Catalytic single-chain polymeric nanoparticles at work: From ensemble towards single-particle kinetics

Abstract

Folding a single polymer chain around catalytically active sites to construct catalytic single chain polymeric nanoparticles (SCPNs) is a novel approach to mimic the activity and selectivity of enzymes. In order to relate the efficiency of SCPNs to their three-dimensional structure, a better understanding of their catalytic activity at an individual level, rather than at an ensemble level, is highly desirable. In this work, we present the design and preparation of catalytic SCPNs and a family of fluorogenic substrates, their characterization at the ensemble level as well as our progress towards analyzing individual SCPNs with single-molecule fluorescence microscopy (SMFM). Firstly, organocopper-based SCPNs together with rhodamine-based fluorogenic substrates were designed and synthesized. The SCPNs catalyze the carbamate cleavage reaction of mono-protected rhodamines, with the dimethylpropargyloxycarbonyl protecting group being cleaved most efficiently. A systematic study focusing on the conditions during catalysis revealed that the ligand acceleration effect as well as the accumulation of substrates and catalytically active sites in SCPNs significantly promote their catalytic performance. Secondly, a streptavidin–biotin based strategy was developed to immobilize the catalytic SCPNs on the surface of glass coverslips. Fluorescence correlation spectroscopy experiments confirmed that the SCPNs remained catalytically active after surface immobilization. Finally, single-SCPN activity measurements were performed. The results qualitatively indicated that fluorescent product molecules were formed as a result of the catalytic reaction and that individual fluorescent product molecules could be detected. So far, no evidence for strongly different behaviors has been observed when comparing individual SCPNs.

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Molecular Systems
Design & Engineering
PAPER
Cite this: DOI: 10.1039/c8me00017d
Received 7th April 2018,
Accepted 24th May 2018
DOI: 10.1039/c8me00017d
rsc.li/molecular-engineering
Catalytic single-chain polymeric nanoparticles at
work: from ensemble towards single-particle
kinetics†
Yiliu Liu,
a
Petri Turunen,
bc
Bas F. M. de Waal,
a
Kerstin G. Blank,
bd
Alan E. Rowan,*
bc
Anja R. A. Palmans *
a
and E. W. Meijer *
a
Folding a single polymer chain around catalytically active sites to construct catalytic single chain polymeric
nanoparticles (SCPNs) is a novel approach to mimic the activity and selectivity of enzymes. In order to re-
late the efficiency of SCPNs to their three-dimensional structure, a better understanding of their catalytic
activity at an individual level, rather than at an ensemble level, is highly desirable. In this work, we present
the design and preparation of catalytic SCPNs and a family of fluorogenic substrates, their characterization
at the ensemble level as well as our progress towards analyzing individual SCPNs with single-molecule
fluorescence microscopy (SMFM). Firstly, organocopper-based SCPNs together with rhodamine-based
fluorogenic substrates were designed and synthesized. The SCPNs catalyze the carbamate cleavage reac-
tion of mono-protected rhodamines, with the dimethylpropargyloxycarbonyl protecting group being
cleaved most efficiently. A systematic study focusing on the conditions during catalysis revealed that the li-
gand acceleration effect as well as the accumulation of substrates and catalytically active sites in SCPNs
significantly promote their catalytic performance. Secondly, a streptavidin–biotin based strategy was devel-
oped to immobilize the catalytic SCPNs on the surface of glass coverslips. Fluorescence correlation
spectroscopy experiments confirmed that the SCPNs remained catalytically active after surface immobiliza-
tion. Finally, single-SCPN activity measurements were performed. The results qualitatively indicated that
fluorescent product molecules were formed as a result of the catalytic reaction and that individual fluores-
cent product molecules could be detected. So far, no evidence for strongly different behaviors has been
observed when comparing individual SCPNs.
Introduction
Folding individual synthetic polymer chains into nano-
particles, which is reminiscent of nature's way of folding
polypeptides into proteins, has emerged as a novel approach
toward attaining defined polymer architectures.
1–6
In general,
these polymers are decorated with pendant groups that are
capable of forming covalent or supramolecular bonds. After
intra-chain (supramolecular) crosslinking between the pendant
groups, the individual polymer chains are folded into single-
chain polymeric nanoparticles (SCPNs). The incorporation of
Mol. Syst. Des. Eng.This journal is © The Royal Society of Chemistry 2018
a
Institute for Complex Molecular Systems, Laboratory of Macromolecular and
Organic Chemistry, Eindhoven University of Technology, P.O. Box 513, 5600 MB,
Eindhoven, The Netherlands. E-mail: a.palmans@tue.nl, e.w.meijer@tue.nl
b
Department of Molecular Materials, Institute for Molecules and Materials,
Radboud University, 6525 AJ, Nijmegen, The Netherlands.
E-mail: alan.rowan@uq.edu.au
c
The University of Queensland, Australian Institute for Bioengineering and
Nanotechnology, Brisbane, Queensland 4072, Australia
d
Mechano(bio)chemistry, Max Planck Institute of Colloids and Interfaces,
Potsdam-Golm Science Park, 14424 Potsdam, Germany
†Electronic supplementary information (ESI) available. See DOI: 10.1039/
c8me00017d
Design, System, Application
A new approach to polymer-based catalytic systems has been recently disclosed, which is based on the folding of a polymer chain around catalytically active
sites covalently bound to the polymer. In contrast to enzymes, these polymer-based catalysts are structurally heterogeneous, so that the catalytically active
sites may reside in different micro-environments. As a result, differences in reactivity may arise due to this diversity of catalytic sites. It is therefore of great
interest to investigate this possible diversity and we here design a system to study the catalytic activity at the single-polymer level. In the design, the single-
chain polymer nanoparticles are connected to the surface, and substrates are transformed from a luminescent, silent state to a strongly fluorescent state.
With these polymers and substrates synthesized it has become possible to study the catalytic activity of individual chains with single-molecule fluorescence
microscopy. The new insights obtained in this work can be used to progress the search to an enzyme-mimic for bio-orthogonal chemistry in complex cellu-
lar media.
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additional pendant groups on the polymers allows the syn-
thesis of SCPNs with a range of different functionalities. Of
particular interest are catalytically active SCPNs, which can
be considered as a first step towards catalysts that show the
activity and selectivity of natural enzymes.
7–14
Organometallic
SCPNs, which are reminiscent of metallo-enzymes, have
attracted significant attention in recent studies. SCPNs
loaded with metal ions, such as those of palladium,
7,14
plati-
num,
8
ruthenium,
9
rhodium,
15
copper,
11–13
show efficient
and selective catalysis in a variety of reactions. For example,
organocopper-based SCPNs were applied in catalyzing the oxi-
dative coupling of terminal acetylenes,
11
hydroxylation of
phenols,
12
and azide–alkyne cycloaddition,
13
in which the re-
action media ranged from organic solvent to aqueous solu-
tion or even an intracellular environment. In this way cata-
lytic SCPNs can be seen as the renaissance of polymer- and
dendrimer-based catalysts that have been studied in great de-
tail a few decades ago.
16–21
Although significant progress has been achieved in
expanding the diversity and versatility of catalytic SCPNs, their
catalytic activity lags far behind the performance of
enzymes. In order to further improve their catalytic function,
it is crucial to gain a deeper insight into the systems currently
developed. Our group has developed water-soluble catalytic
SCPNs by designing polymers comprising water-soluble
oligoIJethylene glycol) side-chains, benzene-1,3,5-tricarboxa-
mides (BTA) supramolecular motifs and catalytic active
sites.
22–24
A number of techniques have been employed to
investigate the BTA-based SCPNs and to improve our under-
standing of how their 3D structures relate to their
functions.
25–28
Circular dichroism (CD) spectroscopy and scat-
tering techniques have been utilized to study their inner struc-
ture and shape;
25,26
single-molecule force spectroscopy has
been introduced to provide information on the folding kinet-
ics and pathways.
27
Recently, Overhauser dynamic nuclear
polarization NMR has been employed to investigate the local
water translational diffusion dynamics at the site where ca-
talysis would occur.
28
All these experiments have provided
detailed information on the properties of SCPNs and the
factors that may affect their catalytic performance. While
these approaches were mostly focused on structural aspects,
methods that are able to directly monitor the catalytic reac-
tion are still lacking. Additionally, ensembles of catalytic
SCPNs are intrinsically heterogeneous as the result of the
molar mass dispersity of the copolymers in combination
with the random incorporation of catalyst sites. As the exact
location of the catalysts cannot be controlled, it may be
envisaged that every SCPN shows a different activity and se-
lectivity. Therefore, a technique able to monitor the catalytic
activity of individual SCPNs is expected to directly reveal if
structural heterogeneities are translated into a different cat-
alytic performance.
Single-molecule fluorescence microscopy (SMFM) com-
bined with fluorogenic substrates provides information on
the activity of individual catalysts, which is inaccessible in
ensemble measurements.
29–41
Although mostly applied for
investigating enzymatic reactions, the fluorogenic substrate
reporter systems also allow for studying non-enzymatic pro-
cesses in real time.
34–38,40,41
Upon cleavage, the initially non-
fluorescent substrate is converted into a highly fluorescent
product molecule. As every turnover yields one fluorescent
product molecule, a detailed and straightforward kinetic
analysis is facilitated. In particular, when following the turn-
over sequence, information about dynamic reaction pro-
cesses is obtained.
42–45
We herein report our first steps to-
ward utilizing SMFM to investigate the catalytic activity of
individual SCPNs (Scheme 1). We have developed an experi-
mental strategy to addresses the following challenges: 1)
obtaining a catalytically active SCPN platform with modular
functionality; 2) synthesizing mono-substituted fluorogenic
Scheme 1 Experimental design for measuring the activity of a single SCPN using a confocal fluorescence microscope. The laser is focused onto
the position of the SCPN immobilized on a glass coverslip. Every fluorophore produced by the SCPN is recorded in real time.
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substrates with different functional groups suitable for en-
semble and SMFM experiments; 3) immobilization of active
SCPNs on a surface, required for the SMFM experiments; 4)
establishing a single-molecule experimental protocol and
data analysis.
Results and discussion
Design and preparation of catalytic SCPNs
Organocopper-based SCPNs were chosen owing to their effi-
cient catalytic performance in a broad range of reactions.
Three ligand-containing polymers (P1–P3) and a catalytically
inactive control polymer (P4) were designed (Fig. 1, Table 1).
These polymers contain water-soluble polyether side-chains
and BTA pendants, which enable them to form SCPNs in
aqueous solution. P1–P3 were modified with phenanthroline
ligands, which can bind Cu ions to form catalytically active
sites, while the control polymer P4 has dodecyl chains in-
stead. To allow surface immobilization, P2 and P3 were deco-
rated with 1% of biotin-containing side chains. Moreover, P3
was labeled with the fluorescent dye Alexa Fluor®488 to
enable its visualization with fluorescence microscopy. The
polymers were modularly synthesized using a strategy of
post-polymerization modification.
46,47
The precursor polymer
polyIJpentafluorophenyl acrylate) (pPFPA,M
n, SEC
= 18.0 kD,
Đ= 1.28, DP = 120) was synthesized by reversible addition–
fragmentation chain-transfer (RAFT) polymerization and the
thiocarbonyl end group was removed upon heating with
excess azobisisobutyronitrile (AIBN) and lauroyl peroxide
(LPO) (Fig. S1†). P1–P4 were prepared via polymer analogous
reactions of pPFPA by sequential addition of amines (Fig. S2–
S4†). 5-Amino-N-(1,10-phenanthrolin-5-yl)pentanamide (phen-
C4-amine) and the enantiomerically pure, amino-
functionalized BTA unit (BTA-amine) were synthesized, the
other amines were commercially obtained. After full modifi-
cation, the final polymers were obtained via dialysis to re-
move excess amines and byproducts.
Next, the polymers were complexed with Cu
2+
ions. For
this purpose, the polymers were dissolved in water, sonicated
and subjected to a heating–cooling procedure. Then CuSO
4
was added to the solution. Taking P1 as an example, its fold-
ing behavior, complexation with Cu
2+
ions (P1@CuIJII)) and
SCPN formation were characterized using a combination of
techniques. The binding of Cu
2+
ions to the phenanthroline
ligand was studied with UV-vis spectroscopy. As shown in
Fig. 2a, the phenanthroline ligand shows a characteristic
absorption band around 270 nm. Upon addition of CuSO
4
the absorption undergoes a red-shift to around 280 nm. A de-
tailed plot of Cu
2+
ratio versus shift in absorption reveals a 2 :
1 binding stoichiometry between the phenanthroline
ligand and Cu
2+
ions. In the CD spectra of P1, a negative
Cotton effect was present at 223 nm before and after Cu
2+
complexation without a change in value. Moreover, the CD
temperature scans of P1 with or without CuSO
4
overlap
(Fig. S5†). These observations indicate that (i) the BTA pen-
dants form helically stacked dynamic aggregates driving the
folding of polymer chains and (ii) Cu
2+
complexation is not
interfering with BTA aggregation.
Fig. 1 Chemical structure of polymer P1–4.P1–P3 were modified with phenanthroline ligands, while the control polymer P4 has dodecyl chains
instead. To allow surface immobilization, P2 and P3 were decorated with 1% of biotin-containing side chains. Moreover, P3 was labeled with the
fluorescent dye Alexa Fluor®488 to enable its visualization with fluorescence microscopy.
Table 1 Composition and SEC characterization of polymers P1–P4
a
Polymer abcdef nmM
n
(kD) Đ
P1 0.08 0.04 0.10 —0.78 —120 —31.4 1.18
P2 0.08 0.08 0.10 —0.73 0.01 120 45 28.0 1.30
P3 0.08 0.10 0.10 0.01 0.70 0.01 120 45 27.8 1.20
P4 0.08 —0.10 —0.82 —120 —31.7 1.20
a
The values for a–fare determined via
19
F-NMR. M
n
is measured by SEC in DMF with 10 mM LiBr, relative to polyIJethylene oxide) standards.
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Small-angle X-ray scattering (SAXS) and dynamic light
scattering (DLS) were employed to evaluate the size of the
polymeric nanoparticles formed. SAXS measurements indi-
cate that P1 and P1@CuIJII)form nanoparticles with a radius
of gyration (R
g
) of 5.6 and 5.4 nm, respectively (Fig. S6†).
The DLS results of P1 and P1@CuIJII)show a monodisperse
distribution of particles with a hydrodynamic radius (R
h
)of
7.4 and 8.2 nm, respectively (Fig. S7†). The scattering results
are comparable to similar systems reported previously, sug-
gesting that single-polymer chains fold into SCPNs indeed.
P2–P4 were characterized in the same way as P1 and similar
results were obtained (Fig. S7, Table S1†). P3, which is mod-
ified with Alexa Fluor®488, was further studied with fluo-
rescence correlation spectroscopy (FCS). The FCS experi-
ments were performed at a concentration of 100 nM of P3
in aqueous solution. The diffusion of the P3-based fluores-
cent nanoparticles was found to be approximately one order
of magnitude slower than a reference fluorophore
(ATTO488, Fig. S8†). This indicates that the Alexa Fluor®
488 dye was successfully coupled to the polymers. Using the
Stokes–Einstein equation, the R
h
of P3 was determined to be 8.3
± 0.8 nm, which is consistent with the light scattering results.
Design and synthesis of fluorogenic substrates
A series of fluorogenic substrates were designed that are
predicted to be good substrates for the Cu-phenanthroline
functionalized SCPNs. Considering that these substrates
should be suitable for both ensemble and single-SCPN
kinetic experiments, a number of criteria need to be consid-
ered: i) the substrates are stable in aqueous solution, but
can undergo fast cleavage in the presence of CuIJI)-based
organometallic catalysts, catalysts made out of the CuIJII)-
based precursor; ii) the substrates are based on a fluorophore
with high brightness, so that a high signal-to-noise ratio can
be achieved; iii) the substrates contain only one cleavable
group, in order to avoid the formation of reaction intermedi-
ates with different fluorescent properties; and iv) the sub-
strates are hydrophobic and are converted into hydrophilic
products, which could facilitate the SCPN-based catalysis.
With these guidelines in mind, substrates S1–S4 were
designed and synthesized (Fig. 3). Rhodamine 110 (Rh110)
was chosen as the fluorophore owing to its high brightness.
However, it carries two amino groups for further modifica-
tion. To prepare a substrate with a single reactive site, one
amino group of Rh110 was first modified with a morpholine-
carbonyl group to obtain MC-Rh110.
48–50
The urea bond
formed is not reactive and hydrolytically stable. At the same
time, MC-Rh110 remains sufficiently bright for SMFM experi-
ments.
48
Further modification of the remaining amino group
to generate carbamate bonds yields substrates S1–S4, which
contain different alkyne or alkene groups. The carbamate
bond of S1 was previously shown to be hydrolyzed in the
presence of CuIJI),
51
while the carbamate bond in S2 is labile
in the presence of PdIJII) catalysts.
52,53
The hydrolytic stability
of the carbamate bonds in S3 and S4 in the presence of CuIJI)
is not known.
SCPN-based catalysis in aqueous solution
The catalytic performance of SCPNs in the carbamate-
cleavage reaction was first studied at the ensemble level in
Fig. 2 Folding and Cu-loading of the polymers; a) UV-vis spectra of P1 (black, 0.6 mg mL
−1
) and P1@CuIJII)(blue, 0.6 mg mL
−1
, [phen] : [Cu] = 2 : 1);
b) CD spectra of P1 (0.5 mg mL
−1
, black dot: 20 °C, red triangle: 90 °C), P1@CuIJII)(blue square, 20 °C, 0.5 mg mL
−1
, [phen] : [Cu] = 2 : 1).
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aqueous solution. The progress of the reaction was moni-
tored with fluorescence spectroscopy as well as liquid
chromatography-mass spectroscopy (LC-MS) (Fig. S9 and
S10†). Whereas the former provides information about the
catalytic reaction in real time, LC-MS allows exact quantifica-
tion of the substrate conversion at set time intervals. As a
first step, a substrate activity screening was performed.
P1@CuIJI)([phen] : [Cu] = 2 : 1) was chosen as a representative
catalyst. Generally, a stock solution of the substrate (in
DMSO) was added into a P1@CuIJII)solution (2 μM) to reach
a final concentration of 30 μM. The reactions were initialized
by adding sodium ascorbate (NaAsc, 1 mM) to generate
P1@CuIJI)in situ. The fluorescence intensity was recorded at
520 nm for 30 min and substrate conversion was directly ana-
lyzed with LC-MS (Fig. S9b†). The fluorescence–time curves
were normalized relative to the maximum conversion and are
shown in Fig. 4. Substrates S1 and S2, which contain end-
functionalized alkyne moieties, are hydrolyzed, with the ter-
tiary alkyne bearing S1 showing the highest TOF (∼200 h
−1
).
In contrast, S3 and S4, which contain internal alkyne and
Fig. 4 Carbamate cleavage reaction of S1–S4 catalyzed by P1@CuIJI)([P1]=2μM, [CuSO
4
]=10μM, NaAsc = 1 mM, substrate = 30 μM). The
kinetic curves were recorded via fluorescence spectroscopy (ex. 495 nm; recorded at 520 nm) followed with normalization according to the
conversions obtained from LC-MS.
Fig. 3 Chemical structures and synthesis of the substrates S1–S4. (a) 4-Morpholinecarbonyl chloride; (b) triphosgene, 2-methylbut-3-yn-2-ol; (c)
propargyl chloroformate; (d) triphosgene, but-2-yn-1-ol; (e) triphosgene, prop-2-en-1-ol.
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alkene moieties, respectively, show almost no reactivity. It
has been proposed that the formation of a Cu-acetylide inter-
mediate is crucial in the catalytic mechanism. The terminal
alkyne moieties of S1 and S2 bind CuIJI)togeneratethe
Cu-acetylide. This leads to the formation of a Cu-stabilized
propargyl cation thus cleaving the carbamate bond. The
methyl groups of the tertiary propargyl moiety in S1 further
stabilize the cation, thereby promoting the cleavage reaction.
51
The ligand-containing SCPNs have several merits toward
aqueous catalysis: α) ligand accelerated catalysis; β) hydro-
phobic interior; and γ) accumulation of catalytically active
sites. Whereas the first feature is determined directly by the
mechanism of the Cu-catalyzed reaction, the other two fea-
tures lead to an enhancement of the local concentration of
active sites and/or substrate molecules. To verify in how far
these three features are responsible for enhancing the kinet-
ics of the hydrolysis reaction, we performed systematic
kinetic experiments on S1 and S2. As catalysts, we selected
P1@CuIJI), which possesses contributions from α,βand γ,
whereas P4 mixed with Phen@CuIJI)only possesses the αand
βcontribution. As reference Phen@CuIJI),P4 mixed with CuIJI)
and only CuIJI) were used (Table 2). The outcome of these
catalysis experiments is shown in Fig. 5 (and Fig. S9c†). The
phenanthroline ligand indeed accelerates the CuIJI)-catalyzed
carbamate-cleavage reaction. Moreover, the accumulation of
catalytic active sites and substrates enhances the reaction to
a large extent. In short, the more characteristics the system
contains, the more effective the catalytic reaction becomes.
Surface immobilization of catalytic SCPNs
Having obtained insights into the substrate specificity and
the kinetics of the SCPN catalyzed reactions, a first step was
taken toward single-SCPN experiments. For observing individ-
ual SCPNs with a confocal microscope, the SCPNs need to be
immobilized on the surface of glass coverslips. We selected
an immobilization strategy based on the well-established
streptavidin–biotin interaction. In detail, the glass surface
was first modified with an amino silane (3-aminopropyl di-
methylethoxy silane). In the next step, a hetero-bifunctional
polyIJethylene glycol) containing an amino-reactive NHS ester
and a biotin group (NHS-PEG
3000Da
-biotin) was used to obtain
a biotin covered surface. The biotin-containing SCPNs were
then immobilized on the surface using streptavidin as a
bridge (Fig. 6a). P3 was employed to test the SCPN immobili-
zation protocol. P3 contains biotin motifs on its side-chains
and is labeled with Alexa Fluor®488, which allows its visuali-
zation with confocal microscopy. Fig. 6b shows confocal im-
ages obtained for different surfaces incubated with P3 at con-
centrations of 50 nM, 5 nM, and 0.5 nM, respectively. Inspection
of Fig. 6b shows that the immobilization is successful, as
indicated by the concentration-dependent density of SCPNs.
The question then arises whether the surface-immobilized
SCPNs are still catalytically active. Using fluorescence correla-
tion spectroscopy (FCS), the carbamate-cleavage reaction was
monitored by following the increase in fluorescent products
that are released from the immobilized SCPNs and diffuse
into the bulk solution (see ESI†for details, Fig. S12).
P2@CuIJII)was chosen for these experiments instead of
P3@CuIJII)to avoid any influence from the Alexa Fluor®488
dye used for labeling the SCPNs. The immobilization of
P2@CuIJII)followed exactly the same protocol as shown for
P3. A solution containing S1 (10 μM) and NaAsc (1 mM) was
added onto a surface with or without P2@CuIJII), respectively.
As shown in Fig. 7, hydrolysis of S1 is clearly visible on the
P2@CuIJI)covered surface. This provides first evidence that at
least a fraction of SCPNs remains catalytically active after sur-
face immobilization.
Fig. 5 A study on the characteristics of SCPN-based catalysis. The conversions of the reactions under different catalytic conditions (Table 2) were
obtained though LC-MS.
Table 2 Catalysts with different characteristics. α)ligandacceleratedca-
talysis; β) hydrophobic interior; γ) accumulation of catalytically active sites
Entry (S1/S2) Catalyst Characteristics
1P1@CuIJI)α,β,γ
2P4 &Phen@CuIJI)α,β
3Phen@CuIJI)α
4P4 &CuIJI)β
5CuIJI)—
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Single-molecule activity measurements
Single-SCPN activity measurements were performed to find
out if individual turnovers of a single SCPN can be followed
over time. In these experiments, P3@CuIJII)was used in com-
bination with S1 as the substrate. As the complex of the
phenanthroline ligand with Cu ions quenches the fluores-
cence of Alexa Fluor®488 (Fig. S11†), this fluorescent label is
used indirectly to visualize Cu-containing SCPNs on the sur-
face. Therefore, we developed the following protocol to local-
ize the SCPNs on the coverslip surface. P3-based SCPNs (20
nM) were first immobilized on the glass coverslip. A surface
area of 10 ×10 μm was scanned before and after incubation
with CuSO
4
solution (concentration 1 mM). Fluorescent spots
that disappeared after the incubation with CuSO
4
were con-
sidered as locations of Cu-loaded SCPNs (Fig. 8a). It is impor-
tant to note that the disappearance of the fluorescent signal
can also originate from bleaching of Alexa Fluor®488 during
consecutive scans. The degree of bleaching was tested and it
was found to be negligible under the experimental conditions
used (Fig. S13†).
Fluorescence time traces were recorded at the positions of
different SCPNs for 1 minute each after delivering a solution
containing 10 μMS1 and NaAsc. One representative time
trace is shown in Fig. 8b. Despite the relatively low signal-to-
noise ratio, which is expected for MC-Rh110, the results sug-
gest that fluorescent product molecules are produced on the
surface and can be detected. When comparing the time
Fig. 6 Surface immobilization of biotinylated SCPNs. a) Experimental design. b) Confocal images showing biotinylated and Alexa Fluor®488-
labeled SCPN on a functionalized coverslip. The images show SCPNs immobilized in various concentrations and a corresponding control measure-
ment where no SCPNs were added. The bar represents 40 and 4 μm in the top and bottom panel, respectively.
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traces recorded at the position of one SCPN with the control
(empty area which does not contain SCPNs), a small increase
in the frequency of fluorescence bursts (with high intensity)
was observed, suggesting that the SCPNs are turning over the
fluorogenic substrate. However, the number of clearly re-
solved turnovers detected appears to be low (Fig. 8b), which
can be rationalized by the relatively low average TOF of the
catalytic SCPN system. Nevertheless, the fact that no individ-
ual SCPNs with a significantly higher activity was observed
(Fig. S14 and S15†), may imply that structural heterogeneities
of the SCPNs do not cause a dramatic diversity in their cata-
lytic performance.
Although a detailed quantitative kinetic analysis proved
difficult, the results above provide a practical platform that
holds potential for further optimization. Catalytically-active
SCPNs with organometallic sites other than copper com-
plexes, e.g. palladium or ruthenium complexes, are possible
candidates, which may have higher TOFs and overcome the
fluorescence quenching problem. Besides developing new
catalysts, we are continuing our efforts on optimizing the ex-
perimental setup to obtain higher SNR and throughput. For
example, the possibility of using a wide-field fluorescence
setup combined with nano-photonic metal structures, such
as zero-mode waveguides (ZMWs),
54–57
is under exploration.
Wide-field fluorescence microscopy, which allows for moni-
toring many individual SCPNs on the surface simultaneously,
may provide insights into possible heterogeneities between
individual SCPNs in a more straightforward manner. Mean-
while, ZMWs are able to reduce the detection volume drasti-
cally, thus allowing the use of higher substrate concentra-
tions while reducing the background signal. In addition,
their ability to enhance the fluorescence signal could increase
the signal-to-noise ratio even further.
Fig. 8 Representative single-SCPN activity measurement. a) Confocal images (10 ×10 μm) showing the Alexa Fluor 488-labeled and biotin-
functionalized SCPNs immobilized on a streptavidin-functionalized glass coverslip before (left) and after (right) the addition of CuSO
4
. The
bar in the panel represents 4 μm. b) Fluorescent intensity time traces and c) corresponding intensity distributions obtained when binning the
data with 1 ms bin size. The data was recorded on the location of one individual SCPN (blue) and on an empty area on the surface (red;
control).
Fig. 7 Fluorescence correlation spectroscopy (FCS) to determine
product accumulation in solution. The graph illustrates the rate of
product accumulation for the sample containing immobilized SCPNs
(blue) and for a control sample without SCPNs (red, auto-hydrolysis).
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Conclusion
We have shown the preparation of SCPNs that are capable of
catalyzing fluorogenic reactions, as well as provide protocols
for their surface immobilization and for monitoring their cat-
alytic activity. The combined design of catalysts and sub-
strates enabled us to obtain novel information about the sub-
strate specificity of SCPNs and allowed to guide the activity of
the catalytic reaction at the ensemble level. Similar to
changes in reactivity as the result of mutations in enzymes,
we expect that not every SCPN will be equally reactive.
58
Therefore, we are highly intrigued by the question on how to
discriminate the diversity in reactivity at the individual level.
The protocol for surface immobilization, which retains the
catalytic activity of SCPNs, allows further SMFM measure-
ments on individual SCPNs. In the course of our investiga-
tion, we identified the experimental challenges, mainly a low
TOF of the catalysts combined with a low signal-to-noise ratio
in SMFM measurements, which will allow us to improve the
system in future experiments. So far, we have not found any
evidence that some SCPNs are more reactive than the others.
However, this work has laid a solid foundation for applying
SMFM to further understanding the structure–function rela-
tion of catalytic SCPNs. We anticipate that these efforts will
assist in directing the future of catalytic SCPNs, e.g. if se-
quence control in the polymer synthesis and regulation of
polymer chain's precision folding are the gateway toward
accessing enzyme-like activity and selectivity.
Conflicts of interest
There are no conflicts to declare.
Acknowledgements
Y. L., A. R. A. P. and E. W. M. acknowledge financial support
from the Dutch Ministry of Education, Culture and Science
(Gravity program 024.001.035) and the European Research
Council (FP7/2007-2013, ERC Grant Agreement 246829). P. T.,
K. G. B. and A. E. R. acknowledge financial support from the
Dutch Ministry of Education, Culture and Science (Gravity
program 024.001.035), the Dutch National Research School
Combination Catalysis Controlled by Chemical Design
(NRSCC grant 2009-10016B, A. E. R.), as well as The Nether-
lands Organization for Scientific Research (NWO; VIDI grant
700.58.430, K. G. B.). Dr. L. Albertazzi is acknowledged for
fruitful discussions. Gijs ter Huurne is acknowledged for as-
sistance with the SAXS measurements. The ICMS animation
studio is acknowledged for providing the artwork.
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