Salen/salan metallic complexes as redox labels for electrochemical aptasensors

Article abstract of DOI:10.1039/c9cc07575e

This work presents the synthesis and characterization of salen/salan metal complexes for their future application as electrochemical labels in affinity sensors. Due to its stability and electrochemical properties, an oxovanadium salan complex was selected

Full text of DOI:10.1039/c9cc07575e

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Salen/salan metallic complexes as redox labels for
electrochemical aptasensors†
Cite this: Chem. Commun., 2019,
55, 12821
abc
abd
c
Amani Ben Jrad,
Thierry Noguer,
Hussein Kanso,
Delphine Raviglione,
ab
ab
Nicolas Inguimbert *c and Carole Calas-Blanchard
*
Received 26th September 2019,
Accepted 2nd October 2019
DOI: 10.1039/c9cc07575e
rsc.li/chemcomm
This work presents the synthesis and characterization of salen/salan In this context and to extend the scope of electroactive salen/
metal complexes for their future application as electrochemical salan complexes, this study proposes to use them for the
labels in affinity sensors. Due to its stability and electrochemical labeling of DNA oligonucleotides, aptamers, which are used as
properties, an oxovanadium salan complex was selected and biomimetic recognition elements in biosensors. Aptamers have
coupled to an estradiol-specific aptamer. The response of the numerous advantages compared to other recognition elements
resulting aptasensor was shown to decrease with increasing estradiol such as antibodies or enzymes as they are specifically synthe-
concentration.
sized in large quantities at moderate cost. The intrinsic stability
of DNA aptamers allows their modification on both sides, one
Salen ligands and their more stable reduced form, salan, are being used for attachment on the electrode surface, while the
popular chelates in chemistry.1,2 Over the last decade, salen other supports the marker.7,8 Traditionally, optical markers,
based metal complexes have been adopted for many applications in especially fluorescent ones such as fluorophores,9,10 are used
catalysis,3 cancer diagnosis,4 and magnetism and for their biological for labeling aptamers.11 Despite their high selectivity, optical
activities.5 The present work proposes to use such complexes as aptasensors have long response times and the detection requires
redox labels for electrochemical affinity biosensing. The tetradentate the use of expensive and bulky instruments.12 Electrochemical
(N2O2) system offers the possibility of chelating a variety of metal detection offers faster response and requires inexpensive and
ions, leading to several combinations for a better sensitivity and compact equipment. Electrochemical aptasensors based on redox
selectivity of electrochemical response. Additionally, the intro- labeling have high selectivity and low detection limits.12,13 Electro-
duction of different substituents on the salicylaldehyde moiety chemical labeling generally involves a redox probe molecule, such as
allows optimizing the electronic characteristics of the metallic methylene blue14–16 or ferrocene,17–19 usually detectable by
active center.1 Such complexes have been already used by our voltammetric techniques.20 The sensing strategy of the resulting
group as electrochemical labels with the aim of developing an electrochemical aptasensors depends on the efficiency of electron
electrochemical immunosensor for estradiol (E2) detection in transfer between the redox marker and the electrode surface. In fact,
water.6 Indeed, E2 has been placed on the European watch list the binding of the target molecule on the aptamer induces a
(EU 2015/495) to prevent its potential hazard. This hormone is conformational change that results in moving the marker closer
considered as an endocrine disruptor requiring monitoring in (Fig. 1A) or farther away from the electrode surface (Fig. 1B). Such
the environment, mainly in water. Therefore, it is essential displacement results in a variation of the measured signal which
to have effective tools for the rapid detection of this molecule. can be correlated with the target concentration.21 For applications
such as environmental pollution monitoring, this type of device that
combines a stable, specific aptamer and compact electrochemical
a
´
Universite Perpignan Via Domitia, Biocapteurs-Analyses-Environnement, F-66860,
transducers to generate an electrical signal offers a simple, accurate
and inexpensive platform. It is the ideal tool for analysis in fields
with miniaturized equipment.13,22 In this work, we report the
synthesis of salen/salan metal complexes and their conjugation to
aptamers for their future application in affinity sensors. As a proof
of concept and for comparison purposes with our previous work, we
chose an E2-specific aptamer23,24 to test the effectiveness of the
labeling of such a molecule with these metallic complexes. In order
to fulfill the above-mentioned aims, we developed a suitable strategy
Perpignan, France. E-mail: carole.blanchard@univ-perp.fr
b
c
´
Laboratoire de Biodiversite et Biotechnologies Microbiennes, USR 3579 Sorbonne
´
´
Universites (UPMC) Paris 6 et CNRS Observatoire Oceanologique, F-66650,
Banyuls-sur-Mer, France
´
USR 3278 CRIOBE, PSL Research University, EPHE-UPVD-CNRS, Universite de
ˆ
Perpignan Via Domitia, Laboratoire d’Excellence ‘‘CORAIL’’, Batiment T, 58 avenue
P. Alduy, 66860 Perpignan, France. E-mail: nicolas.inguimbert@univ-perp.fr
d
´
´
Faculte de Medecine Dentaire, Univ. Libanaise, Campus Rafic Hariri, Hadath, Liban
† Electronic supplementary information (ESI) available: Synthesis and character-
ization of compounds 1–13. See DOI: 10.1039/c9cc07575e
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the spectrum shows three absorption bands at 223, 261 and
354 nm. The first band is assigned to the azomethine group.
The second and third bands are due to the p - p* transitions
of the phenolic chromophores. In the presence of metals,
bathochromic shifts of these bands to 233, 284 and 403 nm
and to 248, 291 and 407 nm were observed for copper and
oxovanadium complexes, respectively, thus confirming proper
metal complex formation (ESI,† S17, Fig. 1A). Lower intensity
absorption bands were observed at 580 and 600 nm for the
copper and oxovanadium complexes, respectively. These bands
were not observed in the salen ligand spectra and were thus
assigned to ligand-to-metal charge-transfer (LMCT) transitions.31,32
The electrochemical behavior of the different products was studied
by cyclic voltammetry (CV). The cyclic voltammogram of the copper
complex 8 showed a quasi-reversible oxidoreduction behavior of
copper, with a half-wave potential (E1/2) of 0.04 V vs. Ag/AgCl. For the
oxovanadium (VO) complex 9, the voltammogram also showed a
Fig. 1 Principle of electrochemical aptasensors based on the recognition
of the target by an aptamer. The increase (A) or decrease (B) of the
electrochemical signal after the detection depends on the aptamer
configuration and the probe position relative to the electrode (screen
printed carbon electrode).
quasi-reversible oxidoreduction behavior of oxovanadium at E1/2
=
0.14 V vs. Ag/AgCl (Fig. 2A). The voltammogram presented a well-
defined shape of the VO-salen reduction peak when compared with
allowing the synthesis of salen 1a or salan 1b ligand functionalized the Cu-salen one. Based on the characterization results of the salen
with a maleimide coupling group for further conjugation to the complexes 8 and 9, oxovanadium was preferred for the salan
aptamer (ESI†). Therefore, the designed compounds 1a and 1b complex. An absorption spectrum of the free salan ligand 1b
(ESI,† S3, Scheme S1) are composed of two parts, a tetradentate exhibited two bands at 225 and 292 nm. These bands were
ligand prepared by condensation of 3-(tert-butyl)-2-hydroxy-5- also observed in the spectrum of the VO-salan complex 10 after
methoxybenzaldehyde and 2,3-diaminopropionic acid, with bathochromic shifts at 282 and 345 nm, and were assigned to
a spacer supporting a maleimide end-group. The spacer is a the p - p* transitions of the aromatic rings. The spectrum of
1,6-hexamethylenediamine moiety that allows segregating the VO-salan complex 10 showed an additional band at 450 nm
electroactive ligands from the aptamer in order to avoid unfavor-
able interactions between these two elements. The synthesis of
compound 1a containing the salen chelate was achieved in 3 steps
with an overall yield of 62% (ESI,† S3, Scheme S2). Spacer 4a was
prepared by adding Boc-hexanediamine 2 with the NHS ester
of the N-succinimidyl 3-maleimidopropionate 3. After Boc
deprotection compound 4b was engaged in a coupling reaction
with Boc-Dap-Boc 5 mediated by oxyma/EDC coupling reagents.25
Boc deprotection of compound 6a and subsequent reaction with
3-(tert-butyl)-2-hydroxy-5-methoxybenzaldehyde 7b at neutral pH
allowed the formation of the Schiff base ligand 1a. Generally,
salan can be obtained by reduction of salen imines.2,26 However,
the reduction of compound 1a with sodium borohydride led to
the reduction of the maleimide group rather than the imine
one.27 Therefore, the maleimide group of 4a was protected
through a Diels Alder reaction with dimethylfuran leading to
compound 4c (ESI,† S9, Scheme S3). The next steps of the
synthesis were identical to the salen one with an additional
deprotection of the maleimide through a reverse Diels–Alder
reaction.28–30 It is noteworthy that the final step leading to
compound 1c was unsatisfactory and thus impacted the global
yield, which was 16% (ESI†).
Metal complexes (compounds 8–10, ESI,† S14 and S15) were
prepared in MeOH with pure functionalized ligands 1a and b
and two different metal salts, copper(II) acetate or vanadyl
acetylacetonate. Characterization of complexes 8–10 was carried
Fig. 2 Cyclic voltammograms measured in 0.1 M KCl for (A) salen 1a
(black), Cu-salen 8 (red) and VO-salen 9 (blue); and (B) salan 1b (black) and
VO-salan 10 (green). Before the experiment, 6 nmol of each compound,
out by UV-Vis spectroscopy. The absorption spectra were recorded
over the wavelength range 200–900 nm. For the salen ligand, carbon electrode. Scan rate: 0.1 V sꢀ1
prepared in MeOH, were adsorbed on the surface of a screen-printed
.
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corresponding to the LMCT transitions (ESI,† S17, Fig. 1B).26,31
CV experiments showed that VO-salan complex 10 displays a
quasi-reversible oxidoreduction wave at E1/2 = ꢀ0.16 V (Fig. 2B).
This low value of E1/2 is expected to impart a better selectivity to
the resulting aptasensor, avoiding non-specific signals due to
potential interfering substances. The promising characterization
results of the salen and salan complexes confirm the potential of
these redox systems for the development of affinity sensors.
Next the labeling strategy based on the reaction between the
maleimide group of the functionalized ligands 1 and the thiol
terminal group of the E2 aptamer sequence was explored.
This type of bioconjugation for labile biomolecules like
proteins and antibodies presents the advantage of being easily
performed without the need for activating agents or harsh
conditions.33 Therefore, the estradiol specific aptamer having
the following sequence 50-AAG GGA TGC CGT TTG GGC CCA
AGT TCG GCA TAG TG-30 and modified by an amine group at
the 50 end and a thiol group at the 30 end was subjected to
reaction with compounds 1. An aptamer in its lyophilized form
was solubilized in sterile water to the desired concentration. In
order to facilitate the coupling step, a heat treatment was
performed to linearize the aptamer sequence (8 min at 90 1C,
4 min at 0 1C and 15 min at room temperature). The coupling
reaction was then carried out using 11 nmol of aptamer
prepared in sterile water and 22 nmol (2 eq.)34,35 of ligands in
MeOH so that the reaction medium will be finally constituted
by 10% water and 90% MeOH. This reaction was carried out to
test the maleimide–thiol coupling and to observe the behavior
of the ligands under the reaction conditions. Proper coupling of
the two parts was assessed by monitoring the reaction using
HPLC coupled to electrospray ionization HRMS operating in
negative mode.
Fig. 3 LC/HRMS of the E2 aptamer (A1 and B1), salan 1b (A2 and B2),
compound 12 (A3 and B3), compound 10 (A4 and B4) and compound 13
(A5 and B5).
Besides the remaining E2 aptamer characterized by peaks at
m/z 2239.16 and 1865.80 (ESI,† S16, A1, B1 and A3, B3-2)
corresponding to the five and six charged states, respectively,
a small amount of compound 14 (ESI,† S15) resulting from the
coupling of the aptamer with a degradation derivative of 1a for expected compound 13 with m/z at 2081.87 and 2402.24
which the two benzaldehyde derived groups are lacking due to (Fig. 3A5 and B5).
the imine hydrolysis was characterized by a mass at m/z =
In order to verify the effectiveness of our redox-labeled
2312.60 and 1926.84 (ESI,† S15, A3, B3-1). The susceptibility of detection system, the aptasensor was set up and tested in the
the salen compounds to hydrolysis drove us to examine the presence of E2. The VO-salan-labeled aptamer was immobilized
salan ligand (1b) as an alternative. The coupling was performed through a peptide bond after EDC/NHS activation of the
under the same conditions and the MS spectra showed peaks at carboxyl groups on the electrode surface.36 Salan-labeled and
m/z = 2389.4520 and 1991.0488 corresponding to the five and label-free aptamers were immobilized by the same method and
six charged states of compound 12 respectively (Fig. 3A3 and used as controls. As shown by Square Wave Voltammetry
B3). The peak corresponding to the starting aptamer m/z = (SWV), in the absence of E2, a significant peak was observed
2239.1659 (Fig. 3A1 and B1) entirely disappeared, showing that only for the VO-salan labeled aptamer, which was attributed to
the totality of the aptamer was labeled by the salan ligand.
oxovanadium electroactivity (Fig. 4A). After incubation of the
A peak at m/z = 750.44 was assigned to the ligand added in aptasensor with 8 mM of E2, this peak was almost switched off
excess for the reaction (Fig. 3A2 and B2). This result confirms and our aptasensor was considered as an on/off system (Fig. 4A).
the superior stability of the salan ligand compared to the salen. This observation can be attributed to the spatial rearrangement
Indeed, the structure of compound 12 is not affected by the of the E2/aptamer assembly compared to the aptamer alone.
presence of 10% water during the coupling reaction. Based on Thus, before the association with E2 the VO-salan complex is
these results, we applied the same reaction conditions by engaging nearby the electrode surface, and a peak with an intensity of
the E2 aptamer and the VO-salan complex 10 (Fig. 3A4 and B4). about 0.1 mA was measured (Fig. 4A).37 After E2 binding to the
LC/MS monitoring allowed identifying the formation of the aptamer, the VO-salan complex moved away from the electrode
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12824 | Chem. Commun., 2019, 55, 12821--12824
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