Antimicrobial Peptides for Detection and Diagnostic Assays - Designing Receptors for the Next Generation of Biosensors - page 93

Fig. 2 CecA (cecropin A); CecB (cecropin B); CeMe (cecropin A (1-8)-melittin (1-18) hybrid);
CecP1 (cecropin P1); Mel (melittin); Indol (indolicidin)
effects of commercial microtiter plate chemistry on AMP immobilization and their
subsequent interaction with bacterial biomarker, LPS. AMPs were immobilized to a
series of commercial microtiter plates—two plates with amine-reactive functional-
ity (Immobilizer Amino ® , maleic anhydride), a maleimide-activated plates for
thiol-directed coupling, and a hydrophobic polystyrene plate (Microfluor I ® ).
We evaluated the effects of the different immobilization chemistries on peptide
presentation and orientation by determining the relative amount of free primary
amines on each peptide. As the primary amines are the potential points of attach-
ment, the fluorescence labeling efficiency of the peptides after surface immobiliza-
tion can provide information on the degree of multipoint attachment as well as the
availability of these amine residues for binding to target analytes. We observed
dramatic differences in the labeling efficiency of surface-bound AMPs that were
both peptide- and immobilization chemistry-dependent (Fig. 2 ). These results are
consistent with previous studies that have shown that the method used for immobi-
lization greatly affects AMPs' functionality in rapid detection assays [ 39 , 52 , 53 ].
Interestingly, we did not observe a correlation between peptide display and LPS
binding activity amongst the different AMPs tested. In addition, we could not
identify optimal immobilization chemistry for target capture that could be applied
to all AMPs. For example, the cecropin A (1-8)-melittin (1-18) hybrid appears to
exhibit the most flexible configuration when coupled to the maleic anhydride
microtiter plate; however, it demonstrates the highest LPS binding affinity when
immobilized to the maleimide-activated microtiter plate (Fig. 3 ). In contrast,
indolicidin exhibits the greatest number of free primary amines when coupled to
the Immobilizer Amino plate yet shows the best binding affinity to LPS when
coupled to the maleic anhydride microtiter plate. Even analogous methods of