Label-Free
Multimodal
Protein/Perylene
Dye
Protease
Detection
Co-assembly
and
Based
on
Enzyme-Triggered
Disassembly
Yiyang Lin, Robert Chapman, and Molly M. Stevens*
Department of Materials, Department of Bioengineering and Institute for Biomedical
Engineering, Imperial College London, Exhibition Road, London SW7 2AZ, UK.
E-mail: m.stevens@imperial.ac.uk
Tel: +44 (0)207 594 6804
Fax: +44 (0)207 594 6757;
1
ABSTRACT
The development of novel assays for protease sensing plays an important role in
clinical diagnostics and therapeutics. Herein, we report a supramolecular platform for
label-free
protease
detection,
based
on
protein/dye
self-assembly
and
enzyme-triggered disassembly. In a typical case, co-assembly of protamine sulfate and
perylene dye via electrostatic attractions and π-π interactions caused significant
colorimetric and fluorescent responses. Subsequent addition of trypsin was found to
cleave the amide bonds of protein, triggering the dissociation of protein/dye
aggregates and the release of perylene dyes. The enzyme-triggered disassembly was
transduced into multiple readouts including absorption, fluorescence, and polarization,
which were exploited for trypsin detection and inhibitor testing. This assay was also
used for turn-on fluorescence detection of Cathepsin B, an enzyme known to be
over-expressed in mammalian cancer cells. The integration of supramolecular
self-assembly into enzyme detection in this work has provided a novel label-free
biosensing platform which is highly sensitive with multimodal readouts. The relative
simplicity of the approach avoids the need for time-consuming substrate synthesis,
and is also amenable to naked eye detection.
Keywords: self-assembly, biosensor, protease, protein, perylene bisimide.
2
INTRODUCTION
Proteolytic enzymes or proteases play central roles in most biological processes
owing to their ability to regulate the physiological functions of many proteins through
initiating hydrolysis at the post-translational level. Aberrant activity of proteases is
also central to major human diseases such as cardiovascular, oncologic,
neurodegenerative, and inflammatory diseases.1-3 The screening of protease-targeted
inhibitors is thus also of high clinically relevance to identify new therapeutic
approaches for these diseases. For example, protease inhibitors against matrix
metalloproteinases (MMPs) have been proposed to control tumor growth.4
Over the past decade, proteases have mostly been detected using immunoassays
which involve antibody or affinity based detection5, have been mostly used to detect
proteases, owing to the high sensitivity and specificity of this approach. However, the
immunoassay
format
always
requires
a
process
of
antibody
identification/generation/isolation, as well as potentially complicated bioconjugation
for antibody immobilization and fluorophore/chromophore labeling. Besides, readout
of direct enzyme activity cannot be achieved from the immunoassay. Another strategy
for protease sensing is based on hydrolysis of the peptide substrate by the target
protease. This approach can provide information about enzyme activity and is used in
diagnostics and drug discovery.6-8 Assaying proteolytic activity can be diversified by
combining short peptide substrates with different detection principles (e.g.
fluorescence resonance energy transfer, FRET) and signal-enhancing reporters (e.g.,
fluorophores and chromophores).9-12 However, this method always requires organic
3
and bioconjugation chemistry to synthesize specific peptide substrates and attach
molecular probes to the substrates. Therefore, the development of novel label-free
approaches for sensitive protease detection is of great interest.
To this end, we have designed a versatile biosensing platform for protease activity
detection based on the non-covalent co-assembly of protein and perylene bisimide.
Protein self-assembly is of great fundamental interest to understand the
physicochemical basis of protein-protein interactions and can be used to design new
to novel biomaterials for disease diagnostics and therapy.13 However, the concept of
integrating organic dyes into protein self-assembly has been rarely reported. On the
other hand, perylene bisimide (PBI) derivatives have been extensively used as
industrial pigments owing to photo/thermal stability, high fluorescence quantum yield,
and inert chemistry.14 More recently, research into PBI derivatives has emerged in
various optical and electronic applications including organic photovoltaic devices,
field-effect transistors, and n-type semiconductors.14,15 Owing to the hydrophobic
effect and π-π interactions, PBI derivatives are mostly water-insoluble and have a
strong tendency to aggregate,16 which results in fluorescence quenching in aqueous
solution. In the past, this has greatly restricted their applications in biosensing and
bioimaging.17-21
Here, a highly fluorescent water-soluble PBI dye was synthesized by attaching
multiple charge groups to perylene bisimide.22 Driven by electrostatic attractions and
π-π interactions, the protein/PBI dye system self-assembled into soft nanoparticles,
which led to a series of optical responses including aggregation-induced fluorescence
4
quenching, polarization, and decrease in UV-vis absorption. The presence of protease
hydrolyzed protein and dissociated the protein/dye self-assembly, which consequently
restored PBI absorption/fluorescence emission and decreased its fluorescence
polarization. In particular, we have used protamine and polylysine as enzyme
substrates to fabricate protease sensors for trypsin and Cathepsin B, respectively. In
comparison with common protease detection methods, our assay utilizes natural
proteins or peptides as substrates which avoids time-consuming substrate synthesis. In
addition, the optical probe is integrated into our assay platform through
supramolecular self-assembly or non-covalent interactions without the need for
complicated fluorophore labeling. The combination of the dynamic nature of
non-covalent self-assembly with the possibility to incorporate a variety of molecules
that can trigger the optical responses in the protein holds great promise for
applications as stimuli-responsive supramolecular systems (e.g., nanocarriers) and
biosensors.
Experimental Section
Materials. Protamine sulfate from salmon, poly-L-lysine hydrobromide, trypsin from
bovine pancreas (TPCK-treated, essentially salt-free, lyophilized powder, ≧10,000
BAEE units/mg protein) and Cathepsin B were purchased from Sigma–Aldrich (UK).
GRP peptide was provided by GenScript (US) and used without further purification.
Water was purified using a Millipore filtration system.
Synthesis
of
water-soluble
PBI
derivative.
5
Aspartic
acid-functionalized
water-soluble perylene bisimide (PBI-Asp) was synthesized according to the
literature.22 In detail, 3,4,9,10-Perylenetetracarboxylicacid bisanhydride (196 mg, 0.5
mmol), aspartic acid (146 mg, 1.1 mmol), zinc acetate (1.83 mg, 0.05 mmol), and 4.0
g of imidazole were heated at 120 °C for 12 h with stirring under argon atmosphere.
The reaction mixture was cooled to 90 °C, and then poured into water in the presence
of argon. The mixture was filtered and the filtrate was acidified to pH 2-3 with
hydrogen chloride solution. The precipitate was filtered, washed with water and dried
under vacuum at 80 °C to give the product.1H NMR (400 MHz, d6-DMSO,) δ: 12.85
(-COOH, 2H), 7.91 (-CH-, 4H), 7.75 (-CH-, 4H), 5.99 (-CH-, 2H), 3.38 (-CH2-, 2H),
2.90 (-CH2-, 2H). FTIR v/cm−1: 3525, 2939, 1750, 1695, 1635, 1590, 1571, 1508,
1435, 1401, 1362, 1341, 1302, 1255, 1172, 1132, 992, 960, 854, 809, and 745.
Sample characterization. TEM micrographs were obtained with a JEOL 2000FX
(working voltage of 200 kV) by negative-staining method with uranyl acetate solution
(1.0 wt%) as the staining agent. One drop of the solution was placed onto a carbon
Formvar-coated copper grid for 3~5 min. The excess liquid was sucked away with
filter paper. After this, one drop of the staining agent was placed onto the copper grid
for 2~5 min. After removing the excess staining agent with filter paper, the sample
was dried in air before TEM observation. Fluorescence spectra were recorded on
Fluorolog®-3 spectrofluorometer. UV-vis absorbance was measured on a Perkin
Elmer Lambda 25 UV-vis spectrometer with the path length of 1.0 cm. Fluorescence
polarization was measured on SpectraMax M5 plate reader and was experimentally
6
calculated from the measurement of fluorescence intensity parallel to the plane of
linearly polarized excitation light (I∥ ), and that perpendicular to the excitation plane
I −(G×I )
(I⊥ ), which was expressed as:𝑃 = I∥ +(G×I⊥), wherein G was an instrumental factor.
∥
⊥
Dynamic light scattering (DLS) and ζ-potential were measured on a Malvern
Zetasizer Nano ZS (Malvern, UK) with a backscattering detection at 173 o, equipped
with a He-Ne laser (λ=632.8 nm).
Trypsin activity assay. Trypsin stock solutions were prepared in 1.0 mM hydrogen
chloride solution and stored at -20 oC. The trypsin activity test was conducted in
384-well plates and monitored by plate reader. In detail, 10 μM PBI-Asp and 15
μg/mL protamine sulfate were dissolved in 5 mM phosphate buffer (pH 8.5) and
transferred to 384-well plates. After that, 5 μL of trypsin stock solution were added to
reach required enzyme concentrations. The kinetics of enzymatic hydrolysis were
monitored by fluorescence intensity (λex=490 nm, λem=550 nm) using plate reader at
room temperature. After enzymatic digestion for 3 hours, the UV-vis absorbance,
fluorescence spectra, and polarization were recorded.
Trypsin inhibitor test. The trypsin inhibitor, benzamidine hydrochloride, with
varying concentrations was pre-incubated with trypsin at 25 oC for 15 min, and then
added into solutions of protamine/PBI-Asp mixture in phosphate buffer (5.0 mM, pH
8.5). The final concentration of PBI-Asp, protamine sulfate, and trypsin was 10 μM,
15 μg/mL, and 10 nM, respectively. The resulting solutions were incubated at room
temperature and the fluorescence intensity (λex=490 nm, λem=550 nm) was recorded
with time.
7
Cathepsin B assay. The stock solution of Cathepsin B (10 U/mL) was added to
activation buffer with a final concentration of 20 mM DTT, 10 mM EDTA and
incubated at 37 °C for 15 min. To the activated enzyme solution, reaction buffer (25
mM sodium acetate, 1 mM EDTA, pH 5.0, pre-warmed at 37 °C) and
polylysine/PBI-Asp solution (1.0 μg/mL and 1.0 μM) were added. The reaction
solution was incubated at 37 °C and the fluorescence emission from PBI-Asp
(λex=490 nm, λem=550 nm) was recorded.
RESULTS AND DISCUSSION
1. Supramolecular self-assembly of protein/perylene dye
A water soluble perylene-derivative named PBI-Asp (Fig. 1a) was synthesized
through condensation reaction of 3,4,9,10-perylenetetracarboxylicacid bisanhydride
and L-aspartic acid. The attachment of aspartic acid to perylene introduced charge
repulsion between multiple carboxyl groups and endowed a high solubility in aqueous
conditions. Three well-resolved absorption peaks (534, 495, and 466 nm) and a weak
broad shoulder around 415 nm were observed in the UV-vis spectrum of PBI-Asp (Fig.
1b), which were ascribed to characteristic S0→S1 transitions with different vibronic
structures. The ratio of 0−0 to 0−1 transition in absorption intensity was calculated to
be 1.56, being close to the normal Frank-Condom progressions (A0−0/A0−1≈1.6) for
non-aggregated PBI.23 This confirmed the existence of free dye monomer in solution.
Meanwhile, the monomeric PBI-Asp displayed a high fluorescence quantum yield
(~50%, Supporting Information) with two emission peaks at 548 and 590 nm (Fig.
8
1c).
Figure 1. (a) Molecular structure of PBI-Asp; (b) UV-vis and (c) fluorescence spectra
of 1.0 μM PBI-Asp solution in 5.0 mM phosphate buffer (pH 8.5). The fluorescence
excitation wavelength was 490 nm.
Protamine sulfate is a small nuclear protein with an arginine-rich sequence and
has the ability to condense plasmid DNA to increase gene therapy transduction rates
by both viral and nonviral mediated delivery mechanisms.24 Herein, protamine was
chosen as a protease substrate because it is trypsin-degradable. Strong interactions
between PBI-Asp and protamine sulfate were demonstrated by multiple techniques.
As shown in Fig. 2a, the absorption intensity of PBI-Asp decreased with the addition
of protamine sulfate, suggesting the protein-induced aggregation of PBI-Asp. When
the protamine concentration reached 10 μg/mL, the absorption peaks at 534 and 495
nm disappeared completely and a new shoulder attributed to the extended oligomers
was observed at 562 nm. The isosbestic point at 552 nm indicated that two species
9
exist in equilibrium between monomers and oligomers. The spectral variations were
accompanied by notable changes in the appearances of the solution (Inset in Fig. 2a).
Simultaneously, when adding protamine sulfate, the fluorescence emission
decreased gradually due to the formation of non-emitting aggregates with forbidden
low-energy excitonic transitions (Fig. 2b).25 The fluorescence emission was largely
quenched (~99.8%) by 15 μg/mL of protamine sulfate (Fig. 2c), which was noticeable
with a conventional UV lamp (Inset in Fig. 2b). Interestingly, further addition of
protamine caused the fluorescence to increase (Inset in Fig. 2c), indicating a
weakening in the aggregation of PBI-Asp. This phenomenon is unexpected and will
be discussed in the next section.
Figure 2. (a) UV-vis and (b) fluorescence spectra of protamine/PBI-Asp. The
concentration of PBI-Asp is 10 μM while the protamine concentration is (from top to
10
bottom): 0, 2.0, 4.0, 6.0, 8.0, and 10 μg/mL. The inset in Fig. 2a shows PBI-Asp
solution without (left) and with (right) protamine; the inset in Fig. 2b shows the same
solutions upon UV irradiation (365 nm). (c) Fluorescence intensity of PBI-Asp
solution (10 μM) (λex= 490 nm, λem = 550 nm) with different amounts of protamine
sulfate. Inset: Intensity profile of PBI-Asp fluorescence when protamine concentration
varies from 15 to 120 μg/mL. (d) Fluorescence polarization of protamine/PBI-Asp
mixtures, indicating the variations of perylene rotation diffusion rate: φ1<φ3<φ2.
Fluorescence polarization was further employed to provide information into
protein/dye self-assembly at the molecular level, especially with regards to perylene
rotation. In principle, when a dye is excited with polarized light, it will emit
fluorescence with the same polarization assuming the dye doesn’t rotate during the
lifetime of the excited state.26 Depolarization occurs when the dye rotates during its
emission lifetime. The faster it rotates, the smaller the fluorescence polarization will
be. As shown in Fig. 2d, the addition of protamine (0~15 μg/mL) greatly enhanced the
fluorescence polarization. This suggests that the rotation diffusion of PBI-Asp was
restricted in the presence of protamine (φ1>φ2, Fig. 2d), owing to the protein-assisted
perylene aggregation. Further addition of protamine (15~200 μg/mL) unexpectedly
decreased the fluorescence polarization, implying a faster PBI-Asp rotation (φ3>φ2,
Fig. 2d). This phenomenon coincided with the fluorescence results in Fig. 2c, in
which a maximum fluorescence quenching effect was observed with 15 μg/mL
protamine.
11
We hypothesized that the optical responses (i.e., absorption decrease, emission
quenching, and fluorescence polarization) originated from protein-induced dye
self-assembly, which were studied by ζ-potential, dynamic light scattering (DLS), and
transmission electron microscopy (TEM) (Fig. 3). It is known that arginine and
aspartic acid can form a salt bridge through guanidinium–carboxylate interaction
which is a key stabilizing structural element in natural systems including RNA stem
loops25 and zinc finger/DNA complexes26. In this case, the guanidinium–carboxylate
interaction between protamine and PBI-Asp became the main driving force for
protein/dye self-assembly (Fig. 4). As shown in Fig. 3a, the ζ-potential decreased
from negative (-14 mV) to neutral as the protamine concentration increased,
indicating the incorporation of cationic protamine into the protamine/PBI-Asp
complex. Zero ζ-potential was achieved with ~10 μg/mL of protamine, consistent with
the results of UV-vis absorbance, fluorescence, and polarization (Fig. 2). These results
suggest the important role of guanidinium–carboxylate charge interaction in
protamine/PBI-Asp self-assembly. The formation of 100-200 nm nanoparticles in
protamine/PBI-Asp solution was demonstrated by TEM and DLS (Fig. 3b, 3c, and
S1). Owing to the protein-assisted aggregation, PBI-Asp was caged (Fig. 4) and its
absorption/fluorescence was strongly quenched owing to π-π stacking (Fig. 2a and 2b).
Meanwhile, molecular aggregation prohibited the rotational diffusion of PBI-Asp and
hence intensified its fluorescence polarization (Fig. 2d). When the protein
concentration exceeded 10 μg/mL, the ζ-potential was reverted to positive (Fig. 3a),
suggesting the incorporation of excess protamine into the protein/dye complex. The
12
increased positive charges on protamine/PBI-Asp particles led to a loose molecular
packing (Fig. 4), which restored the absorption and fluorescence of PBI-Asp (Fig. 2c
and 2d). Owing to the less dense packing of PBI-Asp inside protamine/PBI-Asp
particles, the diffusion rotation of PBI-Asp was enhanced and the fluorescence
polarization decreased when the protamine concentration exceeded 10 μg/mL (Fig. 2c
and 2d).
Figure 3. (a) ζ-potential of protamine/PBI-Asp with varied protamine concentrations,
in which the PBI-Asp concentration is 10 μM. (b) DLS and (c) TEM image of
protamine/PBI-Asp with 10 μM of PBI-Asp and 15 μg/mL of protamine sulfate.
To further illustrate the factors that contribute to protein/dye self-assembly, we
investigated the co-assembly behavior of PBI-Asp and a protamine-analogous peptide.
To
this
end,
a
short
arginine-rich
peptide
with
the
sequence
of
Gly-Arg-Pro-Gly-Arg-Pro-Gly-Arg-Pro (GRP) was synthesized. It is interesting that
no significant fluorescence quenching or UV-vis absorption decrease was observed
when adding GRP peptide to PBI-Asp solutions (Fig. S2). Although GRP is highly
positive and will electrostatically interact with peryelene dyes, the short peptide chain
makes it less efficient at assembling perylene dyes. This indicates the
13
polymer-analogue structure of the protein is indispensible in promoting the self
assembly of protamine/PBI-Asp (Fig. 4).
Figure 4. Schematic illustration of guanidinium–carboxylate electrostatic interaction,
protamine/PBI-Asp self-assembly into positive and negative nanoparticles, and
GRP/PBI-Asp interactions.
2. Trypsin detection based on protein/dye disassembly
As discussed above, the polymer-analogous structure of protamine plays a crucial role
in protamine/PBI-Asp self-assembly, while small peptides are less efficient at
promoting the formation of perylene dye oligomers. It is therefore expected that if
protamine is enzymatically cleaved into short peptides, the self-assembled
protamine/PBI-Asp nanoparticles will disaggregate and the caged PBI-Asp be
released. Based on this hypothesis, protamine/PBI-Asp self-assembled complexes are
expected to be applicable for the detection of protamine-specific proteases.
14
Figure 5. (a) Real-time fluorescence enhancement of protamine/PBI-Asp in the
presence of 0-50 nM trypsin; (b) fluorescence spectra after 3 h enzyme digestion. The
protein/dye concentration in (a, b) is 15 μg/mL protamine and 10 μM PBI-Asp for
0-10 nM trypsin. (c) Emission intensity of protamine/PBI-Asp after incubation with
different amounts of trypsin for 3 h (λex=490 nm, λem=550 nm). The protein/dye
concentrations in Fig. 3c are: (A) 15 μg/mL protamine and 10 μM PBI-Asp, (B) 2.0
μg/mL protamine and 1.0 μM PBI-Asp. (d-g) Appearance of protamine/PBI-Asp
mixtures before (d, f) and after (e, g) trypsin digestion.
Protamine is an ideal trypsin substrate owing to its arginine-rich structure. As the
most important digestive enzyme, trypsin is secreted by the pancreas and plays a key
role in controlling pancreatic exocrine function. It is involved in the digestive enzyme
activation cascade, which induces the transformation of other pancreatic proenzymes
15
into their active forms. Changes in trypsin levels are also closely related to the
presence of some pancreatic diseases.27,28 Here, the real-time detection of trypsin
activity was realized by tracking the fluorescence recovery of protamine/PBI-Asp
solution in the presence of trypsin (λex =490 nm, λem =550 nm, Fig. 5a). A gradual
fluorescence increase was observed dependent on the trypsin concentration, with
higher enzyme concentrations generating faster fluorescence increments. The
fluorescence spectra after 3 h incubation with trypsin were also recorded to evaluate
the enzyme activity (Fig. 5b). The limit of detection (LOD) defined as the lowest
assayed concentration of analyte that yields a signal higher than three times the
standard deviation of the background was determined to be 0.051 nM trypsin which
gave a notable fluorescence rise (Fig. 5b and 5c). Importantly, the enzyme activity
was detectable directly with the naked eye, both under sunlight or a UV lamp, which
is very useful for point-of-care enzyme detection.
Figure 6. (a) UV-vis spectra and (b) fluorescence polarization of protamine/PBI-Asp
solution after incubation with trypsin for 3 h. The results of fluorescence polarization
indicates the perylene rotation diffusion φ1<φ2. The PBI-Asp concentration is 10 μM
and the protamine concentration is 15 μg/mL.
16
The enzyme activity could also be measured by UV-vis absorbance and
fluorescence polarization. As shown in Fig. 6a, the absorption peaks at 495 nm and
534 nm that were suppressed by protamine sulfate were restored after enzyme
incubation. A noticeable increase in the UV-vis absorption was observed with a
minimal trypsin concentration of 0.2 nM. The trypsin activity could be also detected
by fluorescence polarization. As shown in Fig. 6b, trypsin-catalyzed protein digestion
caused fluorescence depolarization, which resulted from the enhanced rotational
diffusion of PBI-Asp. Similarly, the value of fluorescence polarization was found to
plateau with 0.2 nM trypsin. To exclude the contribution of non-specific interactions
between trypsin and protamine/PBI-Asp to fluorescence enhancement, control
experiments were conducted by replacing trypsin with different proteins (Fig. S3). No
significant fluorescence increase was noted in the protamine/PBI-Asp system after
incubating with streptavidin, histone H1, lysozyme, cytochrome c, bovine serum
albumin, and human serum albumin. In addition, trypsin cleavage-induced
disassembly of protamine/PBI-Asp complexes was demonstrated by TEM, wherein no
self-assembled particles were observed after trypsin digestion. The drop of light
scattering intensity from 162 to 2.80 kcps after trypsin incubation confirmed the
dissociation of large particles. The -potential of protamine/PBI-Asp solution after
enzyme treatment was found to be -3.64 mV, also indicating the disassembly of
protamine/PBI-Asp particles.
The detection limit of trypsin by this assay can be further lowered to 0.006 nM
17
when the protamine/PBI-Asp concentration is reduced (Fig. 5c and S4). This
detection limit is much lower than that of commercial kits using FITC-Casein as a
substrate (~0.5 μg/mL). Commercial protease-sensing protocols are based on a
dye-labeled peptide or protein substrate, which means one amide bond cleavage
corresponds to one chromophore or fluorophore release. In our assay, the absorption
and fluorescence signal is quenched by the non-covalent self-assembly of
protein/perylene dye. This means that in principle the enzymatic digestion of an
amide bond is possible to uncage several perylene dyes, which is transduced through
multimodal signals. In addition, the high fluorescence quantum yield, large extinction,
and strong aggregation-induced quenching of the perylene dye also contribute to the
high sensitivity of our assay. Another advantage of this assay is its tunable dynamic
detection range. For example, the dynamic detection range shifted from 0.05-10 nM
to 0.005-2.0 nM when the PBI-Asp concentration is lowered from 10 μM to 1.0 μM
(Fig. 5c).
The development of rapid and simple methods for screening chemical libraries of
potential protease inhibitors is important in the pharmaceutical industry. For this
reason the developed protamine/PBI-Asp self-assembly system was also used to study
the inhibition of trypsin activity by inhibitors. In Fig. S6a, the degree of fluorescence
enhancement for the ensemble of protamine sulfate and PBI-Asp containing trypsin
was retarded by the trypsin inhibitor benzamidine hydrochloride. Based on the plot of
the inhibition efficiency vs. inhibitor concentration (Fig. S5), the IC50 value of
benzamidine hydrochloride toward trypsin was estimated to be 5.0 μM (Fig. S6b).
18
3. Label-free detection of Cathepsin B
Considering the universality of non-covalent self-assembly in protein/perylene dye
systems, we hypothesized that this strategy could be extended to detect different
proteases. Of particular interest are disease-related proteases, such as HIV or cancer
relevant enzymes. To this end, Cathepsin B (CTSB), which is frequently
over-expressed in premalignant lesions, was chosen as a target protease. Increased
expression of Cathepsin B in primary cancers, especially in preneoplastic lesions,
suggests that this enzyme might have pro-apoptotic features. For the sensing of
Cathepsin B, we replaced protamine with polylysine, a highly positive peptide that
has been demonstrated to be a CTSB substrate.29,30 Owing to the strong charge
interactions, electrostatic self-assembly of polylysine/PBI-Asp was expected. Indeed,
upon the addition of polylysine to PBI-Asp, self assembly was observed by UV-vis
absorbance and fluorescence. As before, the absorption peaks at 495 nm and 533 nm
gradually decreased, finally disappearing at 2.0 μg/mL of polylysine (Fig. 7a).
Significant fluorescence quenching (~98%) was observed in the presence of
polylysine (Fig. 7b). It was expected that enzymatic cleavage of polylysine amide
bonds would disrupt the polymer–analogue structure and reduce the positive charges
(via the formation of carboxyl groups), and would consequently trigger the
disassembly of polylysine/PBI-Asp (Fig. 7c). In our experiment, the enzyme-triggered
fluorescence recovery in polylysine/PBI-Asp was recorded after 6 h incubation with
CTSB and notable emission increases were observed at enzyme concentrations of 20
19
mU/mL (Fig. 7d). Unlike previous work, in which fluorophores modified polylysines
have been used for imaging CTSB, here we provide a simple and effective approach
to design a CTSB sensor via a non-covalent strategy without dye labeling.
Figure 7. (a) UV-vis absorbance and (b) fluorescence spectra of polylysine/PBI-Asp
solution (pH 5.0) for polylysine concentrations from 0-2 μg/mL. The inset in Fig. 7b
shows the fluorescence intensity (λex =490 nm, λem =550 nm) of PBI-Asp with
different amount of polylysine. (c) Cleavage of polylysine amide bond by CTSB. (d)
Fluorescence spectra of polylysine/PBI-Asp mixtures after incubating with Cathepsin
B for 6 h at 37 oC for CTSB concentrations of 1.0, 5.0, 10, 20, 50, and 200 mU/mL,
respectively.
The
inset
in
Fig.
7d
gives
the
fluorescence
intensity of
polylysine/PBI-Asp for PBI-Asp concentration of 1.0 μM and polylysine
concentration of 1.0 μg/mL after incubation with CTSB (λex =490 nm, λem =550 nm).
20
CONCLUSIONS
In this work, we have designed a versatile biosensing platform for label-free protease
detection through the non-covalent self-assembly of proteins/perylene dye. The
driving forces of protein/dye self-assembly were ascribed to electrostatic attractions
and π-π interactions, which led to the formation of activatable soft nanoparticles and
significant colorimetric and fluorometric responses. The presence of protease
specifically hydrolyzed the protein, resulting in the disassembly of supramolecular
nanoparticles as detectable by multiple optical readouts (i.e., UV-vis absorbance,
fluorescence, and depolarization). This assay was exploited for sensitively detecting
trypsin and Cathepsin B by using protamine and polylysine respectively as substrates.
Because of its inherent modularity and dynamic nature, the developed non-covalent
biosensing platform is highly sensitive, and can be further explored for the detection
of other proteases. We anticipate that the presented concept of integrating natural
proteins or polypeptides with synthetic dyes via supramolecular self-assembly will
open up a new avenue towards multifunctional biomaterials for smart nanocarriers
and biosensors.
AUTHOR INFORMATION
Corresponding Author
*E-mail: m.stevens@imperial.ac.uk. Phone: +44 (0)20 7584 6804.
Notes
21
The authors declare no competing financial interest
ACKNOWLEDGMENTS
M.M.S. thanks the Engineering and Physical Sciences Research Council (EPSRC)
grant EP/K020641/1. Thanks to Dr. Roberto de la Rica for help in preparing this
manuscript.
ASSOCIATED CONTENT
Supporting Information
Measurement of fluorescence quantum yield, TEM image, effects of different proteins
to protamine/PBI-Asp fluorescence, comparison of GRP and protamine, and trypsin
inhibitor test. This material is available free of charge via the Internet at
http://pubs.acs.org.
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