Biomedical Engineering Reference
In-Depth Information
and PNOS in multi-sensor respirometry, we have demonstrated real-time production
and consumption of H
2
S by several mammalian tissues and cultured cells.
8.3 FABRICATION OF POLAROGRAPHIC H
2
S SENSORS
8.3.1 Macro polarographic H
2
S sensors
The macro PHSS (Fig. 8.2), based on published electrochemistry [42], was designed
with dimensions equal to that of the polarographic oxygen sensor (POS; model 2120
Orbisphere, Geneva, Switzerland) used in the Oroboros Oxygraph respirometer
(Innsbruck, Austria). In this dual chamber respirometer, a PHSS replaced the POS
in one chamber for H
2
S respirometry of marine tissues and mitochondria [36]. The
PHSS housing was machined from polyether ether ketone (PEEK, Victrex U.S.A. Inc.,
Rockford, MI), chosen for its chemical inertness and very low O
2
memory in order
to limit O
2
back diffusion into the solution during experiments under low O
2
condi-
tions (see Victrex PEEK Material Properties Guide, Victrex, Lancashire, UK). PEEK
also coated the stir bars used in the respirometer chambers. Both anode and cathode
were fashioned from platinum wire (1 and 0.5 mm diameter, respectively; Blankinship
Porter, Birmingham, AL) and cemented into the PEEK housing with epoxy (Scotch-
Weld 2216, 3M, St Paul, MN). The electrolyte was held in the sensor tip reservoir with
a two-layer membrane made of an H
2
S-permeable membrane (MEM 213, 25
µ
m thick,
WPI), cemented (Silicone Adhesive RTV 167, GE, Waterford, NY) to a 25
m thick
H
2
S-impermeable membrane. H
2
S-impermeable membranes include saran, mylar,
perfuoroalkyl-tetrafl uoroethylene copolymer (PFA), and fl uorinated ethylene/propyl-
ene (FEP). In our experience, only silicone polymer membranes allow rapid diffusion
of H
2
S, whereas polyethylene and polypropylene permit only minor H
2
S diffusion (see
Table 1 in [36]). The 0.5 mm diameter hole in the impermeable membrane, concentri-
cally located above the 1 mm platinum anode, was covered by the permeable mem-
brane. In this confi guration, the impermeable membrane ring served as a virtual guard
ring, greatly accelerating the sensor response time by preventing the diffusing H
2
S from
equilibrating the entire electrolyte volume. The bilayered membrane was held onto the
PHSS tip between an O-ring and an adaptor ring. H
2
S-dependent changes in anode cur-
rent were converted to proportional voltage with a modifi ed POS meter, and the output
voltage was recorded digitally (Virtual Bench, National Instruments, Austin, TX).
µ
8.3.2 Miniature polarographic H
2
S sensors
The miniature PHSS design was based on the original design of the macro PHSS [41].
The 2 mm diameter PHSS utilized a cylindrical polished-tip platinum anode that was
electrically insulated along the side from a platinum cathode that was coiled around the
core (Fig. 8.3). The core was housed in a 2 mm outside diameter stainless steel cylin-
drical sleeve that served as an electrical shield and to which a 25
m thick H
2
S-perme-
able membrane was fi xed to cover the tip. The reservoir between the sleeve and core
µ
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