been employing enzymes incorporated into self-referencing electrodes. For example, among various enzyme types, the use of oxidases is favored as they produce electrochemically detectable hydrogen peroxide as a result of enzymatic oxidation in the presence of oxygen. In addition we have generated preliminary data on a design simplification using ceramic sol-gel matrices for encapsulation of materials. We anticipate that this design will have wide application to sensor construction, impacting several potentiometric and amperometric approaches. Using such a gel has allowed us to begin using dehydrogenase-based detection with moderate success.

Progress:

During this reporting period we have focused on the detection of glycerol and phosphate.

Glycerol
The glycerol biosensor in development is a 1.1mm/o.d. sol-gel based carbon powder electrode. The carbon powder is doped with the mediator Meldolas Blue (MB) through a process of mixing, heating and drying. The electrode is then coated with a glycerol dehydrogenase (GDh), polylysine and glutaraldehyde coating. NAD, a key component in detection, is added to the bath, rather than the coating, because of its instability in regards to leaching and shelf life. NH4Cl is also added to the bath to increase sensitivity of the probe. The construction of the probe is a four-day process. However, electrodes can be reused and re-coated. Coatings have shown consistent sensitivity for as long as one month if stored in the refrigerator. Re-coating can require as little as 2-3 hours of drying time, although drying overnight is more desirable.  Glycerol biosensors at the BRC have been recoated and used continuously for more than six months, showing no decrease in sensitivity. The sensitivity range of the biosensor is from as low as 50nM to higher than 2mM. Biosensor resolution has been as low as 100nM.

Phosphate
The phosphate biosensor is a 1.1mm/o.d. paraffin oil based carbon paste electrode. The coating is a bi-enzyme system consisting of Nucleoside Phosphorylase (NP), Xanthine Oxidase, and glutaraldehyde. Inosine is added to the bath to aid in the initial reaction with inorganic phosphate (Pi). This bi-enzyme system yields 2mol H2O2 for every mol of Pi, which is then detected at a working potential of +0.6V. This yield magnifies the Pi signal, increasing sensitivity and detection range. Currently, the BRC phosphate biosensor has a detection range from 100nM to more than 1.5mM, making it applicable both physiologically and environmentally. On a flow system, using a control and a 100nM concentration, the speed of reaction was approximated to be three seconds. Phosphate biosensors are very easy to prepare and can be reused and recoated for months at a time without loss of sensitivity.

In both glycerol and phosphate electrodes, miniaturization will be the next step.