Acetylcholinesterase-induced fluorescence turn-off of an oligothiophene-grafted quartz surface sensitive to myristoylcholine

Article abstract of DOI:10.1039/c5tb00679a

Conjugated polyelectrolytes (CPEs) have recently emerged as label-free materials for biosensing due to their intrinsic ability to transduce an amplified optical signal in response to interactions with different analytes. Herein, the conformational change of an anionic oligothiophene is exploited to generate a unique fluorescent response upon interaction with myristoylcholine (MyrCh). The variations observed in spectroscopic signals are explained in terms of a synergistic combination of hydrophobic and electrostatic forces involving the oligothiophene chains and MyrCh molecules, inducing the disassembling of oligothiophene chains. The enzyme acetylcholinesterase (AChE) is able to reverse this effect by catalyzing the hydrolysis of MyrCh; hence, its enzymatic activity can be monitored through the variation of fluorescence emission of the system. The oligothiophene sensing probe retains its conformational sensitivity with regard to the AChE-mediated cleavage of MyrCh upon immobilization onto a quartz substrate, which is accomplished by a "grafting onto" approach based on click chemistry. These results are encouraging for the further development of such a label-free system towards the fabrication of sensing devices that would incorporate CPEs and would be potentially useful for the specific detection of a wide range of bioanalytes. This journal is

Full text of DOI:10.1039/c5tb00679a

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Materials Chemistry B
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Acetylcholinesterase-induced fluorescence turn-
off of an oligothiophene-grafted quartz surface
sensitive to myristoylcholine†
Cite this:DOI: 10.1039/c5tb00679a
a
a
a
a
a
a
G. Grisci, W. Mroz, U. Giovanella, K. Pagano, W. Porzio, L. Ragona,*
´
b
a
a
a
F. Samperi, S. Tomaselli, F. Galeotti* and S. Destri
Conjugated polyelectrolytes (CPEs) have recently emerged as label-free materials for biosensing due to their
intrinsic ability to transduce an amplified optical signal in response to interactions with different analytes.
Herein, the conformational change of an anionic oligothiophene is exploited to generate a unique fluorescent
response upon interaction with myristoylcholine (MyrCh). The variations observed in spectroscopic signals
are explained in terms of a synergistic combination of hydrophobic and electrostatic forces involving the
oligothiophene chains and MyrCh molecules, inducing the disassembling of oligothiophene chains. The
enzyme acetylcholinesterase (AChE) is able to reverse this effect by catalyzing the hydrolysis of MyrCh;
hence, its enzymatic activity can be monitored through the variation of fluorescence emission of the
system. The oligothiophene sensing probe retains its conformational sensitivity with regard to the AChE-
mediated cleavage of MyrCh upon immobilization onto a quartz substrate, which is accomplished by a
Received 13th April 2015,
Accepted 17th May 2015
‘‘grafting onto’’ approach based on click chemistry. These results are encouraging for the further development
DOI: 10.1039/c5tb00679a
of such a label-free system towards the fabrication of sensing devices that would incorporate CPEs and would
be potentially useful for the specific detection of a wide range of bioanalytes.
www.rsc.org/MaterialsB
onto their p-delocalized backbone (hydrophobic domain). The
pendant moieties confer to CPs the water-solubility that is essential
Introduction
In the last decades, conjugated polymers (CPs) have emerged as for operating as sensing materials in aqueous environments that
1,3,4
promising label-free materials for biosensing applications by mimic biological conditions.
taking advantage of their intrinsic ability to transduce an ampli- Among CPEs, it has been possible to envision novel and
fied optical signal in response to interactions with analytes such unexpected applications for polythiophenes (PTs) in the bio-
as ions, small molecules, proteins, enzymes, nucleic acids and sensing field after the pioneering studies performed by Wudl
1
5
cells. Upon recognition, the delocalization of p-electrons along and coworkers on water-soluble PT derivatives. Ever since, a lot
the CP polyunsaturated backbone undergoes a perturbation, of research efforts have been addressed towards the improve-
which runs ultrafast over long distances (resonance cooperative ment of the conformational sensitivity of PTs to external stimuli
effect), resulting in a concurrent change of the optical properties (e.g. type of solvent, pH, temperature, and presence of analytes)
of the whole polymer chain. Thus, because of this structurally by manipulating the side chains incorporated onto the conju-
intrinsic mechanism of signal amplification, CPs behave as gated backbone. For example, Leclerc and coworkers proposed
‘
‘collective’’ entities, which show higher sensitivity in detecting an imidazolium salt PT derivative for transducing the conforma-
1
,2
6
analytes than individual ‘‘receptor’’ molecules.
tional changes of single-stranded DNA, whereas Shinkai and
Conjugated polyelectrolytes (CPEs) are peculiar amphiphilic coworkers successfully employed a cationic PT derivative in
7
CPs that bear ionic side chains (hydrophilic domain) grafted adenosine triphosphate sensing. Indeed, such structural modi-
fications allow the suitable tuning of the optical properties
a
b
of PTs, i.e. UV-visible absorption and photoluminescence, to
Istituto per lo Studio delle Macromolecole (CNR), Via Edoardo Bassini 15,
0133 Milano, Italy. E-mail: laura.ragona@ismac.cnr.it, f.galeotti@ismac.cnr.it
2
achieve an increased efficiency in the transduction of analyte-
8
Istituto per i Polimeri, Compositi e Biomateriali (IPCB) UOS Catania, CNR,
Via Gaifami 18, 95126 Catania, Italy
binding events. In general, upon interaction with the analyte,
the fluorescence output is modulated in terms of a wavelength
†
Electronic supplementary information (ESI) available: Reaction scheme for
9
,10
8,11
1
shift
or emission turn off/on event.
thiophene monomer, H 1D and DOSY NMR, positive ions MALDI-TOF mass
spectroscopy, UV-vis absorption, rhodamine B grafting description, water contact
angle, AFM, XPS and X-ray analysis. See DOI: 10.1039/c5tb00679a
The pronounced tendency of hydrophobic collapse, shown
by water-soluble PTs in aqueous environments, is governed by
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p-stacking interactions established both intra-molecularly (back-
bone folding) and inter-molecularly (aggregation) among fragments
belonging to the same, or different PT chains, respectively. This
feature can be regulated by modifying the pendant side groups, by
varying solvent properties or by adding amphiphilic analytes (e.g.
surfactants) that are able to form electrostatic complexes with
3,8
oppositely charged CPE. Thus, amphiphile-induced conforma-
tional rearrangements of water-soluble PT chains can lead to
significant alterations of the optical properties of the polymer
(
‘‘surfactochromic’’ effect) as a result of complexation phenomena
12
Scheme 1 Enzymatic reaction of AChE on MyrCh.
between the macromolecular probe and the targeted analyte.
Consequently, the choice of a proper side chain functionalization,
combined with the intrinsic ability of PT backbones to reorganize
upon external stimulation, offers the possibility to create new tools
for sensing applications. Recently, also thiophene-based conjugated
oligomers have gained increasing attention as conformational-
the foundations for the development of a label-free functional
sensing platform. For this aim, we designed and synthesized
an alternating carboxyl functionalized oligothiophene (AA-OT)
terminated with a clickable alkyne by a synthetic strategy based
on Suzuki coupling. The pendant carboxylic chains, besides
imparting water solubility, present sites for both molecular inter-
actions and self-assembly enhanced by hydrogen bond formation.
Then, we investigated, by photoluminescence (PL) and NMR studies,
its aggregation behavior in an aqueous environment and the
variations caused by the presence of MyrCh and AChE. Finally,
we demonstrated that the alkyne-terminated AA-OT can be
covalently anchored to an azido-modified quartz surface via
the ‘‘grafting onto’’ approach based on the copper(I)-catalyzed
13,14
sensitive fluorescent probes for biodiagnostic purposes,
after
these oligomers have found extensive usage as a functional semi-
conductor material in organic electronics (e.g. field effect transistors,
15,16
photovoltaic cells, electroluminescent devices).
Targeting the enzyme acetylcholinesterase (AChE) is demon-
strated to be a valid strategy in symptom-treating Alzheimer’s
disease (AD), the most common form of dementia afflicting millions
of people worldwide. According to the so called ‘‘cholinergic
1
7
hypothesis’’, a cerebral deficit of the neurotransmitter acetyl-
choline (ACh) contributes to neural cell death, which along with
the damages caused by the abnormal accumulation of extracellular
b-amyloid plaques and intraneuronal fibrillar tangles, gives rise to
a chronic and progressive neurodegenerative disorder. Following
this hypothesis, cholinergic transmission enhancement, obtained
through drugs inhibiting AChE activity, reduces cognitive impair-
2
5
azide-alkyne cycloaddition (CuAAC) click chemistry reaction.
Remarkably, AA-OT chains retained their conformational sensi-
tivity towards MyrCh even in the bidimensional polymer brush
26–28
‘‘configuration’’.
The results reported here represent a proof
of concept for the viability of our functional ‘‘smart’’ material for
the fabrication of a biosensor-prototype to monitor AChE activity.
18–20
ments in AD patients.
Furthermore, the formation of amyloid
deposits (fibrillogenesis) appears to be facilitated by the AChE-
2
0,21
mediated hydrolytic breakdown of ACh.
Experimental
Materials and methods
Recently, simple and continuous fluorimetric assays for AChE
activity, based on CPEs fluorescence quenching, have been pro-
posed. However, these methods are limited by the time-consuming MyrCh (as the chloride salt) and AChE (from Electrophorus
preventive labelling procedure to mark the AChE substrates with a electricus – electric eel) were purchased from Sigma Aldrich.
22,23
quencher moiety.
To skip the tagging step, Shi and coworkers All the other chemicals and solvents were supplied by Sigma
Aldrich and Alfa Aesar and were used as received, without further
purification. Merck silica gel 60 (0.040–0.063 mm) was used for
column chromatography. Macherey-Nagel precoated TLC sheets
set up a simple colorimetric assay to monitor AChE activity through
analyte-induced conformational perturbations shown by the back-
bone of an anionic regioregular PT derivative. In this assay, the
24
cationic surfactant myristoylcholine (MyrCh) interferes with PT (silica gel 60 with fluorescent indicator UV254) were used for thin
aggregation by building both electrostatic and hydrophobic layer chromatography. The phosphate buffer (PB) solution,
2 4 2 4
interactions whose synergistic combination determines the 0.1 M Na HPO –NaH PO pH = 8, was freshly prepared before
disassembling of polymer aggregates. The enzymatic activity use with ultrapure water (resistivity about 18.2 MO cm at 25 1C)
s
29
of AChE induces the breakdown of the PT:MyrCh complex by obtained with a Millipore Direct-Q 3 system. The cut dimen-
2
sions of quartz slides (20 ꢀ 5.0 mm , 1.0 mm thickness) for
catalyzing the hydrolysis of MyrCh into myristate (Myr) and
choline (Ch, Scheme 1). As a consequence, the polymer chains
re-aggregate. These MyrCh-mediated disassembly–(re)assembly
grafting were suitably assessed to perform the PL characteriza-
tion of the AA-OT-functionalized quartz surface into 10 mm
events trigger surfactochromic variations, mainly in the PT absorp- path length cuvettes.
tion spectra, causing a colorimetric response.
Characterization techniques
Herein, we report the sensing application of a novel oligo-
thiophene-based probe, which tunes its optical properties in GC-MS analysis was performed on an Agilent 7890A combined
response to the AChE reaction on MyrCh, both in solution and with a detector (Agilent 5975C MSD) and electron ionization (EI)
even after its covalent immobilization on a substrate, thus laying source. AFM investigations were performed using a NT-MDT
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NTEGRA apparatus in a tapping mode under ambient condi- in PB were employed after overnight stirring at room temperature.
tions. Fluorescence microscopy images were obtained using a MyrCh was used in all the experiments well below its critical
24
Nikon Eclipse TE2000-U inverted confocal microscope.
micelle concentration (i.e. 2.5 mM). All aqueous samples (stock
Mass analysis. Matrix assisted laser desorption/ionization solutions, respective dilutions and mixtures) were analyzed imme-
time-of-flight mass spectra (MALDI-TOF MS) were recorded in diately after preparation. Optical characterization (absorption and
linear and reflectron modes using a Voyager-DE STR instru- emission properties) was performed at room temperature in
ment (Perseptive Biosystem) mass spectrometer equipped with 2.0 mm path length cuvettes.
a nitrogen laser (l = 337 nm, pulse width = 3 ns) working in
Fluorescence experiments on the AA-OT-modified quartz
positive ion mode at 10 atm. The accelerating voltage was surface were carried out by leaning the functionalized slide
0 kV, grid voltage and delay time (delayed extraction and time against one of the internal sides of the cuvette (path length =
ꢁ8
2
lag) have been optimized for each sample to achieve the highest 10 mm). Using this instrumental configuration, PL spectra were
mass resolution (M/DM). Laser irradiance has been maintained recorded both in dry (without solvent) and wet (with solvent)
0
slightly above threshold levels. Several matrices, such as 2-(4 - conditions (lexc = 360 nm). Excitation light was cut by a long
hydroxybenzeneazo)benzoic acid (HABA), trans-2-[3-(4-tert-butyl- pass optical filter (FGL435S Thorlabs), and in the case of a weak
phenyl)-2-methyl-2-propenylidene]malononitrile (DCTB), and 0.1 M PL signal, the detected spectra were examined to ensure that it
2,5-dihydroxybenzoic acid (DHB) in THF with and without a did not have filter origins.
cationization agent (CF COOK) were used. The best spectra were Structural characterization. Powders inserted into a sealed
3
recorded using the HABA matrix, obtaining a mass resolution of capillary were examined at 25 1C using X-ray diffraction beam-
about 1200–1500 in linear mode and 5000–6000 in reflector mode, line 5.2 at the Synchrotron Radiation Facility Elettra in Trieste
in the mass range from m/z 300 to m/z 1500. In a typical procedure, (Italy). The X-ray beam emitted by the wiggler source on the
ꢁ
1
1
3
mL of AA-OT solution (5 mg mL in THF) was mixed with 1 mL or Elettra 2 GeV electron storage ring was monochromatized by a
mL of a matrix solution; then, 1 mL of each analyte/matrix mixture Si(111) double crystal monochromator, focused on the sample
was spotted on the MALDI sample holder and slowly dried to allow and collimated by a double set of slits giving a spot size of
2
analyte/matrix co-crystallization.
0.2 ꢀ 0.2 mm . The energy adopted was 10.33 keV. Samples were
NMR. NMR spectra were recorded at 25 1C with a Bruker oriented by a four-circle diffractometer with a motorized gonio-
DMX spectrometer operating at 600 and 500 MHz. 1D H and metric head. Data were recorded with a 2M Pilatus silicon pixel
D H-DOSY experiments were performed on AA-OT 2.25 mM X-ray detector (DECTRIS Ltd, Baden, Switzerland) positioned
1
1
2
(in constitutional repeating unit) samples and AA-OT:MyrCh normally to the incident beam at a distance of 200 mm from
mixtures dissolved in 30 mM deuterated phosphate buffer, pH = 8, the sample. Data were calibrated against LaB powder (standard
6
at 25 1C. DOSY spectra were acquired with the convection- reference material 660a of NIST). The integration of 2D patterns
compensated two dimensional double stimulated echo bipolar was performed with the software Fit2D, which corrected the
3
0
36
pulse (DSTE-BPP) sequence. Matrices of 2048 (t2) by 80 points geometry, Lorentz, and beam polarization effects.
t1) were collected. The z axis gradient strength was varied
(
ꢁ
1
Synthetic procedures
linearly from 2% to 98% of its maximum value (53 G cm ).
Water suppression was achieved with the addition of a sculpt-
ing module. Self-diffusion coefficients were derived by fitting following an already reported procedure. To a solution of
Methyl 2-(thiophen-3-yl) acetate. This material was prepared
3
1
37
3
2
NMR data to the Stejskal and Tanner equation. The Stokes– 2-(thiophen-3-yl)acetic acid (Alfa Aesar, 1.0 g, 7.03 mmol) in
Einstein equation correlates the diffusion coefficient of a molecule MeOH (100 mL), a catalytic amount of H SO (0.069 g, 38 mL,
with its hydrodynamic radii, which in turn can be related to 0.70 mmol) was added. The reaction mixture was refluxed for
its molecular weight (M ) through the following proportion: 24 h and then evaporated under reduced pressure. The residue
0.4 mM dioxane was added as an internal was dissolved in EtOAc and the solution was extracted twice
reference to correlate relative diffusion coefficients with the M with a saturated NaHCO solution, twice with brine and finally
2
4
w
(
ꢁ1/3) 33–35
D p (M
w
)
.
w
3
of molecules and their aggregates in solution. This approach three times with water. The organic layer was dried over sodium
also allows the minimization of erroneous D estimates deriving sulfate and concentrated under reduced pressure to obtain the
from temperature fluctuations and changes in sample viscosity expected product as a viscous yellow oil (0.96 g, 6.15 mmol,
and density. The effects of AChE (8U/600 mL) activity on the yield = 87%). The crude product was used in the next synthetic
1
AA-OT (2.25 mM)–MyrCh (0.5 mM) complex were monitored step without further purification. H NMR (CDCl
3
): d 3.66
1
by recording 1D H spectra at regular time intervals (one every (s, 2H), 3.70 (s, 3H), 7.03 (dd, J = 1.2, 4.8, 1H), 7.14 (m, 1H),
minutes for the first 3 hours and one per hour for the 7.28 (dd, 3.0, 4.8, 1H). m/z (EI): 156.0 (M+).
5
following 5 hours). The enzymatic reaction was followed at
7 1C to optimize the AChE activity.
Methyl 2-(2,5-diiodothiophen-3-yl)acetate. A solution of methyl
3
2-(thiophen-3-yl)acetate (0.50 g, 3.20 mmol) in CHCl /CH COOH
3
3
Optical characterization. UV-vis spectra were obtained with a 1 : 1 (25 mL) was cooled to 0 1C and N-iodosuccinimide (Alfa
Perkin Elmer UV/VIS/NIR Lambda 900 spectrometer. PL spectra Aesar, 1.80 g, 8.00 mmol) was added. The reaction mixture was
were collected by a Spex 270M monochromator in conjunction stirred at room temperature for 18 h and then diluted with a
with a liquid nitrogen cooled CCD. The light source for PL was a 10% w/v KOH solution (40 mL). The organic layer was washed
monochromated xenon lamp (lexc = 360 nm). Polymer solutions twice with H
2
O, dried over sodium sulfate and evaporated
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under reduced pressure. The purification of the red residue by This residue was collected by filtration through a cellulose
flash chromatography with hexane–dichloromethane (1 : 1) as nitrate filter (0.2 mm) and dried under vacuum to furnish the
the eluent gave the expected product as a yellow-earth solid AA-OT derivative as a dark red solid (57 mg, 48% in coupling
1
1
(
(
(
0.97 g, 2.37 mmol, yield = 74%). H NMR (CDCl ): d 3.58 efficiency). H NMR (DMSO-d ): d 3.62–3.95 (br m, –CH COOH
3 6 2
1
s, 2H), 3.71 (s, 3H), 7.04 (s, 1H). H NMR (DMSO-d ): d 3.74 and –CRC–H), 4.92 (d, –CH CRC–), 6.84 (d, terminal benzene
s, 2H), 3.75 (s, 3H), 7.26 (s, 1H). m/z (EI): 407.9 (M+).
6
2
ring), 7.07 (d, terminal benzene ring), 7.19–7.40 (m, thiophene
2-(2,5-Diiodothiophen-3-yl)acetic acid. 1 N NaOHaq (1.90 mL, rings of constitutional repeating unit), 7.41–7.43 (m, terminal
1.90 mmol) was added dropwise to a solution of methyl 2-(2,5- benzene ring), 7.45–7.49 (m, terminal thiophene ring), 7.66–7.79
diiodothiophen-3-yl)acetate (0.31 g, 0.76 mmol) in THF (10 mL). (m, terminal thiophene ring), 12.71 (br s, –COOH).
When the starting material disappeared completely, as observed
Fabrication of AA-OT-modified quartz surface
by TLC, the reaction mixture was concentrated under reduced
pressure and the oily residue was diluted with ethyl acetate
Preparation of the (3-aminopropyl)triethoxysilane (APTES)
(
(
10 mL). The resulting mixture was acidified with 1 N HClaq monolayer. The quartz slides were first cut to the desired
2
2.0 mL, 2.00 mmol) and transferred to an extraction flask. The dimensions (20 ꢀ 5.0 mm , 1.0 mm thick) and then cleaned
organic layer was washed three times with 1 N HClaq, dried over in sequence with EtOAc, CH
2
Cl
2
and acetone prior to piranha
sodium sulfate and evaporated under reduced pressure to solution (96% H SO /35% H O 3 : 1 v/v) treatment (30 min at
2 4 2 2
obtain the expected product as a whitish solid in quantitative room temperature) to avoid any violent reactions. Warning: this
yield (0.30 g, 0.76 mmol). The crude product was used in the strong acid mixture has been reported to detonate unexpectedly;
1
next synthetic step without further purification. H NMR thus, handle it very cautiously. The activated substrates were
(
DMSO-d ): d 3.63 (s, 2H), 7.25 (s, 1H), 12.63 (s, 1H). m/z (EI): rinsed three times with ultrapure water and dried by a nitrogen
6
393.9 (M+).
stream immediately before performing APTES monolayer deposi-
1
-Bromo-4-(prop-2-ynyloxy) benzene. This material was pre- tion. All glassware used to covalently fix an APTES monolayer onto
3
8
pared following an already reported procedure. A mixture quartz slides were thoroughly cleaned with a 2% v/v Hellmanex II
of 4-bromophenol (Sigma Aldrich, 1.0 g, 5.78 mmol) in DMF solution (HNO /H SO 1 : 1) in distilled water, rinsed several times
15 mL) containing K CO (2.4 g, 17.4 mmol) and NaI (1.3 g, with ultrapure water, washed with acetone and finally dried in an
.67 mmol) was stirred for 30 minutes at room temperature, and oven at 70 1C overnight.
then propargyl bromide (Sigma Aldrich, 0.77 mL, 8.64 mmol) The preparation of the self-assembled monolayer (SAM) was
3
2
4
(
2
3
8
was added. The resulting mixture was stirred for 18 h at room accomplished by immersing the activated quartz surfaces in a
temperature, diluted with a saturated NH Cl solution and finally freshly-distilled toluene solution of APTES (6 mM) under a dry
4
extracted with EtOAc. The organic layer was dried over sodium nitrogen atmosphere. After 4 h stirring at room temperature,
sulfate and evaporated under reduced pressure. The purification the substrates were withdrawn from the solution, rinsed in
of the residue by flash chromatography with hexane–ethyl sequence with plenty of freshly-distilled toluene (under nitro-
acetate (8: 2) as the eluent gave the expected product as a lemon gen atmosphere), EtOH and CH
2
Cl
2
to remove any physisorbed
1
yellow oil (1.16 g, 5.50 mmol, yield = 95%). H NMR (CDCl
d 2.52 (t, J = 2.4, 1H), 4.66 (d, J = 2.4, 3H), 6.86 (d, J = 9.0, 2H), according to a previously reported procedure.
3
): contaminants and finally dried under a nitrogen stream,
3
9
1
7
.39 (d, J = 9.0, 2H). H NMR (DMSO-d ): d 3.47 (m, 1H), 4.89
Preparation of the azido-modified quartz surface. In a
6
(d, J = 2.0, 2H), 7.09 (d, J = 8.8, 2H), 7.58 (d, J = 8.8, 2H). m/z (EI): 4.0 mL capped vial equipped with a magnetic stir bar, a solution
2
10.0 (M+).
of 14-azido-3,6,9,12-tetraoxatetradecanoic acid (OEG-4, 0.030 mmol)
0
Oligo-[2-(thiophen-3-yl)acetic acid-co-thiophene] (AA-OT). To in CH
2
Cl
2
(1.5 mL) containing N-(3-dimethylaminopropyl)-N -
a Schlenk flask under nitrogen, 2-(2,5-diiodothiophen-3-yl)acetic ethylcarbodiimide hydrochloride (EDCꢂHCl, 9 mg, 0.047 mmol),
acid (100 mg, 0.25 mmol), thiophene-2,5-diboronic acid bis- 4-(dimethylamino)pyridine (DMAP, catalytic amount) and triethyl-
(pinacol) ester (Alfa Aesar, 101 mg, 0.30 mmol) and 3.0 mL of amine (TEA, 0.006 mL, 0.044) was stirred at 0 1C for 30 min before
dry toluene were added. After the complete dissolution of the immersing the APTES-functionalized substrates. After 24 h of
reagents, 1.5 mL of a degassed 2 M K CO solution, [1,3-bis(2,6- stirring at room temperature, the substrates were withdrawn from
2
3
diisopropylphenyl)imidazol-2-ylidene](3-chloropyridyl)palladium(II) the solution, rinsed in sequence with plenty of fresh CH Cl ,
2
2
2 2
dichloride (PEPPSIt-IPr, Sigma Aldrich, 3.5 mg, 0.005 mmol), EtOH and again CH Cl to remove any physisorbed contaminants
and one drop of Aliquat 336 were added under nitrogen to the and finally dried under a nitrogen stream.
mixture. The reaction was heated at 80 1C with stirring for 48 h
Preparation of the bidimensional oligothiophene brush by
and then 1-bromo-4-(prop-2-ynyloxy)benzene (53 mg, 0.25 mmol) CuAAC. In a 4.0 mL capped vial equipped with a magnetic stir
was added as an end-capping agent. Finally, after further 24 h bar, a solution of AA-OT (1.7 mg, 0.0077 mmol in constitutional
of reaction, thiophene-2-boronic acid bis(pinacol) ester (Sigma repeating unit) in DMF (1.5 mL) was stirred at room temperature
Aldrich, 53 mg, 0.25 mmol) was added as the other end-capper. overnight. Then, N,N-diisopropylethylamine (DIPEA, 0.001 mL)
The resulting mixture was allowed to react for another 24 h, and a catalytic amount of CuI were added. The azido-modified
then cooled to room temperature and transferred to an extrac- quartz slides were immersed and the resulting mixture was
tion flask. To the recovered aqueous layer, 1 N HClaq (8.0 mL, stirred at room temperature for 24 h. Subsequently, the sub-
8.0 mmol) was added and a brick red solid was precipitated. strates were taken out from the solution and sonicated in
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4
sequence with plenty of fresh DMF, 28–30% NH OHaq, 1 N
HClaq, ultrapure water, EtOH and CH Cl to remove any physi-
2 2
sorbed contaminants. The grafted AA-OT brush samples were
dried under a nitrogen stream and finally placed under inert
atmosphere for further characterization.
Results and discussion
Synthesis and properties of AA-OT
The design of the oligothiophene derivative AA-OT as a conforma-
tionally sensitive fluorescent probe for MyrCh detection was
inspired by similar anionic OT and PT derivatives, whose
1
0,40
backbones were found to be responsive to peptide analytes.
Generally, alternating conjugated copolymers are obtained by
palladium(Pd)-catalyzed polycondensations based on either
41
Suzuki or Stille cross-coupling reactions, which allows for easy
capping of the growing chains with the desired end-group. The
synthesis of PTs showing an alternation of substituted and unsub-
stituted thiophene rings along with the backbone has been com-
Scheme 2 Reaction scheme for AA-OT.
42–44
monly achieved by applying the Stille method.
examples have been reported about the Suzuki route to copoly-
merize two different thiophene building blocks carrying comple- required to covalently graft AA-OT to the azido-modified quartz
Conversely, few
45
mentary functionalities or to assemble bithiophene monomers surface by the CuAAC click chemistry reaction.
46
for polymerization. This discrepancy is mainly due to the hydro-
Our SPC synthetic protocol furnished AA-OT as an oligomeric
lytic deboronation of thiophene boronic acids/esters prior to C–C material. This statement was first suggested by NMR analysis
bond formation, which is a limiting feature that may be overcome and then corroborated by MALDI-TOF MS experiments. In fact,
41
1
by developing new effective Pd catalysts. Nevertheless, the Suzuki the H NMR spectrum of AA-OT in DMSO-d6 (Fig. 1S, ESI†)
coupling appears more convenient than the Stille one to plan a displays a mix of sharp and well-framed signals overlapping with
synthetic strategy in terms of chemical handling, width of func- broader components both in the aromatic region (B6.80–8.00 ppm)
tional groups tolerance, availability of purification methods from and in the downfield aliphatic region (B3.60–4.00 ppm), which is
41
undesired compounds and toxicity of byproducts.
ascribable to the presence of oligomer species showing both
Analogous metal-catalyzed coupling reactions have also been different lengths and regiorandom enchainments.
4
7
applied to obtain aryl oligomers. In particular, with the aim of
These findings are in agreement with the MALDI results.
developing more ‘‘user friendly’’ synthetic methods, great efforts The spectra of AA-OT were recorded using HABA as the matrix and
have been spent in implementing Suzuki approaches for the they confirmed the absence of oligomers longer than 10 aromatic
4
8
preparation of oligothiophenes.
residues (n = 4). The most intense peaks are attributable to species
Thus, AA-OT was built by adapting a previously reported terminated with both the expected thiophene and alkyne func-
method for obtaining similar terthiophene oligomers to our tionalities. All the other lower signals can be related to undesired
4
0
Suzuki polycondensation (SPC) conditions (Scheme 2). In the products originated from side reactions (e.g. deiodination
4
9
presence of the PEPPSIt-IPr catalyst and K
newly-synthesized di-iodo thiophene acetic acid derivative was and 3S, ESI†).
reacted with thiophene-2,5-diboronic acid bis(pinacol) ester. After On the basis of the consistent matching between the NMR
8 h, backbone growth was terminated by adding 1-bromo-4-(prop- and MALDI outcomes, we concluded that our synthetic strategy
2 3
CO base, the and deboronation) occurring in our SPC conditions (Fig. 2S
5
0
4
2
-ynyloxy)benzene and thiophene-2-boronic acid bis(pinacol) was successful in capping one of the two extremities of the
ester in sequence, which were used as end-cappers for chain AA-OT backbone with an alkyne group, which is crucial for
termination.
further click-grafting on a quartz surface.
The di-iodinated monomer was prepared starting from the
Therefore, we can refer to AA-OT as an oligothiophene deriva-
commercially available 2-(thiophen-3-yl)acetic acid, which was tive whose repeating unit consists of a bithiophene equipped with
protected as the methyl ester by Fischer esterification before an acetic pendant group. It can be regarded as a monotelechelic
iodination with N-iodosuccinimide. Basic hydrolysis of the oligoelectrolyte because it shows carboxylic side chains (hydro-
iodinated ester afforded the expected acid in a quantitative philic domain) hanging from a conjugated alternating thiophene-
yield (Scheme 1S, ESI†).
based backbone (hydrophobic domain), which bears a clickable
propargyl end-group.
3
8
Obtained by referring to an earlier published procedure,
the propargyl terminating agent was selected with the aim of
This peculiar structure based on a regiorandom sequence of
inserting on the oligothiophene scaffold, the alkyne end-group substituted–unsubstituted thiophene rings, accounts for the
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amphiphilic nature shown by AA-OT. In fact, it can be dissolved AA-OT in aqueous solution both in the absence and presence of
both in some organic solvents, such as DMSO, DMF and THF, different amounts of analyte.
and in basic aqueous environments. Longer polythiophene
First, we prepared six AA-OT samples with diverse concentra-
derivatives with more than 10 aromatic rings (not shown) were tions ranging from 0.045 mM to 2.25 mM by suitably diluting an
ꢁ1
found to be poorly soluble in PB and therefore, were not further aliquot of a freshly-prepared AA-OT stock solution (1.0 mg mL
developed. AA-OT is also partially soluble in other organic media 4.5 mM in constitutional repeating unit) with PB (pH 8). Then, we
i.e. MeOH and CHCl ) and displays a decreasing water-solubility recorded the absorption and the fluorescence emission spectra of
trend as the pH value is lowered. This observation is consistent each sample at room temperature (Fig. 1).
,
(
3
with the above-mentioned AA-OT precipitation upon the addi-
The absorption maximum in the UV-vis spectra of AA-OT
tion of 1 N HCl solution and can be explained in terms of a solutions in PB is centered at 390 nm, regardless of the concen-
progressive fall in the degrees of protonation of acetate side tration (Fig. 1A). This absorption is consistent with the oligo-
ꢁ
groups (–CH COO ) in response to the increase of acidity of meric nature of the AA-OT derivative, as demonstrated by the
2
1
AA-OT aqueous solutions. Thus, we assumed that AA-OT water- above discussed H NMR and MALDI results. Conversely, a
solubility is pH-dependent inasmuch regulated by the balance comparison of the normalized PL spectra highlighted an inter-
between the number of protonated carboxylic moieties and that esting concentration-dependent trend (Fig. 1B). Especially at
1
0
of the salified ones, along with oligothiophene chains. In low concentrations, the emission spectra of AA-OT samples
addition, the alternating side-chain substitution along the exhibited at least three main components, which were centered
AA-OT backbone can facilitate the reciprocal twisting of thio- around 450, 500 and 550 nm. As the concentration increases to
phene rings in limiting the steric congestion upon the solvent about two orders of magnitude, from 0.045 mM up to 2.25 mM,
change, which makes the system more sensitive to external we observed a corresponding change to the mutual intensity
stimuli. This intramolecular conformational ‘‘adjustment’’ (fold- of each component. Such spectral changes reflect a different
ing) can enhance p–p interactions among different oligothiophene population of conformations as a function of AA-OT concen-
chains (confirmed by solid state X-ray diffraction analysis, Fig. 11S, tration. The calculated Commission Internationale de l’Eclairage
ESI†), leading to aggregation. In basic aqueous environments, (CIE) chromaticity coordinates varied from (0.236, 0.296) for the
this tendency can further be strengthened by the establishment most diluted sample, to (0.289, 0.408) for the most concentrated
of H-bonds that bridge together the acetate side groups and the one, as displayed by Fig. 1C. This remarkable variation in
protonated ones.
fluorescence properties of AA-OT is related to the establishment
Thus, we thoroughly studied the aggregation behavior of AA-OT of multiple noncovalent interchain interactions (i.e. p-stacking
in water by performing spectroscopic investigations through parallel and H-bonds), which progressively drive AA-OT into self-
PL and NMR studies in a PB solution at pH 8, which was selected to aggregation. In addition, as shown in the inset of Fig. 1B, PL
strike a satisfying balance between AA-OT solubility and optimum intensity increases more significantly passing from 0.045 mM
pH value for AChE activity.
to 1.35 mM than from 1.35 mM to 2.25 mM. This observation
led us to suppose that H-bonds among acetic acid/acetate side
residues represent the primary driving force for the formation
of AA-OT aggregates.
Optical characterization of AA-OT interacting with MyrCh in
phosphate buffer solution
To evaluate the ability of the anionic oligothiophene derivative
As sample concentration increases over 1.35 mM, p-stacking
AA-OT to interact with the cationic surfactant MyrCh by form- hydrophobic forces become more intense and bring thiophene
ing an electrostatic complex, we analysed the optical features of backbones closer, strengthening aggregation and inducing PL
Fig. 1 Absorption (A) and emission (B) spectra of AA-OT samples in PB (pH = 8). In frame B, the arrows indicate the trend of spectral change with
increasing concentration; in the inset, the variation of PL intensity with AA-OT concentration is reported (intensity values taken from as-recorded spectra,
l = 525 nm). (C) Resulting variation in the CIE coordinates of PL spectra passing from the lowest to the highest concentration measured. Excitation was at
3
60 nm in photoluminescence experiments.
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quenching. Thus, AA-OT aqueous samples in PB at room tem- sensitivity for our thiophene-based sensing material in detect-
perature are dispersions of soluble aggregates. Isolated chains are ing MyrCh, about one order of magnitude higher than analo-
ꢁ5
24
probably present only at concentration lower than 10 M.
Then, we prepared three AA-OT samples (0.45 mM) contain-
gous colorimetric assays.
Finally, we assessed the ability of the label-free AA-OT:MyrCh
ing different amounts of MyrCh (i.e. 0.01, 0.05 and 0.10 mM) by system in real-time monitoring AChE activity by performing a
mixing aliquots of AA-OT and MyrCh stock solutions and ‘‘mix-and-detect’’ assay. We prepared a PB mixture containing
diluting with PB. These samples did not exhibit any chromatic AA-OT and MyrCh using the MyrCh:AA-OT stoichiometry ratio of
variations visible to the naked eye compared to the reference, B0.2 and then we added AChE in the required amount to obtain
ꢁ1
0
.45 mM MyrCh-free sample in AA-OT. No wavelength shift of a final enzyme concentration of 1.5 U mL . The time course of
the absorption maximum was recorded when the corresponding the resulting sample was immediately followed by recording PL
UV-vis spectra were compared (Fig. 4S, ESI†), confirming the spectra at room temperature at regular time intervals (Fig. 2C).
unaided eye observation and the impossibility to apply our AA- As the incubating time proceeded from 0 to 180 min, we observed
OT derivative in a colorimetric assay for MyrCh detection.
a gradual increase of ratio between the intensities of spectral
On the other hand, the comparison of PL spectra showed components centered around 550 and 450 nm, corresponding to
intriguing changes (Fig. 2A). In fact, as the MyrCh concentration a considerable variation of CIE chromaticity coordinates: from
increased, we observed a gradual decrease of the ratio between (0.255, 0.355) to (0.297, 0.415), as displayed in Fig. 2D.
the intensities of spectral components centered around 550 and
Indeed, the addition of AChE to the AA-OT:MyrCh mixture in
450 nm. This variation led the spectrum of sample with the PB at pH 8 was demonstrated to produce an opposite effect with
highest concentration of MyrCh (0.10 mM) to be perfectly super- respect to the surfactochromic variations induced by MyrCh.
imposable with that recorded for 0.045 mM in AA-OT, repre- This was associated to the catalytic hydrolysis of MyrCh ester
senting the most diluted MyrCh-free conditions (Fig. 2B). We bond performed by AChE.
interpreted this observation as the consequence of the dis-
The enzymatic charge reversal of the cationic MyrCh into the
assembling effect of MyrCh, whose interaction with oligo- anionic Myr not only caused the collapse of the AA-OT:MyrCh
thiophene chains is able to promote AA-OT disaggregation by electrostatic complex, but also the ‘‘zip-up’’ of AA-OT chains
competitive interference against the forces stabilizing AA-OT into aggregates (re-assembling effect). Moreover, the absence of
aggregates. Presumably, our oligothiophene derivative behaves any relevant time-dependent changes in the PL spectrum after
similar to the anionic PT zipper sensors previously described by 2 hours allows us to assume that the hydrolytic breakdown of
5
1
McCullough and coworkers. MyrCh is a quaternary ammonium MyrCh was almost completed.
cation bearing a bulky tetradecanoyloxy ethyl group. To accom-
The conformational sensitivity shown by AA-OT in detecting
modate the steric hindrance of this large counterion, p-stacked both the presence and the AChE-triggered degradation of MyrCh
and H-bonded AA-OT chains are induced to adopt a more was further investigated by NMR studies.
disordered phase by unzipping. Therefore, a molecular recogni-
tion event involving AA-OT and MyrCh can be assumed to occur
based on the synergistic interplay of electrostatic and hydro-
NMR characterization of AA-OT aggregation state
phobic interactions.
NMR diffusion ordered spectroscopy (DOSY) is a powerful tool
The smallest significant changes of AA-OT emission proper- to analyse complex mixtures, allowing for the identification of
ties, in response to the presence of different amounts of MyrCh, molecular components through chemical shift analysis, and
were observed when the MyrCh:AA-OT stoichiometry ratio was to simultaneously derive information on their diffusion coeffi-
5
2,53
about 0.02 (MyrCh 0.01 mM). This value suggested a good cients.
Diffusion coefficients are sensitive to structural
Fig. 2 (A) Comparison of the emission spectra of 0.45 mM AA-OT solutions in PB (pH 8) with increasing amount of MyrCh; the arrows indicate the trend
of spectral change with increasing MyrCh concentration. (B) Superposition of the emission spectrum of a 0.45 mM AA-OT + 0.10 mM in MyrCh solution
with 0.045 mM AA-OT solution. (C) Time course of photoluminescence spectrum of a 0.45 mM AA-OT + 0.10 mM in MyrCh solution in the presence of
ꢁ1
AChE (1.5 U mL ) at room temperature; the arrows indicate the trend of spectral change for increasing incubation time. (D) Resulting variation in CIE
coordinates of PL spectra after AChE incubation. Excitation was at 360 nm for all the analyses.
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properties of the observed species such as weight, size, shape as indicates that, upon MyrCh cleavage, the equilibrium reverts
well as binding and aggregation phenomena.
back to the assembled oligomers state.
In the present study, DOSY was used as a method to monitor
the distribution of AA-OT aggregate sizes and to follow the
disassembly process mediated by MyrCh. The diffusion coeffi-
Fabrication and characterization of the AA-OT-functionalized
quartz surface
cients (D) obtained for a 2.25 mM AA-OT sample were in the To date, robust, efficient, and reproducible synthetic approaches
ꢁ
10
2
ꢁ1
range of 3.2–1.4 ꢀ 10
m s , corresponding to a distribution for obtaining surface-grafted conjugated polymer brushes have
of molecular weights between B700 Da and 8 kDa (Fig. 3A). been scarcely reported. This depended on the strict reaction
When the sample was titrated with MyrCh, an increase of D conditions required to avoid CP degradation during the grafting
values was clearly observed for AA-OT resonances (Fig. 3A). This procedure. Recently, Gopalan and coworkers optimized a
is consistent with the onset of lower molecular weight species, versatile ‘‘grafting onto’’ methodology in which the CuAAC
highlighting the disaggregating ability of MyrCh. Conversely, click chemistry reaction was employed as a mild and high-
no significant differences in both the AA-OT chemical shifts yielding synthetic tool to covalently bind an ethynyl-terminated
and diffusion coefficients were observed when the oligomers polythiophene derivative onto oxide surfaces. We were inspired by
were titrated with Ch (Fig. 5S, ESI†). These data suggest the this work to fabricate a bidimensional AA-OT brush. Our ‘‘grafting
relevant role of the Myr hydrophobic chain in interacting with onto’’ approach is based on a three step synthetic pathway, culmi-
54
AA-OT and dissociating its aggregates.
nating with the click coupling between the alkyne-terminated
NMR was also used to monitor the effects of AChE enzymatic AA-OT and a suitably azido-modified quartz surface, as depicted
activity following the time-dependent cleavage of MyrCh and in Scheme 3.
the re-assembly of AA-OT. A series of 1D spectra was acquired
A preliminary ‘‘piranha solution’’ treatment was performed
on the AA-OT : MyrCh 1 : 0.2 sample after enzyme addition. to activate the clean quartz slides to increase the number of
The onset and increase of free Ch resonances is a simple and exposed surface hydroxyl groups. Quartz was selected as a flat
straightforward way to follow the enzymatic cleavage of MyrCh substrate for immobilizing our oligothiophene-based sensing
due to AChE activity. The change of Ch methyl resonance probe because of its suitability for optical studies. The sub-
intensity (right inset of Fig. 3B) is plotted as a function of time sequent SAM of APTES was deposited according to literature to
3
9
in Fig. 3C (black diamonds). Simultaneously, AA-OT signals conclude step one. Then, the desired azido group was easily
shift back towards the resonances observed for the oligomer in inserted on the APTES functionalized substrates by linking
the absence of MyrCh. In particular, the shift of the oligomer the –NH groups protruding from the surface with the –COOH
2
resonance at B7.5 ppm in the AA-OT : MyrCh 1 : 0.2 sample group of the bifunctional tetraethylene glycol derivative
towards the signal typical of the AA-OT aggregated state (AA-OT 14-azido-3,6,9,12-tetraoxatetradecanoic acid via amide bond
without MyrCh) as a function of time (Fig. 3C, grey diamonds) formation (step two). Finally, AA-OT was grafted on the result-
mirrors the onset of Ch. Taken together, all the NMR data ing azido-modified quartz surface by CuAAC in the presence of
Fig. 3 (A) Overlay of 2D DOSY spectra of AA-OT in the presence of MyrCh at 0 (black), 0.1 (blue), and 0.2 (red) MyrCh:AA-OT molar ratios, at 25 1C,
1
6
00 MHz. Aromatic resonances region is displayed. (B) Overlay of H 1D spectra recorded at 37 1C of AA-OT : MyrCh 1 : 0 (black), AA-OT : MyrCh 1 : 0.2
(red), and AA-OT : MyrCh 1 : 0.2 after 9 min (orange), 30 min (yellow), 1 h (green), 3 h (cyan), and 8 h (blue) after the addition of AChE enzyme. The inset on
the right displays the increase of the Ch methyl resonance. (C) The increase in the intensity of the free Ch methyl groups (black diamonds, units on left
axis) is plotted vs. time. On the same graph, the change of the oligomer resonance (Dd) at ca. 7.5 ppm in the AA-OT : MyrCh 1 : 0.2 sample with respect to
the AA-OT resonance in the absence of MyrCh is plotted as a function of AChE reaction time (gray diamonds, units on right axis).
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Scheme 3 ‘‘Grafting onto’’ approach for fabricating an AA-OT bidimensional brush onto quartz slides via CuAAC.
CuI/DIPEA as the catalytic coupling agents (step three). The UV lamp excitation. A satisfying homogeneity in surface cover-
conditions for this last step were first optimized using a propargyl- ing was assumed by comparing the images reported in Fig. 4,
55
functionalized rhodamine B, prepared as already reported, and and further confirmed by AFM analysis (Fig. 8S, ESI†). Never-
selected as a model fluorescent probe in the place of AA-OT theless, microscope analysis also suggested that the average
(
Fig. 6S, ESI†).
The thorough ultrasound treatment with plenty of various be related to the collapsed conformation adopted by the
solvents (i.e. DMF, NH OHaq, HClaq, water, EtOH and CH Cl block arms of the brush in a ‘‘dry’’ state. The oligothiophene
emission of the ultrathin AA-OT film is rather weak. This could
4
2
2
)
performed during work-up procedures allows us to exclude the segments are ‘‘frozen’’ in a stacked situation in which their
physisorption of undesired materials on the functionalized proximity partially quenches the emission of the fluorescent film.
slides. Their stability after incubation in good solvents, such No significant PL emission could be recorded even in ‘‘wet’’
as DMSO, and exposure to ultrasounds for 60 min at 37 1C conditions, i.e. after the incubation of the functionalized sub-
further corroborated this statement. In both the cases, no strates in a good solvent such as DMSO.
fluorescent traces were detectable in solutions where substrates
With the aim of overcoming this drawback and improving
were incubated. Water contact angle measurements indicated the poor wettability of the AA-OT film by perturbing the
that surface wettability increases after APTES functionalization aggregation state of the grafted chains, we tried to employ the
and again after OEG-4 grafting (Fig. 7S, ESI†). AFM analysis above-discussed disassembling effect of MyrCh to make a
attested that RMS roughness gradually increases after each breach in this locked system. We incubated the functionalized
grafting step (Fig. 8S and 9S, ESI†). XPS analysis showed the slides in a 0.5 mM MyrCh solution in DMSO, in a cuvette,
gradual shift towards lower binding energy values of the overnight, and then collected PL spectra by keeping the sub-
nitrogen signal (N1s), in agreement with the chemical trans- strates immersed (‘‘wet’’ conditions). This procedure triggered
formation of the N-containing groups (i.e. amino into amide the appearance of a signal centered at around 600 nm, which
and azide into triazole ring), and the appearance of sulfur
signals (S2p and S2s) in the last grafting step (Fig. 10S, ESI†).
All these evidences supported the inference that our ‘‘click-
grafting’’ approach was successful in covalently anchoring
AA-OT onto quartz. Such a tailored surface can be considered
a bidimensional brush showing a block structure in which a
hydrophilic and flexible oligoethylene glycol spacer is bound to
a more rigid and hydrophobic segment by a triazole ring. This
di-block architecture should impart the freedom of movement
to AA-OT segments, which is necessary to retain the sensing
response characteristic of AA-OT in solution.
The AA-OT brush samples were preliminary characterized by
fluorescence microscopy analysis. A grafted quartz slide was
placed adjacent to a pristine slide and observed in this configu-
Fig. 4 (A) Direct comparison between pristine (left) and grafted (right)
quartz substrates as seen by optical microscopy. (B) Same view taken
ration both in transmission and fluorescence modes. The
under fluorescence microscopy. The pristine substrate appears dark. The
functionalized sample provided a brightly colored image under scale bar is 40 mm.
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demonstrate that the sensing probe partially retains its recep-
tivity to the enzyme activity, even when AA-OT is anchored on
quartz.
Even though such a response should be further optimized
through careful synthetic design, our ‘‘grafting onto’’ approach
certainly demonstrates important progress towards the goal
of realizing a real-time sensing platform based on grafted
AA-OT.
Indeed, we expect that by increasing the wettability/solubility
of the thiophene-based oligomers, combined with a further
reduction of the Mw dispersion in the oligomeric mixture, a
sensing response suitable to monitor AChE activity can also be
achieved in the bidimensional brush configuration.
Conclusions
In summary, we developed a sensing system based on the con-
formational changes of the anionic oligothiophene AA-OT, both
in solution and in a ‘‘grafted’’ state. This functional material is
Fig. 5 PL characterization of the AA-OT grafted brush. All the samples
were analyzed after incubation in a MyrCh solution. The curves in the characterized by a pH-dependent solubility and concentration-
main plot were smoothed for clarity; the magnified plot shows the
as-recorded spectra. The cartoon on the top represents the stacked/open
dependent aggregation behaviour in aqueous environments.
Upon interaction with MyrCh in solution, AA-OT chains dis-
assemble. This effect, observed by DOSY NMR experiments,
state of the grafted AA-OT arms in response to AChE and MyrCh action,
respectively.
induces a detectable PL change in mutual intensities of the main
spectral components, ascribed to different conformational states
populated by the oligothiophene probe. By catalyzing the hydro-
did not disappear after drying the substrates under a nitrogen
lysis of MyrCh, the AChE enzyme is able to restore the AA-OT
stream (light blue curve in Fig. 5). Most likely, the significant
aggregated state, thus allowing enzymatic activity to be monitored
increase in emission intensity was due to the opening of the
by means of fluorescence spectroscopy.
stacked system upon interaction with MyrCh. The surfactant may
AA-OT was designed as an alkyne-terminated oligomer to be
induce the separation of the grafted AA-OT chains by triggering
covalently anchored on a surface by the CuAAC click chemistry
the same phenomena observed in the above-discussed PL experi-
reaction. Hence, it was successfully grafted onto an azido-
ments performed in solution.
modified quartz support. We demonstrated that the fabricated
Thus, this event of fluorescence ‘‘turn-on’’ proves that our
bidimensional brush not only exhibits a clear fluorescence
oligothiophene-based probe is able to retain its conforma-
turn-on response in the presence of MyrCh, but also preserves
tional sensitivity, even in the configuration of a bidimensional
the AA-OT conformational sensitivity towards the detection of
brush.
AChE-mediated cleavage of MyrCh. The ability of AA-OT to operate
To evaluate the sensing properties of our platform for
in pH conditions that are close to physiological ones, suggests its
monitoring AChE activity, we moved this ‘‘turned-on’’ quartz
potential applications in biosensing. Upon appropriate material
slide into a cuvette filled with PB solution. MyrCh (1.0 mM) was
optimization, we envision to address our bidimensional brush to
also added because pure PB solution could wash away some
the fabrication of device-prototypes (biosensors) to be employed
MyrCh molecules already present on the sensing brush. As
as reliable diagnostic ‘‘tools’’ in drug discovery for the high-
shown in Fig. 5 (teal curve), a fluorescent peak is still detectable
throughput screening of AChE inhibitors. Furthermore, we plan
in the aqueous medium. The evident reduction of this PL signal
to investigate the effectiveness of such biosensors in discriminat-
compared to the one detected in DMSO is probably due to
ing between native and aggregated states of fibrillogenic proteins,
the incomplete wettability of the brush in PB, which leads the
10,14,40
as suggested by recent studies in this field.
AA-OT arms to partially pack themselves, similar to what happens
under ‘‘dry’’ conditions. Nevertheless, we were able to test the
enzyme activity on the grafted AA-OT.
Acknowledgements
In fact, by adding AChE to the system, we could clearly
detect the disappearance of the weak emission peak after This work was financially supported by the MIUR-CNR project
about 1 h of incubation (violet curve in Fig. 5). This event of ‘‘Nanomax N-CHEM’’, MIUR-CeRICT scrl PON 04a2_C project
fluorescence ‘‘turn-off’’ (60% quenching by ratio of integrated ‘‘SMART HEALTH 2.0’’. S.T. and K.P. acknowledge the Alzheimer’s
PL spectra area) is attributed to the AChE-triggered hydrolytic Association (NIRG-14-321500) for funding. K.P. gratefully
breakdown of MyrCh, which causes the system to assume acknowledges the De Marco Foundation for financial support.
a more stacked conformation (see cartoon in Fig. 5). The data Thanks are due to Dr L. Barba of IC-CNR (Trieste) for
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