Information Technology Reference
In-Depth Information
the bioreporter have a shorter path and fewer obstructions to the light-sensing
portion of the BBIC.
Lyngberg et al. [41] immobilized
E. coli
HB101 (
mer-lux
) as part of a mer-
cury biosensor. A preparation of 1.2 g cell paste was resuspended in 0.3 ml 50%
glycerol and 1 ml Rovace SF091 (Rohm and Haas, Philadelphia, PA). This was
spread onto a polyester support using a 26-mil-wire Mayer bar to a thickness
of 30
m) of latex was
applied as a top coat. Using fresh cells in this biosensor, 0.1-10,000 nM HgCl
2
was detected. These latex films were also stable at
µ
m. After curing this first layer, a second layer (47
µ
20°C for 3 months.
A disadvantage of the latex approach is the requirement for rigid curing
protocols. To maintain porosity but promote cell retention, the latex spheres
must be partially cured under controlled humidity and temperature. Full curing
will yield a solid film with no porosity and deprives the bioreporter cells of
water.
−
Cellulose-binding Domain
Cellulose-degrading bacteria produce an extracellular enzyme complex called
cellulases [6, 8]. One feature of the cellulases is the cellulose-binding domain
(CBD), a subunit that physically binds to cellulose. In some species, binding
by the CBD is irreversible [60]. CBD has been used as an affinity tag for the
purification/immobilization of proteins and antibodies [5, 50, 53, 59] as well
as for immobilization of whole cells [62]. CBD incorporation into bacterial
cells and using cellulose as a support matrix has several advantages over the
previous methods: (1) cellulose is an abundant, inexpensive material that comes
in many forms; (2) a uniform monolayer of cells can be achieved putting the
bioluminescent bioreporters closer to the light-sensing portion of the integrated
circuit; (3) binding is rapid and essentially irreversible; (4) there are no pro-
longed curing steps or curing agents that may inhibit cellular activity; and (5)
CBDs are stable and resistant to denaturation and proteolytic degradation [60].
Current Approaches
Previously our work has focused on either alginate or agarose for immobilizing
bioreporters [31]. The advantage of alginate or agarose beads is that they are
easy to prepare and large quantities can be made and stored at 4°C for long
periods (weeks to months) with no loss of activity. However, these natural
polysaccharides may not be suitable for IC applications. Most preparations are
by extrusion of cells in alginate from a syringe into a solution or onto a solid
surface. This does not generate films of uniform thickness, and the films are
relatively thick (hundreds of microns).
We have begun to explore the use of latex as an immobilization agent similar
to Lyngberg et al. [39, 41]. As previously mentioned, the latex can be rolled
into ultrathin sheets (10-100
µ
m) of uniform thickness. Using
Pseudomonas
