Chemistry Reference
In-Depth Information
In this chapter, fluorescent sensors based on FRET between CPEs and organic
fluorophores are introduced. In particular, DNA biosensors are described in details
to illustrate the assay design and operation mechanism. Considering that FRET is a
crucial process that generates the signal and determines the signal intensity, factors
influencing FRET between CPEs and organic fluorophores are discussed using
DNA biosensor as an example. To provide a brief view about how to obtain high
FRET efficiency and in turn maximum detection sensitivity, molecular design
of CPEs with respect to dye-labeled DNA is also elucidated. After acquiring
knowledge of these vital issues in CPE-based FRET sensors, other representative
protein assays are depicted in a case-by-case manner. This chapter intends to draw
attention from researchers working with fundamental fluorescence technologies in
chemistry and biology, and also to guide new researchers into this booming field. It
is also expected that this chapter would help to inspire the specialists to have an
evolutional critical thinking about how to further optimize and advance such
powerful sensory systems.
2 DNA Biosensor
Reliable methods for DNA detection are under intense investigation because of
their vital applications in clinical diagnosis, environmental monitoring, forensic
analysis and antiterrorism [
44
]. Effective DNA sensors require selectivity to iden-
tify single-nucleotide polymorphism (SNPs), and sensitivity to provide information
from the small quantities of naturally occurring DNA molecules. A variety of DNA
assays have been developed based on different transduction mechanisms, including
fluorescence [
45
], electrochemical [
46
], microgravimetric [
47
], enzymatic [
48
], and
nanostructure based methods [
49
]. In general, DNA detection is constrained by the
levels of targets available in a particular sample. Thereby, the assay has to rely on
the aid of enzymatic sample replication (polymerase chain reaction) to increase the
concentration of specific nucleic sequences to detectable levels or turn to complex
labeling steps (dye multiplicity) or enhanced optical (or instrumentation) systems.
Such remedies are often reagent and instrumentation intensive, inciting high levels
of complexity and cost. With these considerations, CPE-based DNA sensors have
been developed to show reasonably high detection sensitivity with minimal DNA
modifications.
2.1 Assay Design
The working mechanism of cationic conjugated polymer (CCP) based DNA assay
using FRET protocol is illustrated in Scheme
3
.[
43
]. This assay comprises two
ingredients: (a) a light-harvesting CCP and (b) a probe oligonucleotide attached by
a fluorescent signaling chromophore (C*). Emission of light characteristic of the
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