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perylene dye loading of 3 mol %. A 3.3-fold increase at the main
chain emission maximum (454 nm) and a 2.3-fold increase at
the perylene emission maximum (604 nm) were observed after
incubation with trypsin (Figure 4a). These first assays proved
Figure 5. (a) Reversible swelling effect in non-cross-linked NP
suspensions of PPE-NB (0.5% dye loading) before (left) and after
(right) addition of THF. The figure shows a red emissive collapsed
NPs suspension (Dh: 200 nm) and a blue emitting swollen NP
suspension (Dh: 450 nm) under irradiation at 365 nm. (b)
Fluorescence spectra of a 3 mL aqueous NP suspension of PPE-NB
(3% dye loading) with increasing amount of THF. IAD/I is represented
in parentheses (λex: 410 nm).
Figure 4. Trypsin sensing assays. Fluorescence spectra of shell cross-
linked NP suspensions before (red) and after (blue) incubation with
trypsin for different polymers at a core dye loading of 3 mol %: (a)
PPE-NHS and (b) PPE-NHS-TEG.
the feasibility of the proposed model but improvements were
needed. The main drawback is the large numbers of linkages
that need to be cleaved by the protease to provoke a significant
luminescent response. For this reason, a modification on the
polymer design was made to lower the number of succinimidyl
ester groups (NHS block) without lowering the hydrophobic/
hydrophilic ratio of the polymer. The resulting ABCBA-
pentablock PPE-NHS-TEG copolymer has two polynorbor-
nene blocks with different functionalities: the “B” block
contains succinimidyl esters and the terminal “A” block is
functionalized with tetraethylene glycol (TEG). Because of the
spatial proximity of the reactive succinimidyl esters to the
luminescent core of the NP, this approach allows an effective
quenching of the luminescence of the NPs with a lower cross-
linking degree and therefore a faster cleaving process. The
assays performed with PPE-NHS-TEG at a core perylene dye
loading of 3 mol % under identical experimental conditions
confirmed the merits of this new design and showed a 15-fold
increase at the main chain emission maximum (454 nm) and a
12-fold increase at the perylene emission maximum (604 nm).
To confirm that the changes are the result of the cleavage of
the peptide chains, we performed control experiments wherein
the ABCBA-pentablock PPE-NHS-TEG copolymer was cross-
linked with 1,2-bis(2-aminoethoxy)ethane. In this case,
exposure to trypsin resulted in no changes in the emission
(see the Supporting Information).
The expansion of the particles upon cleaving of the
constraining cross-linking peptide chains by the protease can
be mimicked by the addition of THF to a suspension of non-
cross-linked NPs of PPE-NB. The addition of THF produces a
reversible swelling of the nanoparticles with an increase in
diameter from 200 to 450 nm and a concomitant change of
their emission properties. These changes are visually observable
upon irradiation at 365 nm (Figure 5a). Addition of other water
miscible solvents (acetone, methanol, ethanol) in which the
copolymer is insoluble did not produce a similar effect.
Furthermore, addition of THF to the shell cross-linked NPs
also produced a negligible effect on the size and emission
properties of the NPs, confirming that the cross-links constrain
the particles in a collapsed state. Figure 5b represents the
emission spectra of a 3 mL aqueous suspension of non-cross
linked NPs of PPE-NB with different amounts of THF.
Swelling of the NPs increases the intensity of both the main
chain and dye maxima. Nevertheless, the enhancement of both
maxima is different so the IAD/I ratio decreases as the NPs
swell. This can be explained by considering that swelling with a
good solvent like THF has two opposite effects on the emission
properties. It enhances the total emission intensity by solvating
the chains and reducing interpolymer π−π interactions that
produce self-quenching. However, it also reduces the energy
transfer to the perylenes by separating the polymer chains. We
have previously shown that energy transfer to low energy
emissive sites is enhanced by polymer aggregation.17 The
balance of these opposite effects determines the ratio of both
maxima and explains the observed behavior.
In summary, tunable highly emissive organic NPs have been
developed as selective nanoprobes for protease sensing. The
design takes advantage of the amplification of the fluorescence
emission of the dye by ET of the light harvested by the
backbone of the conjugated polymers and provides a double
read-out of the fluorescence emission. An exquisite tuning of
the fluorescent emission intensity of NPs was performed by
means of aggregation induced quenching by cross-linking using
a peptide as a shell cross-linking agent. Our results suggest that
the polymer cross-linking effectively strains the NP into a more
tightly aggregated and quenched state. The sensory response is
ascribed to a swelling-like (strain-release) mechanism in
response to the protease cleavage of the cross-links, which
results in a “turn-on” fluorescence response. Sensing assays
under physiological conditions provide a 15-fold increase of the
luminescence intensity after incubation with the protease.
ASSOCIATED CONTENT
* Supporting Information
■
S
Experimental details including synthetic procedures, character-
ization of all compounds. This material is available free of
AUTHOR INFORMATION
Corresponding Author
■
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We are appreciative of support from the Air Force Office of
Scientific Research (FA9550-10-1-0395). Carlos Cordovilla
recognizes a postdoctoral grant from the MICINN.
6934
dx.doi.org/10.1021/ja301259v | J. Am. Chem. Soc. 2012, 134, 6932−6935