Fluorescence Reporting Based on FRET Between Conjugated Polyelectrolyte and Organic Dye for Biosensor Applications - Advanced Fluorescence Reporters in Chemistry and Biology - page 437

mixture as compared to that of buffer [ 81 ]; dilution of dye-labeled probe using
unlabeled ssDNA or cationic surfactant can relax the compaction of the complex
structures, leading to minimized acceptor self-quenching [ 82 ]; addition of nonionic
surfactant increases the quantum yields of donors and reduces donor self-quenching
upon complexation with DNA as a result of the incorporation of CCP molecules
into the surfactant micelles [ 83 , 84 ]; and utilization of silica nanoparticles (NPs) as
the sensing venue to immobilize DNA can further reduce acceptor quenching and
also allow excess DNA probes on the NP surface to capture CCPs, leading to
increased local concentration of donor units [ 85 , 86 ].
3 Protein Biosensor
Since many diseases do not have a specific genetic signature, but rather have a
variation in protein expression, biosensors for protein detection are of particular
significance in medical diagnostics and pathogen recognition [ 87 ]. However, pro-
teins are much more complex and sensitive than oligonucleotides, which merit
additional severe considerations in the course of assay design [ 88 ]. Immunoassays
are conventional methods based on specific antibody-antigen recognition for pro-
tein detection. Enzyme immunosorbent assay (ELISA) is the most widely used
immunoassay in clinics, which requires antibodies to be immobilized on the
substrate to capture antigens and the secondary antibodies [ 89 ]. The enzymes
attached to the secondary antibodies serve as the catalyst to generate detection
signals. Despite its high sensitivity, ELISA demands tedious protein modification,
surface immobilization, blocking and washing and is limited by the availability of
commercial antibodies. Thus, there is an ever-growing and urgent demand for
developing new protein detection strategies that are simple and economical, with
high selectivity and sensitivity.
CPEs have been explored for protein detection based on nonspecific interaction
induced perturbation in their photophysical properties. Fluorescence quenching of
CPEs in the presence of proteins via electron transfer or aggregation mechanisms
allowed protein discrimination according to the pattern of Stern-Volmer quenching
constants [ 90 - 92 ], and a protein sensor array has been built with six water-soluble
poly( p -phenylene ethynylene)s (PPEs) by Bunz's group [ 93 ]. These works provide
important fundamental information about how proteins interact with CPEs and in
turn affect the polymer fluorescence, which form a reference basis to be consulted
during the design of CPE-based protein sensor involving FRET protocols.
3.1 Antibody-Antigen Based Sensor
FRET-based protein biosensors have been developed using CPEs as the light-
harvesting donors in conjugation of “lock-key” recognition. Streptavidin is a