Biomedical Engineering Reference
In-Depth Information
for making arrays of different recognition molecules to capture multiple tar-
gets [6,7]. However, the biotin-avidin approach may have disadvantages if the
recognition molecules used for capture are very small; in this case, a spacer
may be required between the biotin and the capture molecule to make sure
that it extends out of the binding pocket on the avidin and into the solution.
Another situation where immobilization via an avidin bridge may not be the
optimum approach is where the density of the capture molecule is critical for
e
cient binding - as has been documented with sugars [8] and antimicrobial
peptides [9].
Assays with biological recognition molecules for fluorescence biosensors
are configured in four basic formats: direct binding, sandwich, competition,
and displacement assays. The direct binding format is by far the simplest to
implement as it involves only the capture molecule and the target. However,
it only works if either the target is inherently fluorescent or if it has somehow
been prelabeled, as is the case in many DNA hybridization assays. Direct-
binding assays are often used with prelabeled target standards for several
reasons: to select the best capture molecules for a particular application by
characterizing a
nity and specificity of the binding function; to optimize the
assay conditions for sensitivity, speed, and reproducibility; and to provide
positive controls to monitor assay performance. A variation of the direct-
binding assay utilized molecular beacons (reviewed by Yao et al. in [10]). In
these assays, a fluorescence energy transfer donor is immobilized on one area
of a capture molecule, while an acceptor is immobilized on an area that is
adjacent only when no target is bound. When the target is bound, the two
fluorophores separate, and a positive signal is generated.
The sandwich assay is used for detection of targets with at least two bind-
ing sites, including large molecules such as proteins, oligonucleotides, bacte-
ria, and viruses. In the sandwich assay, the target binds the capture molecule
(usually immobilized) and a fluorescent tracer molecule, usually in that order.
The formation of the resultant fluorescent complex is measured, while free flu-
orescent tracer molecules are either removed or optically excluded from the
sensing region. Sandwich assays are described in the majority of publications
on optical biosensors.
For small molecules with only a single-binding site, the formation of such a
sandwich is not possible and either a competitive or displacement assay must
be utilized (Fig. 11.1). Two versions of the competitive assay are widely used.
In one version, the capture molecules are immobilized, labeled target is added
to the sample solution, and the labeled and unlabeled target molecules com-
pete for the binding sites on the immobilized capture molecule. In the second
version, a target molecule is immobilized, labeled antibody is added to the
sample solution, and any target free in solution prevents the binding of the
labeled antibody to the immobilized target. In both these versions, the fluo-
rescent signal at the surface decreases with the increase in target in the sample
solution. Although the fluorescence in solution can also be measured, the flu-
orescent component in solution is generally in su
ciently high concentration
