Hybrid nanoreceptors for high sensitivity detection of small molecules by NMR chemosensing

Article abstract of DOI:10.1039/d0cc07559k

“Nanoparticle-assisted NMR chemosensing” combines magnetization transfer NMR techniques with the recognition abilities of gold nanoparticles (AuNPs) to isolate the NMR spectrum of relevant organic species in mixtures. The efficiency of the magnetization transfer is crucial to set the detection limit of the technique. To this aim, a second generation of nanoreceptors obtained by the self-organization of 2 nm AuNPs onto the surface of bigger silica nanoparticles shows better magnetization transfer performances, allowing the detection of analytes in water down to 10 μM concentration using standard instrumentation.

Full text of DOI:10.1039/d0cc07559k

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Hybrid nanoreceptors for high sensitivity
detection of small molecules by NMR
chemosensing†
Cite this: Chem. Commun., 2021,
57, 3002
Received 17th November 2020,
Accepted 27th January 2021
Federico De Biasi, ‡*ab Daniele Rosa-Gastaldo, ‡*a Fabrizio Mancin
and
a
a
Federico Rastrelli
DOI: 10.1039/d0cc07559k
rsc.li/chemcomm
‘‘Nanoparticle-assisted NMR chemosensing’’ combines magnetization target species.4–6 Nanoparticle-assisted NMR chemosensing
transfer NMR techniques with the recognition abilities of gold nano- makes no exception, as it exploits the recognition abilities
particles (AuNPs) to isolate the NMR spectrum of relevant organic of thiol-protected gold nanoparticles (AuNPs) to extract the full
species in mixtures. The efficiency of the magnetization transfer is 1H-NMR spectrum of the target analyte that binds to the coating
crucial to set the detection limit of the technique. To this aim, a monolayer.5 Since the NMR signals carry unambiguous infor-
second generation of nanoreceptors obtained by the self-organization mation about the interacting molecule, any interferent species,
of 2 nm AuNPs onto the surface of bigger silica nanoparticles shows even if recognized by the nanoreceptor, can be easily identified
better magnetization transfer performances, allowing the detection as a false positive. Relying on a magnetization transfer driven by
of analytes in water down to 10 lM concentration using standard the nuclear Overhauser effect (NOE),7,8 this method suffers from
instrumentation.
low sensitivity, which stimulated continuous efforts over the
years to decrease its limits of detection (LODs).9–11 On the NMR
Detection and identification of selected substances in complex side, we proposed recently a new approach able to improve the
mixtures are primary quests in chemistry. To this aim, the most detection performances down to 50 mM thanks to a high-power
commonly used procedures rely on chromatography to break water-mediated Saturation Transfer Difference (HP wSTD)
down the complexity of the problem into the analysis of many scheme.12 On the other hand, nanoreceptors optimization,
separate fractions comprising fewer components.1 However, essentially aimed at increasing the affinity for the analytes,
sample manipulation in such protocols is rather time-consuming, provided so far only marginal benefits.11,13
and the possibility to directly analyse untreated samples is still
highly desirable in order to optimize the experimental times and to nanoreceptors that, with no synthetic effort, optimizes the
minimize errors. magnetization transfer efficiency in STD-based experiments,
In this communication, we present a second generation of
On such premises, Nuclear Magnetic Resonance (NMR) spectro- allowing us to break and further push down the LOD of
scopy can be considered one of the most powerful techniques for nanoparticle-assisted NMR chemosensing. All the NMR experi-
the direct investigation of organic molecules. Nevertheless, the ments in this communication were performed at 25 1C on a
1
wealth of information it delivers when applied on plain mixtures Bruker AVANCE III spectrometer operating at 500.13 MHz H
is typically overwhelmed by spectral crowding. For this reason, Larmor frequency and equipped with a 5 mm z-gradient broad-
many advanced NMR techniques (e.g. diffusion-ordered spectro- band inverse (BBI) non-cryogenic probe.
scopy, DOSY)2,3 have been developed in the attempt to separate the
The theory of NMR relaxation predicts that the efficiency of
spectra of the mixtures into those of the single components. In the magnetization transfer increases as the tumbling rate of the
several applications the experimental output is further enhanced by receptor decreases. Consequently, stronger STD signals are
the intervention of an external agent to label the signals of a expected upon increasing the size of the nanoreceptors (Section
S1 of the ESI†).14 Nonetheless, the plain use of larger AuNPs in
a
`
Department of Chemical Sciences, Universita degli Studi di Padova Via Marzolo 1,
NMR chemosensing experiments is not trivial and brings along
several drawbacks. First, the synthetic yields of large AuNPs are
usually low. Second, large sizes and small coating-to-core ratios
make the characterization of the monolayer much more difficult.
Third, considerably higher amounts of large nanoparticles
would be required since analyte binding occurs at the surface.
35131 Padova, Italy. E-mail: daniele.rosagastaldo@unipd.it
b
`
Department of Chemistry, Universita degli Studi di Firenze Via della Lastruccia 3-13,
50019 Sesto Fiorentino, Italy. E-mail: federico.debiasi@unifi.it
† Electronic supplementary information (ESI) available. See DOI: 10.1039/
d0cc07559k
‡ These authors contributed equally.
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Fig. 1 Gold nanoparticles (Section S2 of the ESI†) and analytes used in this
work. 1: negatively charged AuNPs; 2: positively charged AuNPs; 3: serotonin;
4: homovanillic acid; 5: L-phenylalanine; 6: dopamine; 7: L-serine. The
1H-NMR spectrum of each analyte, complete of signals assignation, is
reported in Section S6 of the ESI.†
Fig. 2 TEM micrographs of the gold–silica supramolecular nanoconju-
gates. (a) 1-AuNPs on LUDOXs CL NPs. (b) 2-AuNPs on LUDOXs HS NPs.
Gold nanoparticles appear as black spots while the larger SiO2NPs as grey
circles.
Fourth, the affinity of the nanoreceptors for the analytes can be
negatively affected by the decreased surface curvature.15
the analyte recognition process), PEG was chosen also because its
Our hypothesis was that the self-organized nanoconjugates 1H-NMR spectrum in water only consists in a rather sharp singlet
obtained from the spontaneous assembly of a corona of small at 3.6 ppm. DLS experiments in the presence of PEG2000 con-
AuNPs on the surface of large inert nanoparticles could combine firmed the formation of nanoconjugates with similar size and
the benefits of both large and small nanoreceptors. To this aim, z-potential, without any sedimentation for at least 5 h, exceeding
we chose two AuNPs previously used to detect designer drugs, the time needed for sample preparation and collection of the
biogenic amines and carboxylates (1-AuNPs and 2-AuNPs, NMR experiments (about 4 h). We further confirmed the self-
Fig. 1).10,11 Since these are coated with charged thiols, they organization of the AuNPs on the surface of the silica nano-
should stick to the surface of bigger inorganic nanoparticles of particles via TEM analysis. From the images reported in Fig. 2, it
opposite charge. As a result, their tumbling rate in solution would appears how the gold nanoparticles (black dots) assembled onto
be significantly lowered without affecting the local monolayer the surface of the silica nanoparticles (grey circles) form a scattered
dynamics that is responsible for their binding affinity.16
Commercially available colloidal amorphous silica nano-
corona.
Another evidence of the formation of the AuNPs@SiO2NPs
particles (SiO2NPs; LUDOXs HS, negatively charged, and nanoconjugates, together with information about their com-
LUDOXs CL, positively charged) appeared to best fit our scope position, was obtained by H-NMR experiments (Section S5 of
1
as they are significantly larger (about 20 nm) than the typical the ESI†). Small 1 mL aliquots of a 1% w/w colloid of silica
2 nm AuNPs used in chemosensing applications.
nanoparticles suspended in a 0.01% w/w aqueous solution of
As a first step, we investigated the self-organization of the PEG2000 were sequentially added to an NMR tube containing
AuNPs@SiO2NPs nanoconjugates. The colloidal silica suspensions 500 mL of 200 mM phosphate buffer and 100 mM (in coating
in phosphate buffer were analysed using Transmission Electron thiols) AuNPs in H2O : D2O = 90 : 10. The 1H-NMR spectra of the
Microscopy (TEM) to determine the size of the SiO2NPs. We sample, recorded after every addition, showed a progressive
measured an average particle diameter of 17.1 Æ 2.5 nm for the disappearance of the signals of the coating monolayer due
LUDOXs CL and 16.9 Æ 2.3 nm for the LUDOXs HS. Dynamic to the enhanced transverse relaxation as a result of their self-
Light Scattering (DLS) was used to assess their hydrodynamic assembly onto the surface of the SiO2NPs. The NMR titrations
diameter and z-potential, obtaining a value of 41.1 Æ 0.7 mV for showed also that free gold nanoparticles are not in chemical
the LUDOXs CL and À30.1 Æ 0.8 mV for the LUDOXs HS exchange with those bound to the SiO2NPs, since after each
(Section S4 of the ESI†). Upon the addition of oppositely addition of the silica colloids the residual AuNPs signals exhibited
charged AuNPs, the z-potentials shifted to opposite values only decreased intensities and not progressive broadening. Titra-
(À22.5 Æ 2.32 mV for 1-AuNPs@LUDOXs CL and 34.1 Æ tion of 2-AuNPs with LUDOXs HS is reported in Fig. 3 as an
1.0 mV for 2-AuNPs@LUDOXs HS), suggesting that the AuNPs illustrative example. In both the examined cases, completely
were successfully assembling on the silica surface. DLS measure- formed nanoconjugates were characterized by an AuNPs/SiO2NPs
ments revealed a slight increase of the average particles size, as ratio of around 33, corresponding to a footprint of about
detailed in Section S4 of the ESI.† Accordingly, we noticed the 27 nm2 per gold nanoparticle and an exclusion radius of about
sedimentation of the nanoconjugates within a few minutes after 3 nm, which agrees well with the AuNPs hydrodynamic radii
the addition of the AuNPs. These effects, likely due to the cross- measured via DLS (4.0 Æ 1.3 nm for 1-AuNPs and 4.2 Æ 0.4 nm
linking ability of the gold nanoparticles or to the overall charge for 2-AuNPs).
neutralization of the AuNPs@SiO2NPs aggregates, were prevented
In the NMR titrations, sedimentation was observed after the
by adding to the solution a little amount of polyethylene glycol addition of an excess of 1% w/w LUDOXs suspension, even in
(PEG2000). Besides its known ability to sterically stabilize silica the presence of higher PEG2000 concentrations. This behaviour
colloids17 and the absence of charge (which avoids interferences in was different from that observed in the DLS experiments,
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detected in the absence of additives and in the presence of
LUDOXs CL only. More remarkably, at this analyte concentration,
also 1-AuNPs alone were unable to produce signals above the LOD
(set as the signal intensity being larger than three times the
standard deviation of the baseline noise). Only in the presence
of the 1-AuNPs@LUDOXs CL nanoconjugates (panel e of Fig. 4),
all the serotonin signals appeared in the final STD spectrum.
Nicely enough, signals of L-phenylalanine (which has no affinity
for 1-AuNPs) were not detected in any spectrum, confirming
that the analyte binding selectivity of 1-AuNPs was not altered
by the adsorption onto the colloidal silica for what concerns
our experiments.
The same set of experiments was repeated using the system
with opposite charges. In this case, 2-AuNPs@LUDOXs HS
were used to detect 10 mM homovanillic acid, a typical catechol-
amine metabolite, again in the presence of 10 mM L-phenylalanine
as a representative interfering species.18,19 Note that, for example,
homovanillic acid concentration above 10 mM in children’s urine
can be related to the occurrence of neuroblastoma.20 In this case,
two artefact singlets stemming from homovanillic acid do appear
also in the control spectrum with no additives (panel b of Fig. 5), as
well as in all the other spectra. This is because such singlets
resonate too close to the water signal, and receive an extra
irradiation from the fringe of the saturating field employed in
HP wSTD experiments.12,21 Nonetheless, all the homovanillic acid
resonances were detected only in the presence of the supramolecular
nanoconjugates. Again, selectivity over non-recognized analytes
Fig. 3 1H-NMR spectra of 100 mM (in coating thiols) 2-AuNPs and 200 mM
phosphate buffer in H2O : D2O = 90 : 10 after subsequent additions of
a
suspension of LUDOXs HS silica nanoparticles. Asterisks denote
impurities while the circle indicates the PEG2000 signal. Numbers on
the right report the estimated SiO2NPs/AuNPs ratio after every addition
(Section S5 of the ESI†).
where gold nanoparticles were present in slight excess after their
addition and no sedimentation was observed. This suggested that
stable nanoconjugates require, beside the presence of PEG, also
the formation of a complete AuNPs corona on the surface of the
silica nanoparticles.
Having observed the formation of stable nanoconjugates, we
investigated their ability to detect selected analytes at low
concentrations. 1-AuNPs@LUDOXs CL were used to analyse a
mixture of 10 mM serotonin and 10 mM L-phenylalanine, 5 times
lower than the previously established detection limit, by means
of HP wSTD experiments.12 Results are reported in Fig. 4, along
with other HP wSTD spectra acquired in the absence of nano-
particles or including either AuNPs or SiO2NPs. In agreement
with theory and previous results, analyte signals were not
Fig. 5 (a) 1H-NMR spectrum of 10 mM homovanillic acid (4) and 10 mM
L-phenylalanine (5) in H2O : D2O = 90 : 10 with phosphate buffer (200 mM,
pH = 7). (b-e) HP wSTD spectra (4k scans, 4 h of acquisition time)
performed on the same sample presented in (a) in the following conditions:
(b) in the absence of nanoparticles; (c) in the presence of 2-AuNPs; (d) in
the presence of LUDOXs HS nanoparticles; (e) in the presence of the
nanoconjugates. A plus symbol is used to indicate overly enhanced
resonances. Asterisks denote impurities while the circle indicates the
resonance of PEG2000 used to stabilize the aggregates. When present,
2-AuNPs concentration was 20 mM in coating thiols. The broad signals in
(c) and (e) indicated by the triangles stem from the monolayer of 2-AuNPs.
Fig. 4 (a) 1H-NMR spectrum of 10 mM serotonin (3) and 10 mM L-phenylalanine
(5) in H2O:D2O = 90 : 10 with phosphate buffer (200 mM, pH = 7); (b-e) HP
wSTD spectra (4k scans, 4 h of acquisition time) performed on the same sample
presented in (a) in the following conditions: (b) in the absence of nano-
particles; (c) in the presence of 1-AuNPs; (d) in the presence of LUDOXs CL
nanoparticles; (e) in the presence of the nanoconjugates. Asterisks denote
impurities while the circle indicates the resonance of PEG2000 used to
stabilize the aggregates. When present, 1-AuNPs concentration was 20 mM
in coating thiols.
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of NMR chemosensing to 10 mM on non-cryogenic probes. The use
of charged nanoparticles in the analysis of complex matrixes like
urine and biofluids might not yet be straightforward, as many
analytes can be detected at once because of the untargeted nature
of the interaction involved in the recognition process.22,23 None-
theless, the modularity of the proposed method allows an easy
modification and fine-tuning of the sensing systems.
This work was partially supported by P-DiSC#09BIRD2017-
`
UNIPD and P-DiSC#07BIRD2020-UNIPD granted by Universita
degli Studi di Padova and by PRIN 2015 RNWJAM granted by
MIUR. F.M. and D.R.-G. thank the Italian Association for
Cancer Research (AIRC) for financial support (IG ‘‘25003’’).
Fig. 6 (a) 1H-NMR spectrum of a mixture of dopamine (6), homovanillic
acid (4), L-phenylalanine (5) and L-serine (7) all 20 mM in H2O : D2O = 90 : 10
with phosphate buffer (500 mM, pH = 7); (b-c) HP wSTD spectra (4k scans,
4 h of acquisition time) performed on samples similar to that presented in
(a) but in which the concentration of only the binding candidate was
lowered down to 10 mM (dopamine for (b) and homovanillic acid for (c)).
Both HP wSTD experiments were performed in the presence of the
nanoconjugates (1-AuNPs@LUDOXs CL in (b) and 2-AuNPs@LUDOXs
HS in (c)). In both HP wSTD experiments, the AuNPs concentration was 20
mM in coating thiols. A figure showing the entire NMR sweep is reported in
Section S7 of the ESI.†
Conflicts of interest
There are no conflicts to declare.
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