A Photochemical Ligation System Enabling Solid-Phase Chemiluminescence Read-Out

Article abstract of DOI:10.1002/chem.201901858

The peroxyoxalate chemiluminescence (PO-CL) reaction is among the most powerful and versatile techniques for the detection of hydrogen peroxide (H₂O₂) and has been employed in various biological and chemical applications over the past 50 years. However, its two-component nature (peroxyoxalate and fluorophore) limits its use. This contribution introduces an innovative and versatile photochemical platform technology for the synthesis of inherently fluorescent PO probes by exploiting the nitrile imine-mediated tetrazole-ene cycloaddition (NITEC) reaction. In the presence of hydrogen peroxide, the pioneered “2-in-1” molecule emits either yellow or blue light, depending on tetrazole (Tz) structure. Even in the absence of base, the emitted light remains visible and H₂O₂ could be detected in the nanomolar range. Critically, the PO-Tz can be readily incorporated into polymeric materials. As a first application of this promising material, a tailor-made PO-Tz is grafted on poly(divinylbenzene) (PDVB) particles to enable solid-phase chemiluminescence on microspheres.

Full text of DOI:10.1002/chem.201901858

A Journal of
Accepted Article
Title: A Photochemical Ligation System Enabling Solid-phase
Chemiluminescence Read Out
Authors: Laura Delafresnaye, Christian W. Schmitt, Leonie Barner, and
Christopher Barner-Kowollik
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To be cited as: Chem. Eur. J. 10.1002/chem.201901858
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10.1002/chem.201901858
Chemistry - A European Journal
Article type: Full Paper
A Photochemical Ligation System Enabling Solid-phase Chemiluminescence Read Out
Laura Delafresnaye, Christian W. Schmitt, Leonie Barner* and Christopher Barner-Kowollik*
Dr. L. Delafresnaye, C. W. Schmitt, Prof. Dr. L. Barner, Prof. Dr. C. Barner-Kowollik
School of Chemistry, Physics and Mechanical Engineering, Institute for Future Environments,
Queensland University of Technology (QUT), 2 George St, Brisbane, QLD 4000, Australia.
Email: leonie.barner@qut.edu.au, christopher.barnerkowollik@qut.edu.au
Prof. Dr. C. Barner-Kowollik
Macromolecular Architectures, Institut fꢀr Technische Chemie und Polymerchemie, Karlsruhe
Institute of Technology (KIT), Engesserstrasse 18, 76131 Karlsruhe, Germany
Institut für Biologische Grenzflächen, Karlsruhe Institute of Technology (KIT), Hermann-von-
Helmholtz-Platz 1, 76344 Eggenstein-Leopoldshafen, Germany.
Keywords: chemiluminescence, peroxyoxalate, tetrazole, microsphere, fluorescence.
Abstract
The peroxyoxalate chemiluminescence (PO-CL) reaction is among the most powerful and
versatile techniques for the detection of hydrogen peroxide (H2O2) and has been employed in
various biological and chemical applications over the past 50 years. However, its two-
component nature (peroxyoxalate and fluorophore) limits its use. This contribution introduces
an innovative and versatile photochemical platform technology for the synthesis of inherently
fluorescent PO probes by exploiting the nitrile imine-mediated tetrazole-ene cycloaddition
(NITEC) reaction. In the presence of hydrogen peroxide, the pioneered “2-in-1” molecule emits
either yellow or blue light – depending on tetrazole’s (Tz) structure – visible to the naked eye.
Even in the absence of base, the emitted light remains visible and H2O2 could be detected in the
nanomolar range. Critically, the PO-Tz can be readily incorporated into polymeric materials.
As a first application of this promising material, a tailor-made PO-Tz is grafted on
poly(divinylbenzene) (PDVB) particles to enable solid-phase chemiluminescence on
microspheres.
1
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1. Introduction
Chemiluminescence (CL) reactions – the generation of light as a result of a chemical
transformation – are powerful and versatile detection techniques that offer the unique read-out
of emitting light in response to specific molecular events. In the realm of chemiluminescence,
a plethora of applications ranging from analytical methods such as (immuno)essays,[1]
pharmaceutical,[2] environmental[3] or food analysis[4-6] to biosensors[7, 8] have been developed
over the last five decades exploiting its simplicity, low-cost, high sensitivity and fast dynamic
response. Among the CL reactions, the peroxyoxalate (PO) reaction is of particular interest
since it is possible to tune the color of light emitted as exemplified by the well-known glow
sticks. The reaction can be classified as indirect or sensitized chemiluminescence as the excited
species cannot emit light by itself but rather transfers its excess of energy to a fluorophore.
Typically, hydrogen peroxide (H2O2) reacts with a peroxyoxalate to form an unstable energy-
rich intermediate (1,2-dioxetanedione), which rapidly decomposes into carbon dioxide and
releases energy. In the presence of an appropriate fluorescence dye, the chemical energy of the
intermediate is converted to electronic excitation energy. The relaxation of the fluorophore from
its excited state to its ground state releases photons and generates light. Even if the PO-CL
mechanism has been studied in-detail, the chemiexcitation sequence is still not fully understood,
yet it is assumed to proceed via the chemically induced electron exchange luminescence
(CIEEL) mechanism.[9-11] The chemiluminescence quantum yield (i.e. the number of photons
emitted per reacting molecule) mainly depends on the PO structure, its proximity with the
fluorophore, the electronic nature of the fluorophore, the pH level and the oxalate ester leaving
group.[12] Therefore, the parameters and the chemicals components can be advantageously
adjusted to fulfil the requirements of the targeted applications. Since H2O2 plays a crucial role
in regulating fundamental biological processes, CL sensors for H2O2 determination have found
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interests in many applications. Indeed, H2O2 can be employed as an indicator for enzyme
detection as its over expression is often a sign of serious disease (diabetes, cancers). Thus, the
development of in vivo and in vitro H2O2 sensors hold great potential.[13] Critically, the PO
reaction not only represents an efficient tool for imaging and monitoring enzymatic light
emission in real time, yet also to evaluate the proliferation of tumor cells.[14] Lee et al.
synthesized poly(ethylene glycol)-b-poly(ε-caprolactone) (PEG-PCL) micelles in the 50 nm
range containing fluorescent dyes and oxalate esters in the hydrophobic core.[15, 16] Due to their
nanomolar sensitivity and their small size, these advanced contrast agents found numerous
applications for imaging H2O2. Employing nanoparticles as carriers further enables the loading
of reactive molecules[17] or drugs.[18] However, all these processes are based on a three-
component CL reaction, which requires a methodology for confining the peroxyoxalate and the
fluorophore in close proximity in vivo. To overcome the limitation of a two-component system
(e.g. leaching, distance) and elevate the PO reaction to an entirely new level, we herein
introduce a “2-in-1” molecule. Surprisingly, since the first report of the peroxyoxalate reaction
in the early nineteen sixties,[19, 20] the potential of combining the peroxyoxalate moiety and a
fluorophore in the same molecule has barely been investigated. Motoyoshiya et al. observed
that some non-fluorescent diaryl oxalates with electron-donating groups emitted weak light
thanks to the emission from the excimer of the eliminating group.[21] The authors hypothesized
that the emitting species would be formed by an intra- and intermolecular electronic interaction
with a high-energy intermediate. To the extent of our knowledge, this contribution is the only
report of a PO-CL reaction using a single molecule and the present work aims to introduce a
new platform technology in which an inherently fluorescent PO molecule can emit strong light
but also be readily grafted onto surfaces and incorporated in a wide variety of materials. This
strategy relies on the nitrile imine-mediated tetrazole-ene cycloaddition (NITEC), which
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produces a fluorescent five-membered pyrazoline ring.[22] Using either UV or visible light
irradiation, tetrazoles (Tz) form highly reactive nitrile imines under release of nitrogen. The
intermediate is capable of undergoing cycloaddition reactions with a vast array of
dipolarophiles such as maleimides, acrylates and fumarates. Thus, the synthesis of an oxalate-
based molecule bearing a maleimide functionality as dipolarophile will lead to a fluorescent
moiety at the PO’s vicinity (Figure 1). Our group has developed a wide library of UV and
visible light sensitive tetrazoles bearing (meth)acrylate,[23] carboxylic acid or alcohol
functionalities, enabling them to be incorporated into soft matter materials[24] and
(bio)surfaces[25] of different nature. As one possible application of our new class of PO, we
herein describe light emitting chemiluminescent polymeric microspheres (MS). Indeed, their
large surface area as well as non-demanding handling, and long-term storage stability make
microspheres highly attractive scaffolds. Solid-phase chemiluminescence presents a sensitive,
fast and effective detection method and has shown promise for the detection of biological threat
agents, water or food contaminants.[26] Current techniques rely on fluorogenic-
chemiluminescence (FCL) and electro-chemiluminescence (ECL), which depend upon the
isolation of the targeted analyte from the sample by magnetic separation employing magnetic
microspheres as a support. Van Zoonen et al. have addressed the possibility of covalently
immobilizing fluorophores onto controlled pore glass beads to enable solid-phase
peroxyoxalate chemiluminescence in high-performance liquid chromatography (HPLC),
resulting in a sensitive, cost-effective and simple H2O2 detection system in rain water.[27-29]
Applying a similar approach, Pontén et al. compared six amino-based luminophores employing
porous and non-porous methacrylate beads.[30-32] These authors concluded that porous materials
were much more sensitive due to a higher surface area and degree of functionalization. To the
best of our knowledge, this study from the mid-nineties is the only reported MS solid phase
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luminophore. Since then, very little attention has been paid to solid-phase peroxyoxalate
chemiluminescent polymeric microspheres, most likely because of the inherent difficulty of
molecular anchoring the three reactive components in close proximity. Herein, we close this
critical gap by grafting our new system onto the surface of hydrophilic microspheres.
Figure 1. NITEC reaction of the MDCPO with tetrazole and simplified chemiluminescence activation pathway of
the PO-Tz molecule. Pictures of the samples after addition of H2O2 are displayed next to the corresponding
tetrazole.
2. Results and Discussion
2.1. Design of tetrazole-peroxyoxalate molecule
Initially, a suitable maleimide-PO was tailored. Peroxyoxalate esters are generally synthesized
by reacting the respective functionalized phenol with oxalyl chloride under triethylamine (TEA)
catalysis or with oxalic acid mediated by 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide
(EDC) and a base. Due to the high reactivity of oxalyl chloride employed in the last step, the
aromatic compound needs orthogonal functional groups to be able to incorporate maleimides
as reactive double bond for the NITEC reaction. The most frequently used POs –
bis(trichlorophenyl)
oxalate
(TCPO),[33]
bis(2,4-nitrophenyl)oxalate
(DNPO),[33]
bis(pentafluorophenyl) oxalate (PFPO)[19] or divanillyl oxalate (DVO)[34] – do not present
orthogonal pre or post-modifiable functionalities in their structure, requiring alternative
synthetic pathways. Accordingly, a maleimide-PO compound (MPO) based on commercially
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available 4-amino phenol was synthesized in a simple three-step procedure. 1H and 13C NMR
spectroscopy confirmed the successful synthesis (for detailed analytical information, refer to
Figures S2-S3) and its applicability in CL reactions was assessed in further experiments and
compared to TCPO and DVO. Following a typical protocol, the MPO was dispersed in ethyl
acetate (EA), together with a common fluorophore, here 9-10 diphenylanthracene (DPA). The
light emitted was very weak, in contrast to the conventional PO which exhibited a very bright
light, visible to the naked eye (Figure S16). We hypothesized that the maleimide deactivated
the phenol as leaving group in subsequent CL reactions. Therefore, the second generation of
maleimide-PO was tyramine-based (Figure S4-5) in order to introduce a small alkyl chain as
spacer between the maleimide and the aromatic ring (MEPO). Unfortunately, the resulting CL
intensity was even lower compared to the first generation MPO. Since aromatic substituents
have a significant influence on the light emission intensity and electron-withdrawing groups
such as halogens can decrease the pKa value of the respective phenol-group, their leaving group
properties are enhanced.[12] As a consequence, the third generation was based on 4-amino-2,6-
dichloro phenol, leading to bis(2,6-dichloro-4-N-maleimido)phenyl) oxalate (MDCPO)
(Figure S6-9). The light intensity was strongly improved and a bright blue light was readily
observable. Compared to TCPO, the light intensity was lower – TCPO contains 6 Cl whereas
the MDCPO contains only 4 Cl – yet it was 3 times higher than DVO. The subsequent challenge
was to assess the unconventional use of a NITEC adduct to act as a fluorophore in a PO-CL
reaction employing a 2/1 molar ratio. As already noted, the PO-CL reaction requires a
fluorophore, which is usually an organic dye such as 9,10- diphenylanthracene, rubrene,
aminopyrene or Rhodamine B. Considering that the fluorescence quenching effects can be
offset by too high concentration, fluorophores are generally employed in low concentrations,
typically with a 0.05/1 molar ratio relative to the PO. In our strategy, the PO bears two
6
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maleimide moieties, consequently leading to two fluorophores per PO after the reaction with
the tetrazole. In a preliminary study, two UV-B reactive tetrazoles were synthesized, one
bearing a methoxy phenyl substituent (Tz1) and one only bearing a phenyl group adjacent to
the tetrazole (Tz2) (Figures S10-S14). Both tetrazoles were subsequently dissolved in 3 mL of
THF or EA in the presence of MDCPO and irradiated with UV-B light (300 nm) for 30 min. 1H
NMR confirmed that the NITEC reaction proceeded with almost full conversion (94 %) after
only 30 min (Figure S17) as resonances associated with the maleimide double bond protons
(7.77 ppm and 7.04 ppm) disappeared while two doublets arose between 5.21 and 5.44 ppm.
We further observed that aromatic proton resonances of the newly formed PO-Tz shifted upfield.
Absorbance and fluorescence spectra (Figures S18-S19) additionally evidenced the successful
formation of the highly fluorescent pyrazoline adduct with an emission wavelength of 525 nm
for Tz1 and 480 nm for Tz2 (λex = 390 nm). Chemiluminescence was subsequently triggered by
adding 50 μL of a saturated urea hydrogen peroxide aqueous solution and 10 μL of a saturated
sodium acetate solution. Indeed, CL reactions are usually conducted in the presence of a base
which can catalyze nucleophilic H2O2 attack at the PO and increase the light intensity.[35] Both
samples exhibited strong light emissions that were readily observable by the naked eye as
shown on Figure 2. Tetrazole 1 emitted yellow light with a wavelength centered at 525 nm,
while the second tetrazole emitted at 480 nm (blue light). In terms of intensity, tetrazole 1
exhibited a maximum irradiance at 0.024 uW cm-2 nm-1 whereas tetrazole 2 maximum
irradiance was recorded at 0.014 uW cm-2 nm-1. In the visible range, the total irradiance was
4.93 and 3.93 uW cm-2 nm-1 and the corresponding photosynthetically photon flux density
(PPFD) was then calculated at 0.15 and 0.07 μmol m-2 s-1 for PO-Tz1 and PO-Tz2, respectively
(Figure 2A). These experiments not only constitute the first report of a pyrazoline adduct being
used as a fluorophore in a PO-CL reaction, but it is also the first functional PO-CL system that
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does not require additional fluorophores. Remarkably, the substituent near the tetrazole allows
to access different emission wavelengths and colors as it is permitted with conventional dye. It
is worth-noting that the PO-Tz was completely soluble in THF and only partially in EA.
Nonetheless, the light intensity was higher in EA for both tetrazoles. Diethyl phthalate (DEP)
– the solvent used in the glow sticks – was also investigated (Figures S20-S21 and 2B). The
MDCPO and Tz were partially soluble, but even if the emitted light intensity was slightly lower
than in THF, it was sufficiently bright to be observed by the naked eye. Interestingly, due to the
high viscosity of DEP, the emission lasted for 45 min instead of a few min when conducted in
THF or EA (Figure S21). Taking advantage of the viscosity of DEP, the experiment performed
in a 50/50 mixture DEP/EA lasted more than 10 min with a light intensity almost equivalent to
neat EA. Changing the PO-Tz concentration led to higher light intensities with no linear
correlation, most likely due to the moderate solubility of the PO-Tz in EA (Figure S22). The
CL remained visible with a minimal concentration of 0.35 mmol L-1.
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A
B
3000
2500
2000
1500
1000
500
0.05
EA
THF
DEP
EA/DEP
PO-Tz1
PO-Tz2
0.04
0.03
0.02
0.01
0.00
400
0
400
500
600
700
800
500
600
700
800
Wavelength / nm
Wavelength / nm
C
D
PO-Tz1
PO-Tz2
2400
2100
1800
1500
1200
900
PO-Tz1 - base
PO-Tz2 - base
PO-Tz1 - no base
PO-Tz2 - no base
600
300
No
Base
No
Base
0
Base
Base
0
5
10
15
20
25
30
Time / min
Figure 2. Chemiluminescence emission spectra of PO-Tz (Tz1, λmax = 525 nm; Tz2, λmax = 480 nm). Spectra were
recorded in 3 mL of solvent in the presence of PO-Tz (4 mmol L-1), 50 μL of urea hydrogen peroxide aqueous
solution (6 mol L-1) and 10 μL of sodium acetate (4 mmol L-1). (A) Irradiance measurement (spectroradiometer,
EA). (B) Influence of solvent (spectrophotometer set in medium voltage). (C) Kinetics of the light emitted in
presence or absence of sodium acetate (medium voltage, EA). (D) Pictures of the PO-Tz after addition of H2O2.
We then moved to assess the peroxide detection limit of this new system by gradually
decreasing the added amount of H2O2, whereby a weight detection limit (DLW) of 10-6 mol was
determined (Figure S23B). This threshold corresponds to the detection limit of the
spectrophotometer and can still be observed by the naked eye. Classical PO-CL analytical
methods state a DLW between 10-11 and 10-14 mol[36, 37] with much more sensitive instruments.
Therefore, an even lower detection limit could be measured, yet this is beyond the scope of the
present work.
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As previously established, employing different moiety adjacent to the tetrazole led to distinct
colors and we were interested in investigating the effect of various functionalities on the CL.
Hence, a library of tetrazoles bearing small (C2) or long alkyl chains (C11), hydroxyl or
carboxyl groups as well as acrylates were assessed. Similarly to the initial Tz employed, all PO-
Tz molecule exhibited a bright light, visible to the naked eye (Figure S24), demonstrating the
versatility of our platform technology. In our aim to simplify the PO-CL reaction even further,
we only employed the PO-Tz molecule with hydrogen peroxide without the use of any base
(Figures 2C-D). Surprisingly, we observed that the light intensity slowly decayed for more
than one hour where it lasted few minutes in the presence of a base. Several studies have
stressed the importance of a catalyst in the CIEEL sequence and PO-CL reactions performed in
the complete absence of base led to inconsistent results with a very weak light intensity.[38, 39]
However, even if the light intensity was lower compared to the experiments conducted with
sodium acetate, the emitted light remained very bright, which demonstrates that CL reactions
can be triggered with a very simple two-component setup (PO-Tz and H2O2). Then, a plate
reader was used for that experiment – employing acetonitrile as solvent – and hydrogen
peroxide was detected down to 100 nmol L-1 (Figure S23C).
In summary, we introduce a 2-in-1 PO-Tz molecule which emits very bright light in the sole
presence of H2O2. Further, various parameters such as solvent, employed Tz, or the
concentration can be tuned depending on the final application. One key feature of our platform
technology is its ability to use a tetrazole that can be readily incorporated into a polymer
backbone due to its additional functional synthons.
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2.2. All-in-one chemiluminescent microspheres
Driven by the potential of this new molecule, we functionalized the surface of polymeric
microspheres (MS) to produce, for the first time, “all-in-one” CL MS (Figure 3A). Polymeric
microspheres in the 0.1 to 2000 μm range can be synthesized by a variety of processes, yet are
generally prepared by heterogeneous polymerization techniques such as (mini)emulsion,
suspension, dispersion or precipitation polymerization.[40] Due to its simplicity we decided to
employ the precipitation polymerization technique, which does not require any surfactant or
stabilizers that can eventually hinder the surface’s functionalization. The classical approach to
synthesize microspheres by precipitation polymerization is to utilize a monomer and a
crosslinker with a similar structure (e.g. styrene and divinylbenzene) in a near Θ-solvent (e.g.
acetonitrile). During the initiation step, oligomers grow until they reach a critical length – after
which they are no longer soluble in the solvent – precipitate and form spherical nuclei. The
nuclei then grow by adsorbing further monomers and oligomers from solution to finally form
narrow disperse polymeric particles. Thanks to the simplicity of this method, various
morphologies (e.g. highly cross-linked, porous) can be achieved by tuning the reaction
conditions respectively (solvent, monomer ratio, porogen...).[41] Based on well-established
processes,[42] poly(divinylbenzene) (PDVB) MS were first synthesized as a highly crosslinked
seed particle. However, this material is very hydrophobic and is not compatible with (aqueous)
solutions of hydrogen peroxide. Therefore, in a second step, the residual double bonds of the
seed particles were used to grow a shell of poly(2-hydroxyethyl methacrylate) (PHEMA) in
order to make them suitable for polar systems. Using the residual hydroxyl group, the
microspheres were functionalized with the tetrazole carboxylic acid under Steglich conditions.
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The subsequent photoligation with the MDCPO was ultimately conducted with the
aforementioned protocol to lead to so-called “all-in-one” chemiluminescent microspheres.
A
MDCPO
R = OMe (Tz1), H (Tz2)
AIBN
ACN
24h
AIBN
ACN
24h
DCC / DMAP
THF
UV-B
30 min
THF
80 %
20 %
24h
PDVB
PDVB-PHEMA-Tz-PO
D
DVB80
PDVB-PHEMA
C
PDVB-PHEMA-Tz
B
1000
PDVB
PDVB-PHEMA
PDVB-PHEMA-Tz1
PDVB-PHEMA-Tz2
PDVB-PHEMA-Tz1-PO
PDVB-PHEMA-Tz2-PO
800
600
400
200
0
R = OMe (Tz1)
E
B
C
2 μm
R = H (Tz2)
400
500
600
700
Wavelength / nm
Figure 3. (A) Synthetic pathway for the formation of inherently fluorescent microspheres. (B) SEM pictures of the
final PDVB-PHEMA-Tz2-PO. (C) Fluorescence spectra of the microspheres after each step (solid probe, λex
=
390 nm, medium voltage). (D-E) Pictures of the PDVB-PHEMA-Tz1-PO and PDVB-PHEMA-Tz2-PO particles
under a TLC hand-held lamp (365 nm).
Since the tetrazole and MDCPO were used in excess to ensure full conversion during
esterification and NITEC, the microspheres were thoroughly washed during each step to
remove any unreacted species. Scanning electron microscopy (SEM) revealed spherical PDVB
particles with an average diameter of 1.61 μm and a polydispersity (U) of 1.16 (Table 1,
Figure S25). The diameter increased to 2.31 μm after the PHEMA shell grafting (Figure S26).
As expected, the next steps only led to a minor increase in diameter since the grafting of small
molecules (i.e. the tetrazole and the MDCPO) should not strongly affect the size
(Figures S27-29, Figure 3B). The final particles were considered narrow disperse (U = 1.04)
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and exhibited a diameter of 2.52 and 2.72 μm for MS-Tz1-PO and MS-Tz2-PO, respectively.
The microsphere’s surface composition was further analyzed by X-ray photoelectron
spectroscopy (XPS) (Table 1) and revealed the presence of oxygen after the incorporation of
the PHEMA chains and, most importantly, the presence of chlorine at 1.32 and 3.51 atom%
only after the last step for MS-Tz1-PO and MS-Tz2-PO, respectively. Note the increase of the
nitrogen content from 0.13 to 7.49 at% after the incorporation of tetrazole, followed by a slight
decrease to 5.09 at% due to the release of N2 during the NITEC reaction (MS-Tz2). Particles –
after the final ligation step – were still fully dispersible in water which is critical for future
applications in biological systems. Fluorescence was directly measured on the microsphere
powder using a solid probe to confirm their intrinsic fluorescent nature (Figures 3C-E). The
PDVB-PHEMA-Tz core-shell particles are slightly fluorescent and emitted at 443 nm, but a
clear shift to higher wavelengths was observed after the NITEC reaction.
Table 1. SEM, XPS and Fluorescence analysis of the microspheres after each synthetic step.
SEMa
Dn
XPSb
Fluorescencec
Samples
Dw
U
C 1s
N 1s
O 1s
Cl 2p
λem
I
[μm]
[μm]
[at%]
[at%]
[at%]
[at%]
[nm]
[a.u]
PDVB
1.87
2.52
2.51
2.52
2.63
2.72
1.61
2.31
2.60
2.59
2.45
2.61
1.16
1.09
1.04
1.03
1.07
1.04
97.52 ± 0.09
74.39 ± 0.30
75.94 ± 0.39
74.02 ± 0.44
76.05 ± 0.25
73.90 ± 0.15
0.17 ± 0.17
0.13 ± 0.13
9.73 ± 0.12
9.43 ± 0.14
7.49 ± 0.15
5.09 ± 0.21
2.31 ± 0.08
25.49 ± 0.18
14.34 ± 0.27
15.24 ± 0.21
16.47 ± 0.11
17.51 ± 0.43
0.00 ± 0.00
0.00 ± 0.00
0.00 ± 0.00
1.32 ± 0.10
0.00 ± 0.00
3.51 ± 0.07
443
443
443
532
443
475
274
339
201
240
339
730
PDVB-PHEMA
PDVB-PHEMA-Tz1
PDVB-PHEMA-Tz1-PO
PDVB-PHEMA-Tz2
PDVB-PHEMA-Tz2-PO
aDw represents the weight-average particle diameter, Dn represents the number-average particle diameter, and U
represents the polydispersity index (Dw/Dn). bXPS measurements were performed on carbon tape and values were
c
calculating using the average of two locations. Fluorescence measurements were recorded using a solid probe
with an excitation wavelength of 390 nm (medium voltage).
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Consistent with the preliminary small molecule study, the MS-Tz1-PO emitted at 525 nm and
the MS-Tz2-PO at 475 nm. Finally, chemiluminescence measurements were performed on the
MS-Tz-PO. The light emission was recorded at 544 nm (I = 170 a.u.) for Tz1 (Figure S30). The
light was not observable to the naked eye, therefore a photomultiplier suitable for solid samples
(Lumipol 3) was employed to follow the kinetics. The material was loaded in an aluminium pan
(10 mg) and 50 μL of a 2.10-2 mol L-1 solution of solid urea hydrogen peroxide in acetonitrile
(equivalent to 1 μmol of hydrogen peroxide) was simply dropped on the powder. The light
intensity of MS-Tz1-PO started at 1.5 107 counts then decreased to 4.104 counts within 30 min
(Figure 4A). MS-Tz2-PO exhibited a similar behavior, with a light intensity of 2.5 107 counts
– almost two times higher. The higher light intensity can be correlated to a higher chlorine
content and, consequently, to more PO on the particle’s surface. The loading content had a
significant impact as weights of 2.0 and 5.0 mg exhibited lower intensities (Figure S31-32).
The detection limit of hydrogen peroxide was assessed by successive dilution of the urea
hydrogen peroxide solution (Figure 4B - Figure S33). A linear correlation between the quantity
of H2O2 and the photon count was maintained to a concentration of 2.10-4 mol L-1, although the
light intensity was recorded until 2.10-6 mol L-1 (equivalent to 100 pmol of H2O2 for 5 mg of
MS-PO).
14
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10.1002/chem.201901858
Chemistry - A European Journal
A
B
MS-Tz1-PO
MS-Tz2-PO
No H2O2
1e+8
1e+7
1e+6
1e+5
1e+4
1e+3
1e+7
1e+6
1e+5
1e+4
1e+3
1e+2
0
5
10
15
20
25
30
1e-5
1e-4
1e-3
1e-2
1e-1
H2O2 / mol L-1
Time / min
Figure 4. (A). Kinetics of the light emitted by MS-Tz1-PO and MS-Tz2-PO in presence of urea hydrogen peroxide:
10 mg MS-PO, 50 μL of a 2 10-2 mol L-1solution of urea hydrogen peroxide in ACN. (B). Light intensity versus
the hydrogen peroxide quantity (MS-Tz1-PO: 5 mg, 50 μL of a hydrogen peroxide solution in ACN varying from
2 10-2 to 2 10-6 mol L-1, logarithm scale) (Lumipol). The dashed line is a guide to the eye.
3. Conclusion
We herein present a new class of molecule that combines a peroxyoxalate and a fluorophore.
Undetermined by a robust chemiluminescent reaction, the effectiveness of this platform
technology is based on the synthesis of a maleimide-PO that can subsequently react with a
tetrazole under UV-B light to form a fluorescent pyrazoline adduct. By simply adding H2O2,
the PO-Tz emits a very bright light visible by naked eyes. Furthermore, blue or yellow light are
obtained by judiciously choosing the moiety adjacent to the tetrazole. Development of new
tetrazole is attracting much attention and the colour of emitted light could be further tuned.
Additionally, by using red-shifted tetrazole and thereby avoiding detrimental UV light, our
platform technology could be employed in biological systems. Hence, this fluorescent
peroxyoxalate molecule represents an innovative alternative to conventional multicomponent
CL systems and has the potential to outperform current PO-CL reactions. Of fundamental
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10.1002/chem.201901858
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interest is the possibility of incorporating the molecule into various materials but also playing
with its solubility in different media due to the end-group functionality of the tetrazole. In the
present study, we employed an esterification, but a wide library of functional tetrazoles exists,
enabling the PO-Tz to be readily tailored to the targeted application. Here, we grafted the PO-
Tz onto the surface of polymeric microspheres resulting in solid-phase chemiluminescence.
These “all-in-one” microspheres are highly fluorescent and easy to handle and store. The
addition of hydrogen peroxide triggers CL and high photon counts are recorded even at low
peroxide concentrations.
Supporting Information
Supporting Information is available from the Wiley Online Library or from the author.
Acknowledgements
L.D. and C.W.S. contributed equally to this study. C.B.-K. acknowledges the Australian
Research Council (ARC) for funding in the form of a Laureate Fellowship underpinning his
photochemical research program as well as the Queensland University of Technology (QUT)
for key continued support. C.B.-K. and L.B. acknowledge the ARC for funding in the context
of a Linkage project. The authors acknowledge the facilities and the scientific and technical
assistance from the Australian Microscopy and Microanalysis Research Facility (AMMRF) at
the Central Analytical Research Facility (CARF) operated by the Institute for Future
Environments (IFE).
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A Photochemical Ligation System Enabling Solid-phase Chemiluminescence Read Out
Laura Delafresnaye, Christian W. Schmitt, Leonie Barner* and Christopher Barner-Kowollik*
Keywords. Chemiluminescence, Peroxyoxalate, Tetrazole, Microsphere, Fluorescence.
H2O2
Fluorophore
Peroxyoxalate
Functionality
A versatile platform technology combining a fluorescent pyrazoline-peroxyoxalate adduct
is introduced. Upon addition of hydrogen peroxide, the 2-in-1 molecule emits a yellow or blue
bright light visible to the naked eye. By judiciously selecting the tetrazole, this tailor-made
molecule can be readily incorporated into polymeric materials, enabling solid-phase
chemiluminescence read-out.
19
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