Although previous studies on conjugated polymers demonstrated amplified quenching to allow trace detection of analytes, the systems are limited because the polymers only dissolve in organic solvents. A sensor would be more useful if it operates in an aqueous environment. Recently, water-soluble conjugated polymers (WSCPs) show potential for use as a new class of high-sensitivity rapid-response chemical and biological sensors.62 Through electrostatic attraction, WSCPs could attach quenchers with oppositely charge more closely and their fluorescence can be quenched by those very small amounts of charged molecules (quenchers) that quench the excited state by energy transfer or electron transfer. In 1999, L. H. Cheng first reported the amplification of quenching sensitivity of MPS-PPV (PPV-SO32-) to methyl viologen (MV2+), in which Ksv = 1.2 × 107 M-1.62
This quenching can be adapted to biosensing by coupling a quencher to a biological ligand. In aqueous solution, the photoluminescence (PL) from the polymer is quenched when the quencher–ligand conjugate associates with the polyelectrolyte to form a relatively weak conjugate–polymer complex, as a consequence of electrostatic and hydrophobic interactions. Exposure of the conjugate–polymer complex to a biological receptor results in formation of a biospecific receptor–conjugate complex and release of the polymer with concomitant unquenching of the polymer fluorescence.62 (Figure
1.2.3) In 2002, biosensing of the anti-DNP lgG with a charge neutral complex (CNC) formed in aqueous solution by combining PPV-SO3- and a saturated cationic polyelectrolyte at a 1:1 ratio (per repeat unit) was demonstrated by D. L. Wang et al.63 This modified mechanism could efficiently minimize nonospecific interactions between conjugated polyelectrolytes and biopolymers.
Figure 1.2.3 Diagram illustrating the detection mechanism of conjugated polyelectrolyte for biomolecules
Soon after that report, the light-harvesting properties of cationic conjugated polymers (PF) are used to sensitize the emission of a dye on a specific peptide nucleic acid (PNA) sequence for the purpose of homogeneous, real-time DNA detection by G. C. Bazan et al and detection of target DNA at concentrations of 10 pM was realized successfully.64 Figure 1.2.4 showed the mechanism of such a detection method. This method made it possible to take advantage of the optical amplification of WSCPs to detect DNA hybridization to a singly labeled PNA strand.
conjugated polymer
+ +
+ + + + + + + +
+ + + + + +
ligand quencher
fluorescence quenching
target biomolecule
fluorescence revovery
_ _
_ _ + +
+ + + + + + _ _
Figure 1.2.4 Diagrammatic representation for the use of a water-soluble CP with a specific PNA-C* optical reporter probe to detect a complementary ssDNA sequence.
Based on their potential applications as biosensors in the future, the fluorescence quenching of WSCPs were significantly investigated. Under preliminary studies, it was shown that the amplified sensitivity of WSCPs was highly related to its conjugated length,65 charge of quenching,66 ionic strength,67 substrate,68 complex of WSCPs with oppositely charged surfactant69,70 or polyelectrolytes68,71 and aggregation statutes.72 Studies of PPV-SO3- showed that the higher conjugated length of WSCPs and the greater the opposite charge on the quencher, the more amplified quenching will be obtained.65 By layering fluorescent polyelectrolytes onto oppositely charged surfaces, one could tune superquenching effects, for example, when PPV-SO - was deposited on
the surface of polystyrene microsphere, it can be used to detect quencher with the same charge instead of that with opposite charge.68
The complex, which was formed through combining PPV-SO3- with cationic surfactant, showed higher sensitivity to neutral quenchers (such as trinitrotoluene) while swiftly decreased sensitivity to oppositely charged quenchers.69 More recently, energy transfer between oppositely charges polyelectrolytes has been used to obtain fluorescence superquenching.68 These mixtures offer possibilities of “charge reversal”, where it is also possible to encourage interactions between a given polyelectrolyte and a fluorescence quencher with similar charge.
PPV-SO3- (x = 3, 4) PPE-SO3- OCH2CH2N+(CH2CH3)3
OCH2CH2N+(CH2CH3)3
( )n
PPP-NEt3+
O(CH2)3SO3-
SO3-(CH2)3O
( )n
O(CH2)xSO3-
MeO
( )n
Me3N+(CH2)6 (CH2)6N+Me3
( )n
PF-NMe3+
Figure 1.2.5 Molecular structure of WSCPs used as chemo or biosensors
Although a lot of information related to the quenching behavior of WSCPs has been collected by scientists recently, the relationship between quenching behavior and physical and chemical properties of conjugated polymers is still a major concern.
Meanwhile, most of the above researches were focused on anionic WSCPs, especially on PPV-SO -.65-71 Figure 1.2.5 showed the molecular structures of WSCPs which have
been used to study the quenching behavior.64-70, 72,73 For cationic WSCPs, only M. F.
Rubner reported fluorescent quenching of cationic PPP-NEt3+ by several anionic quencher, such as Ru(phen’)34- and Fe(CN)64- in aqueous solution73 and G.. C. Bazan proposed to detect DNA with cationic conjugated polymer PF-NEt3+.71 Thus, preparation of novel cationic WSCPs and detectation of their quenching behavior are more attractive to develop good biosensors with high sensitivity.