simultaneously then becomes possible. For instance, as SA is
broken down into SU in the liver, the ratio of SA to SU could
be used as an indicator of liver health and function. A dual assay
could be envisioned using [Tb(DO2A)]þat pH 8.4 to detect SU
and [Tb(EDTA)]–at pH 13.5 to detect SA in aliquots taken from
the same plasma or urine sample. Such an assay would be rapid,
cost effective, and minimally invasive, providing valuable infor-
mation with little discomfort to the patient.
We plan to take advantage of ligand-induced enhancement of
dipicolinate binding affinity to improve current bacterial spore
detection technologies in two ways. The first involves appending
terbium(macrocycle) complexes to solid polymer substrates to
improve microscopy-based endospore assays. In a second
method, we will bind lanthanide complexes to silica to concen-
trate dipicolinate from very dilute samples on columns.
Covalently attaching [Tb(DO2A)]þ to polydimethylsiloxane
(PDMS) could significantly improve the microscopic endospore
viability assay (mEVA) that we developed to image bacterial
spores. In this assay, endospores are inoculated onto wells of
agarose doped with TbCl 3 and induced to germinate via the addi-
tion of either L- orD-alanine for aerobic or anaerobic spores,
respectively (169,170). As the bacterial spores germinate and
return to a normal vegetative cycle, their DPA is released and
binds to the Tb^3 þions in the agarose. The resulting“halos”of
Tb(DPA)ncomplexes (n¼ 1 – 3) around each endospore are visible
using time-gated fluorescence microscopy ( 72 ). In the current
protocol, certain endospores are more easily observed than
others, asmEVA only images those capable of germination, and
the rate of germination varies significantly among spore species.
Bacillus spores germinate relatively quickly, on the order of
minutes; Clostridium spores, however, exhibit germination
profiles on the order of hours to days (Fig. 17) ( 171 ). For slow-
germinating species, the rate of DPA diffusion begins to outcom-
pete the rate of germination, and little or no signal results.
PDMS is used as a“coverslip”inmEVA to slow agarose drying,
limit DPA diffusion, and improve image quality. However, we
have just begun to explore the potential of PDMS to serve as a
platform upon which dipicolinate-specific sensors can be cova-
lently bound. By appending the lanthanide(macrocycle)
complex to the PDMS, we may be able to lengthen the residence
time of the DPA proximal to the endospore that released it,
expanding our imaging window.
Another problem encountered withmEVA involves microscopy.
The agarose surface on which the spores sit is often uneven due
to multiple variables in its preparation, meaning that not all
36 MORGAN L. CABLEet al.