A supramolecular dissociation strategy for protein sensing

Article abstract of DOI:10.1039/c5cc07408h

We report a simple, robust, and general strategy for protein detection based on supramolecular dissociation. The simplicity of the design is exemplified by the fact that the host assemblies can be widely varied and that these assemblies can be achieved from commercially available surfactants. An operating mechanism that is consistent with all the data has been proposed.

Full text of DOI:10.1039/c5cc07408h

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DOI: 10.1039/C5CC07408H
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A Supramolecular Dissociation Strategy for Protein Sensing
H. Wang,a J. Zhuang,a K. R. Raghupathia and S. Thayumanavan*a
Received 00th January 20xx,
Accepted 00th January 20xx
DOI: 10.1039/x0xx00000x
www.rsc.org/
We report a simple, robust, and general strategy for protein
detection based on supramolecular dissociation. The simplicity of
the design is exemplified by the fact that the host assemblies can
be widely varied and that these assemblies can be achieved from
commercially available surfactants. An operating mechanism that
is consistent with all the data has been proposed.
Development of new biosensors for recognizing biological
analytes and reporting their presence is important due to the
implications in proteomics, medical diagnostics, and pathogen
detection. Approaches to developing new sensors can be broadly
classified into two categories, both of which are inspired by nature:
(i) array-based sensing, where several less-specific receptors are
used to develop a response pattern for each analyte;1 and (ii)
sensing based on ‘lock-and-key’ design, where specific receptors are
needed to selectively bind the analytes of interest.2 The former
method has the advantage of being simple as the receptor design is
less intense and provides convenient opportunities to transduce the
analyte recognition. In contrast, the latter approach has the
promise of being specific to the target analyte even when the
analyte mixture becomes complex. An approach that captures the
simplicity of the former and the specificity of the latter would
certainly be desirable for ultimately implementing these strategies
in practical systems. Here, we disclose a simple supramolecular
dissociation approach to protein sensing based on specific ligand-
analyte interactions.
Figure 1. Schematic illustration of the supramolecular dissociation based protein
sensor. The difference in the equilibrium concentrations (between the interior of the
micelle and the bulk aqueous phase) of the pristine fluorescent probe and the probe-
protein complex affords a simple, turn-on fluorescent sensor.
absence of the protein, but disassemble in its presence. We
envisaged a simple supramolecular dissociation strategy that
obviates this rigorous design requirement (Figure 1). Briefly, in this
approach, a probe is generated by simply tethering the protein-
specific ligand moiety to a hydrophobic fluorophore, which is non-
covalently incorporated into a micelle along with a corresponding
hydrophobic fluorescence quencher. The micelle itself is chosen
such that it is known to exhibit good guest exchange dynamics with
the bulk solvent, i.e. the aqueous phase.5 Here, since the probe
molecule is hydrophobic, its thermodynamic distribution coefficient
will dictate that most of it is present inside the micelle, where the
fluorescence is quenched due to its co-confinement with the
quencher. We hypothesized that in the presence of the analyte
protein, the binding event between the ligand moiety in the probe
and the protein should cause the complex to decidedly favor the
A sensor system requires two important elements, viz. specific
recognition of the target analyte and generation of a signal that
transduces the recognition event. Enabled by the efforts in drug
discovery to impact pharma, specific ligands have been developed
for many important proteins.3 However, methods that utilize these
ligand discoveries to develop protein sensors are scarce, as these
are hampered by strategies that transduce these binding events.
Prior approaches that utilize these ligands require a supramolecular
disassembly event to occur in response to a specific ligand-protein
binding event.4 This strategy therefore requires a design strategy
that requires the ligand-containing molecule to assemble in the
Figure 2. (a) Chemical structures of probe 1, quencher and micelle. (b) Fluorescence
spectra of probe 1 (1 µM) and benzophenone (5 mM) in Brij 35 (1 mM) Tris buffer
solution (25 mM, pH 7.4) treated without or with HCA (5 µM) or with HCA (5 µM) and
EZA (100 µM). All spectra were taken after 3-hour HCA incubation at room
temperature.
a.Department of Chemistry, University of Massachusetts Amherst, Amherst,
Massachusetts 01003. E-mail: thai@chem.umass.edu.
† Electronic Supplementary Information (ESI) available: [synthetic procedures and
characterizations of the probes]. See DOI: 10.1039/x0xx00000x
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DOI: 10.1039/C5CC07408H
Figure 3. Fluorescence spectra of probe 1 (1 µM) and benzophenone (5 mM) in Brij 35
(1 mM) Tris buffer solution (25 mM, pH 7.4) treated with (a) 5 µM of proteins; (b)
different concentrations of HCA. The inset shows fluorescence intensity at λem = 379
nm. All spectra were taken after 3-hour protein incubation at room temperature.
Figure 4. Fluorescence spectra of probe 1 (1 µM) and benzophenone (5 mM) treated
without or with 1 µM HCA in different surfactant solutions: (a) 1 mM Brij 35; (b) 5 mM
Brij 35; (c) 20 mM Brij 35; (d) 4 mM Triton X-100; (e) 1 mM Tween 20; (f) 1 mg/mL PEG-
dodecyl random copolymer. All experiments were carried out in Tris buffer solution (25
mM, pH 7.4). All spectra were taken after 3-hour protein incubation at room
temperature.
HCA,9 caused the fluorescence to go back to the original
fluorescence level, i.e. significantly quenched. In addition to
supporting the design hypothesis outlined in Figure 1, this
observation also suggests that the micellar assembly itself is intact
and that the BP quencher stays encapsulated in this assembly to
cause the re-encapsulated fluorophore to be quenched.
bulk solvent. This anticipation is because, in contrast to the probe
by itself, the probe-protein complex should not have the
hydrophobic driving force to re-bind to the interior of the micellar
host. This specific protein-driven dissociation of the probe from the
micelle also drastically decreases the proximity of the fluorophore
and the quencher, which can be conveniently read as fluorescence
increase, as schematically illustrated in Figure 1. The extent of this
dissociation should also be dependent on the concentration of
analyte protein.
To test our design hypothesis, we chose human carbonic
anhydrase I (HCA) as the target analyte because of its disease
relevance.6 This protein also has several well-established ligand
moieties.6,7 Brij 35, a commercially available surfactant, is used as
the micelle-forming macromolecule because its non-ionic head
group, composed of polyethylene glycol unit, helps reduce
nonspecific interactions. Benzenesulfonamide, which is used as a
specific ligand to bind HCA,6,7 is tethered to pyrene as the
fluorophore using a simple hydrophobic linker to obtain the
fluorescent probe 1 (Figure 2a).
To further confirm that the fluorescence enhancement was
indeed caused by the specific protein-ligand interaction, an
unmodified pyrene fluorophore, which lacks the sulfonamide ligand
moiety, was encapsulated along with benzophenone in Brij 35
micelle assemblies. Upon exposure to HCA, no fluorescence
enhancement was observed (Figure S3). Similarly, to test whether
the probe design is specific to HCA, solutions of BP quenched probe
1 in Brij 35 micelles were exposed to four different proteins with
different isoelectric points (α-chymotrypsin, avidin, β-galactosidase,
For the design to be functional, it's pivotal that probe 1 is and pepsin) that have no known binding affinity toward
incorporated into the micellar assemblies. To test this, probe 1 was benzenesulfonamide. As shown in Figure 3a, none of these proteins
dissolved in a buffer solution (pH 7.4) in the presence and absence
caused a significant change in pyrene fluorescence. In addition, we
of Brij 35 in the solution. Absorption spectra of the solutions also found that the HCA-induced fluorescence enhancement
indicated that while the solution without Brij 35 has negligible occurred in a 50% fetal bovine serum solution (Figure S4). Even
absorbance at 340 nm, the solution with Brij 35 exhibits strong though the background fluorescence signal was much higher
absorption peaks that correspond to pyrene (Figure S1a). This
compared to that in buffer solution, the fluorescence enhancement
indicates micellar assemblies help solubilize the hydrophobic probe after adding HCA was also higher in 50% serum solution. This is
1, which otherwise has limited solubility in aqueous solution. Thus, possibly due to the decreased stability of micelles in serum and
this observation also provides the support that the majority of the nonspecific binding of probe to serum proteins. Further serum
probe in the solution will be present inside the Brij 35 micelle, optimization experiments should be done in the future to minimize
compared to the bulk solvent. To achieve a fluorescence “OFF”
this background fluorescence. Nevertheless, the initial serum study
state,
a
known photoinduced electron transfer quencher suggests that this sensing strategy has the potential to be translated
(benzophenone (BP))8 was co-encapsulated in the micelle. This co- to a complex milieu containing many biological macromolecules.
encapsulation indeed causes the pyrene fluorescence to be
predominantly quenched (Figure S2a).
We were also interested in investigating the sensitivity of this
sensing strategy. If our mechanistic hypothesis is correct, then we
Next, we examined the possibility of utilizing this assembly to should have a linear increase in fluorescence with increase in
sense the presence of the analyte protein. Accordingly, HCA was
concentration of the protein. This is because, the inherent
added to the Brij 35 micellar assembly containing probe 1 and BP. preference for the probe is to be within the micellar assembly and
Indeed, upon addition of the protein, the fluorescence of pyrene therefore exhibit fluorescence quenching. However, the bound
increased dramatically, as shown in Figure 2b. To check if this probes have the opposite preference, i.e. to stay in the bulk
fluorescence enhancement is indeed due to the ligand-protein aqueous phase where there is no fluorescent quenching. Since
binding mechanism illustrated in Figure 1, we added a competitive
increasing concentrations of protein should afford higher amounts
ligand to HCA. The presence of the competitive ligand should of bound probes, the fluorescence increase should be linear.
displace the fluorescent probe from the protein. Since the probe is Indeed, we found that the fluorescence increased linearly with
hydrophobic, it should be re-encapsulated into the micelle upon increasing HCA concentrations (Figure 3b). We found that a
release from the protein to cause the fluorescence to be quenched
again. As shown in Figure 2b, addition of 6-Ethoxy-2- nM concentration of the protein and an appreciable change starts
benzothiazolesulfonamide (EZA), a strong competitive inhibitor of occurring in the protein above 200 nM HCA. Overall, the sensitivity
reproducible change in the fluorescence can be achieved even at 50
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of this surfactant-probe-quencher combination is in the high nM 20 was found to be the least sensitive system. Although we do not
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DOI: 10.1039/C5CC07408H
understand the reasons for these differences at this time, this
range.
observation suggests that
relationship study in the future could provide an even more
sensitive system.
a
systematic structure-property
While investigating the factors that could affect the sensitivity of
this method, we found that the sensitivity of the probe was affected
by the concentration of the surfactant. As indicated in Figure 4a-c,
fluorescence enhancement becomes less obvious as the
Finally, to test whether this design principle can be applied to
detect other proteins, we synthesized a structurally similar probe to
target avidin (probe 2, Figure 5a). Biotin, which has extraordinary
binding affinity toward avidin, was linked to the pyrene fluorophore
in the place of benzenesulfonamide. Similar to that observed with
probe 1 and HCA, the fluorescence intensity was very weak when
probe 2 and benzophenone were co-encapsulated in Brij 35. On
the other hand, the fluorescence was dramatically enhanced upon
the addition of avidin (Figure 5b). As biotin binds strongly and
irreversibly to avidin, the displacement method using a competitive
inhibitor for probe 1 would not be viable for probe 2. Therefore, to
test the recognition-driven dissociation mechanism, a solution of
BP-quenched probe 2 in Brij 35 micelles was pre-incubated with 20-
fold excess biotin, which can compete with probe 2 to bind to the
protein. As expected, upon exposure to avidin, very little
fluorescence enhancement was observed for above solution
compared to the one without the free biotin ligand (Figure 5b).
Interestingly, in addition to the increase in the monomeric pyrene
emission peak, a new peak appears around 485 nm, which
correspond to the pyrene excimer emission. Since high fluorophore
concentration is required for excimer formation, we attribute this
to the tetravalent binding sites of avidin and possible hydrophobic
association. Probe 2 is capable of detecting avidin concentration
even at 5 nM concentrations (Figure 5c), likely due to the high
binding affinity of biotin avidin interaction. To further demonstrate
the generality of this sensing strategy, we also showed that probe 2
can also be used to detect avidin using the random copolymer
micelles (Figure 5d).
Figure 5. (a) Chemical structure of probe 2. (b) Fluorescence spectra of probe 2 (1 µM)
and benzophenone (5 mM) in Brij 35 (1 mM) solution treated without or with avidin (1
µM) or with avidin (1 µM) and biotin (20 µM). Fluorescence spectra of probe 2 (1 µM)
and benzophenone (5 mM) treated with different concentrations of avidin in (c) Brij 35
(1 mM) solution; (d) PEG-dodecyl random copolymer (1 mg/mL) solution. All
experiments were done in Tris buffer solution (25 mM, pH 7.4). All spectra were taken
after 3-hour protein incubation at room temperature.
concentration of Brij 35 increases. In these cases, the probe and
quencher concentrations were kept constant. These observations
seem to be consistent with our mechanistic hypothesis that the
probe binds to the protein in the bulk solvent, as it has some
propensity to partition to the bulk solvent. It is reasonable to
suggest that the probe partitions between the micellar core and
bulk solvent with the equilibrium largely favoring the micelle core.
Increasing the surfactant concentration increases the effective
micelle concentration, which should shift the equilibrium even
further toward the micellar core. This should render the availability
of the probe for protein binding even lesser and therefore the
sensitivity of the probe becomes lesser.
In summary, we have developed
a new supramolecular
dissociation based sensing strategy to detect specific proteins with
turn-on fluorescence signals. The sensing approach is based on non-
covalent encapsulation of ligand-tethered fluorophore/ quencher
pair in the micellar assemblies, where the fluorescence is in the ‘off’
state. Protein-binding induced dissociation of the ligand-
fluorophore combination away from micelle turns the fluorescence
to the ‘on’ state, since its proximity with the quencher is
compromised. The versatility of this approach lies in its simplicity:
(i) well-established and commercially available surfactants can be
used; (ii) other than being hydrophobic, the fluorophore-ligand
combination does not have to exhibit inherent self-assembly
features and therefore does not require extensive molecular
design; (iii) the strategy is conveniently extendable to any
fluorophore-quencher combinations to modulate the colour of
detection; and (iv) the approach is potentially extendable to most
target protein analytes. Overall, we anticipate that the design
principle has the potential to open up fundamentally new avenues
in supramolecular chemistry for generation of fluorescent signal or
even other spectroscopic signals in response to specific protein-
ligand recognition events.
Next, we were interested in testing the broad utility of this
approach. From the perspective of varying the fluorophore and the
quencher, it is perhaps obvious that other combinations can be
utilized for this purpose. However, we were interested in testing
the versatility of this approach by investigating whether it will
accommodate variations in the micellar assembly and the target
protein. Instead of Brij 35, we investigated other charge neutral
micelles generated from Triton X-100, Tween 20 and an amphiphilic
random copolymer based on PEG-acrylate and dodecyl-acrylate.
First of all, we were gratified to find that in all these cases the
protein-induced fluorescence change can be observed (Figure 4d-f).
Next, we also tested whether the sensitivity to the concentration of
the surfactants would be different in each of these cases. All
surfactants exhibited the same trend in that the sensitivity
decreased with increasing surfactant concentration (Figure S6),
suggesting similar operating mechanism. It is however interesting
that the relative sensitivity itself was different at concentrations
above their respective critical micelle concentration. Triton X-100
was found to provide the most sensitive sensing system and Tween
Support from Army Research Office (63889-CH) and the National
Institutes of Health (GM-065255) are acknowledged.
Notes and references
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