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Nanoparticle-assisted NMR spectroscopy: Enhanced detection of analytes by water mediated saturation transfer. Federico De Biasi, Daniele Rosa-Gastaldo, Xiaohuan Sun, Fabrizio Mancin, and Federico Rastrelli J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.8b13225 • Publication Date (Web): 20 Feb 2019 Downloaded from http://pubs.acs.org on February 21, 2019

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Journal of the American Chemical Society

Nanoparticle-assisted NMR spectroscopy: Enhanced detection of analytes by water mediated saturation transfer. Federico De Biasi,‡ Daniele Rosa-Gastaldo,‡ Xiaohuan Sun, Fabrizio Mancin and Federico Rastrelli* Department of Chemical Sciences, Università degli Studi di Padova, via Marzolo 1, 35131 Padova (Italy). ABSTRACT: Nanoparticle-assisted “NMR chemosensing” is an experimental protocol that exploits the selective recognition abilities of nanoparticle receptors to detect and identify small molecules in complex mixtures by NOE magnetization transfer. While the intrinsic sensitivity of the first reported protocols was modest, we have now found that water spins in long-lived association at the nanoparticle monolayer constitute an alternative source of magnetization that can deliver a remarkable boost of sensitivity, especially when combined with saturation transfer experiments. The approach is general and can be applied to analytenanoreceptor systems of different compositions. In this work we provide an account of the new method and we propose a generalized procedure based on a joint water-nanoparticle saturation to further upgrade the sensitivity, which ultimately endows selective analyte detection down to the micromolar range on standard instrumentation.

INTRODUCTION Nuclear magnetic resonance (NMR) is an undisputed powerful tool in the analysis of complex mixtures owing to the minimal requirements on the sample treatment, the detailed chemical information provided on the analytes and the possibility to detect several species simultaneously without any prior separation. However, notwithstanding the increasingly sophisticated instrumentation available, the advantages of NMR in analytical chemistry have been inevitably paralleled by a rising complexity of the systems to be investigated. In the experimental practice, most of the interesting mixtures (e.g. biofluids) frequently result in largely crowded – if not overlapping – patterns of resonances, and the general problem of assigning each signal in the spectrum to a particular species still poses important challenges. The availability of higher magnetic fields has led to significant advances in sensitivity and resolution, both of which can be further enhanced by means of sophisticated experimental schemes or “pulse sequences”. In the common practice of modern NMR, improvements in the spectral resolution are generally pursued by switching from 1D to 2D techniques, while sensitivity demands are addressed by cryogenic probes. Remarkably, though, spectroscopists have often resorted to the external assistance of chemical species to push NMR beyond its limits. Notable examples of “molecule-assisted” NMR range from the early use of shift reagents up to the most recent advances of dynamic nuclear polarization,1 a method that can provide orders of magnitude in signal enhancement with the external intervention of paramagnetic species. Among the “molecule-assisted” NMR experiments lies “NMR chemosensing”, a technique where nanoparticles (NPs) act as a source of magnetization to be selectively transferred − via the Nuclear Overhauser Effect (NOE) − only to specific classes of molecules, thus enhancing the signals of some analytes over others.2-4 To do so, NMR

chemosensing exploits some distinctive possibilities offered by nanoparticles. First, NPs passivated with a proper monolayer can form homogeneous solutions suitable for solution-state NMR spectroscopy. Secondly, because of their small size, (diamagnetic) nanoparticles do not introduce any significant perturbations in the magnetic field homogeneity and are hence compatible with high-resolution NMR experiments. Last and most important, the versatile chemistry of NPs, particularly those made from gold, allows either the grafting of molecular receptors onto their surface or the spontaneous formation of self-organized binding sites.5 This last aspect marks a profound difference with other molecule-assisted NMR techniques. Matrix-assisted diffusometry, for example, relies on non-specific interactions between the analytes and the surface exposed by chromatographic supports, surfactants or polymers.6 On a similar principle, large (20 nm) silica nanoparticles are used as linebroadening agents, with the advantage that they can be converted from anionic to cationic receptors by doping with alumina.7 At difference with the aforementioned methods, though, the Au-S chemistry in gold nanoparticles provides a unique route for turning virtually any supramolecular receptor into a nanoreceptor. In doing so, the functional headgroups that bind specific analytes are generally known and need not be extensively modified. The chemical challenge then mainly rests in grafting such headgroups upon the surface of the nanoparticle, something that is most conveniently achieved through an alkyl-thiol tether. On top of that, the formation of gold NPs is essentially a self-assembly process, offering an effective shortcut to the realization of complex systems with a limited synthetic effort. On the spectroscopic side, NMR chemosensing capitalizes on 1H experiments such as the NOE-pumping8 or the saturation transfer difference (STD),9 respectively based on the inversion or saturation of the spin populations in a macromolecular receptor. In the NOE

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pumping experiment, a diffusion filter initially dephases the magnetization of all the small, fast diffusing species in the sample while retaining that of the macromolecule. This residual magnetization is then transferred via NOE only to the analytes interacting with the macromolecule, and the resulting signals are detected. In the STD experiment, one spectrum is first acquired by saturating the macromolecule resonances through the joint action of a radiofrequency (rf) field and spin diffusion, and a second reference spectrum is acquired with the same rf field applied far from the proton spectral region. In the former case, the signals of the interacting analytes experience a change in intensity due the partial saturation transfer that results from their binding to the macromolecule. The difference between the two spectra finally displays resonances of only those analytes that have effectively bound to the macromolecule. In the context of nanoparticles, it has been shown that STD combined with NPs of 2 nm core can effectively reveal low-concentrated analytes, albeit at the price of long acquisition times on standard instrumentation.10 Chart 1. Sketch of gold nanoparticles (AuNPs) protected with thiols 1 3 and analytes used in this work. 4: serotonin, 5: N-methyl-phenethylamine, 6: phloretic acid, 7: phenylalanine.

overcoming this stumbling block we have once again resorted to the analogy between nanoparticles and biomacromolecules. Namely, in much the same way as a protein surface in solution is surrounded by water molecules with long residence times,11 we have reasoned that, in aqueous systems, water molecules in long-lived association at the NPs monolayer could provide an additional source of magnetization to be transferred to the analytes. In this paper we present an implementation of this idea, showing how it can provide remarkable improvements in terms of sensitivity with respect to early chemosensing experiments.

RESULTS AND DISCUSSION In the context of biomolecular NMR, the idea of exploiting water molecules that reside at a receptor surface lies at the basis of the water-LOGSY experiment.12 Unlike NOE-pumping or STD, the source of magnetisation in water-LOGSY does not come from the spins of the receptor itself, but from those of water molecules either in solution or bound to the macromolecule. On such premises, we first selected a suitable aqueous system of NPs and binding candidates to test if water magnetization could be effectively transferred to the interacting analytes via water-LOGSY. Among other examples, we have already demonstrated that 1-AuNPs outlined in Chart 1 recognize (protonated) tyramine and possibly other phenethylamines in water.13 In this case, the effective binding is likely driven by the concurrence of H-bonding of the protonated primary amine to the 18-crown-6 moiety and hydrophobic interaction of the aromatic portion of the analyte with the inner alkyl region of the monolayer. Based on this evidence, we started our investigations from a system consisting of 6.3 M 1-AuNP (corresponding to 0.5 mM thiol 1) with 0.5 mM serotonin (4) as the binding candidate, complemented with 0.5 mM N-methylphenethylamine (5) and 0.5 mM phloretic acid (6) as the non-interacting species (Chart 1). The interest in serotonin is justified by its biological role as neurotransmitter and by its involvement in cancer cell migration, metastatic processes and as a mediator of angiogenesis.14 The solvent medium was H2O:D2O = 90:10 buffered with 10 mM phosphate at pH = 7.0. We will refer to this system as “0.5 mM sample 1” henceforth. In the remainder of the work, we will also refer to the nanoparticles concentration in terms of grafted thiols rather than gold cores. NMR spectra were recorded at 25°C on a Bruker AVANCE III spectrometer operating at 500.13 MHz 1H Larmor frequency and equipped with a 5 mm z-gradient broadband inverse (BBI) non-cryogenic probe. The pulse sequence used for water-LOGSY experiments was the NOE-ePHOGSY provided in the Bruker library,15-16 consisting in a selective 1D-NOESY17 (on water) with additional solvent suppression modified into a double pulsed field gradient (DPFG) perfect-echo (PE, see paragraph “water-STD” for details). The relaxation delay was set to 5 s, and the mixing time (2 s) was tested among

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The average gold core of 1-AuNP is 1.9±0.4 nm and the estimated formula is Au212SR79. The average gold core of 2-AuNP is 1.6±0.4 nm and the estimated formula is Au127SR44. The average gold core of 3-AuNP is 1.8±0.2 nm and the estimated formula is Au180SR54.

Indeed, the low sensitivity of NMR chemosensing still prevents its application to highly diluted samples, and even samples of moderate concentration require hours of acquisition to provide meaningful spectra. In the quest for 2


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Journal of the American Chemical Society In this experiment, the S/N ratios of serotonin resonances are no larger than those obtained with standard STD experiments. In addition, the overlap of positive and negative signals prevents a clear identification of the binding analyte. A deeper examination of the spectra in Figure 1 also reveals some details on the interaction mode of serotonin, whose estimated binding constant to 1AuNP is Kb ≈ 100 M-1 (see SI). Upon saturating the signals of the crown ether moiety (Figure 1b), all the six signals of serotonin are detected with relative intensities close to the equilibrium 1H spectrum. On the other hand, saturation of the nanoparticle alkyl signals (Figure 1c) allows a clear detection only of the indole protons, while those of the methylenes are almost undetected. This confirms that the 18-crown-6 plays a prominent role in the recognition event, but also that the aromatic portion of the analyte is in contact with the hydrophobic inner portion of the monolayer.

different values to provide the maximum signal enhancement. Water-LOGSY and STD. The water-LOGSY spectrum resulting from the 0.5 mM sample 1 is presented in Figure 1d (see Figure S11 in SI for the reference spectrum recorded in the absence of 1-AuNPs), along with the plain 1H spectrum (Figure 1a) and two “standard” STD spectra (Figures 1b and 1c) recorded at different saturation frequencies. Similar to the case of bio-macromolecules, the resonances of binding and non-binding analytes in our water-LOGSY spectrum appear with opposite signs depending on the interaction of the analytes with bulk water18-19 or with water in long-lived association at the NP monolayer. For this reason, the signals of serotonin 4 are positive20 (confirming its interaction with the monolayer) and those of 5 and 6 are negative, as expected in the case of negligible NP interaction. The signal at about 10 ppm belongs to the slowly exchanging NH proton of the indole moiety in 4: this resonance only appears in spectra where the signal of water is inverted or saturated.

Figure 1. 1H NMR spectra of 0.5 mM serotonin (4), 0.5 mM N-methyl-phenethylamine (5), 0.5 mM phloretic acid (6) and 0.5 mM 1AuNP. a. Plain 1H NMR spectrum with DPFG-PE (W5) solvent suppression. b and c: standard STD spectra with 2 s saturation at the frequencies highlighted by the arrows. d. water LOGSY with 2 s mixing time. The conventional phase of the signals has been reversed to facilitate the comparison with STD spectra. e. water-STD featuring 180° Gaussian pulses (B1 = 24 Hz) and 2 s saturation at the frequency of H2O. f: Same as e, but with 5560° high-power Gaussian pulses (B1 = 750 Hz). g: 1H NMR reference spectrum of serotonin. Spectra b f were acquired with 64 total scans, corresponding to a duration of about 10 min each. Circles denote the accidental superposition of resonances belonging to interacting and non-interacting analytes (see text).

Water-STD. Having confirmed that 1-AuNPs can act as macromolecular receptors for serotonin, and that watermediated magnetization transfer is effective in the system under investigation, we focused our efforts on increasing the sensitivity of the method. To this aim, our first attempt was to saturate the water signal instead of the NP resonances, a strategy that had been suggested also by Dalvit et al. in the context of water-LOGSY as an alternative to the transient NOE.12 The pulse sequence employed for this experiment was a modified version of

the Bruker pulse program for acquiring STD spectra. Specifically, the experiment consists in the acquisition of multiple spectra interleaved between on- and offresonance saturation of water, achieved by a train of Gaussian-shaped radiofrequency pulses of 50 ms duration. Note that, when the power of such pulses is calibrated to deliver a 180° rotation, only the resonances close to the rf carrier are affected and a “selective” saturation is achieved. Solvent suppression was incorporated in the pulse sequence as a DPFG-“perfect 3



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LOGSY and water-STD, the HP water-STD experiment delivers an impressing 300% enhancement on the signals of serotonin in only 10 minutes, and no interfering resonances from non-interacting species, as expected. For the sake of comparison with early NMR chemosensing experiments, we also acquired an NOEpumping spectrum of the same sample using the same amount of time as the HP water-STD. Not surprisingly, the resulting spectrum showed no analyte signals at all (Figure S13). Comforted by this result, we lowered the concentration of the analytes in sample 1 down to 50 M keeping the concentration of 1-AuNP unchanged, and we repeated the HP water-STD experiment with a predetermined acquisition time of about 60 minutes. The spectrum resulting from this “50 M sample 1” is provided in Figure 2d, along with the plain 1H spectrum (2a), the standard STD (2b) and the water-LOGSY (2c) for comparison. Again, the resonances of serotonin are clearly visible with an average S/N ratio of 9.49 in the spectral region 6.5 7.5 ppm. In contrast, the same signals are only barely detected both in the water-LOGSY spectrum and in the STD spectrum obtained by saturating the oxymethylene resonances.

echo” (PE) with W5 clusters as the refocusing element:2122 the elimination of antiphase magnetization by the PE results in a better lineshape and, ultimately, in smaller subtraction artefacts between on- and off-resonance spectra. The spectra resulting from this experiment, which we dub “water-STD” (water-Saturation Transfer Difference), are presented in Figure 1e: the signals of the binding serotonin 4 display similar enhancements compared to the water-LOGSY experiment of the same duration. While the two approaches are conceptually similar, however, the STD can potentially provide stronger signals because, on account of the steady state, any binding event generates the same enhancement when the monolayer magnetization is saturated. In contrast, the magnetization transfer in water-LOGSY is efficient for analytes binding to the monolayer soon after the inversion of its magnetization (early binding) but, due to relaxation, it drops significantly for late binding events. High-power saturation. One potential limitation of both water-LOGSY and water-STD in the screening of complex mixtures is that the signals of the analytes in the final spectra will have different signs depending on whether or not they interact with the monolayer. As a result, when the signals of interacting and non-interacting analytes accidentally overlap, they tend to cancel each other and generate possible false negatives. In our system, this circumstance is well epitomised by the resonances marked with circles in Figures 1d and 1e. While this may not be a crucial problem in targeted ligand-protein screening, it can severely mask a true response in untargeted NMR chemosensing of an unknown mixture. To alleviate the drawback of conflicting positive and negative signals, we increased the power of the Gaussian pulses in the STD experiment. In doing so, not only each pulse delivers a rotation angle much larger than 180°, but it also loses selectivity, generating a “leak” of the rf field at larger offsets.23 Eventually, such a leaked rf field can reach the signals of the analytes and allow their partial saturation. However, while the small signals of noninteracting analytes (generated by NOEs from bulk water) are largely reduced by the leaked field, the signals of interacting analytes (generated by NOEs from bound water) are little affected. A rationale of this phenomenon is provided in section 5 of the SI, with the aid of SpinDynamica24 simulations. In addition, the same leaked rf field can also saturate the spins of the NP monolayer, thus adding a further important source of saturation to be transferred to the interacting analytes. Notably, since the spins of the monolayer and those of bound water molecules share the same slow tumbling regime, the two sources provide concurrent effects in the final STD spectrum. To test the efficacy of high-power (HP) saturation we first optimized the acquisition protocol on the 0.5 mM sample 1 without nanoparticles, reducing the signals of all the analytes as much as possible (Figure S12). Keeping this optimized set of parameters, the experiment was then repeated on the 0.5 mM sample 1 with nanoparticles to obtain the spectrum in Figure 1f. With respect to water-

Figure 2. 1H NMR spectra of 50 M serotonin (4), 50 M Nmethyl-phenethylamine (5), 50 M phloretic acid (6) and 0.5 mM 1-AuNP. a: Plain 1H NMR spectrum with DPFG-PE (W5) solvent suppression. b: standard STD spectrum with 2 s saturation at the frequency highlighted by the arrow. c: water-LOGSY with 2 s mixing time. d: HP water-STD with 2 s saturation at the

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frequency of H2O. The total acquisition time for spectra b the same (60 min).

d is

water-STD spectra, care must be taken to avoid an excessive saturation of the analytes, some which may otherwise appear as false positives. To verify this potential issue in our case, we repeated the water-STD experiment on the 0.5 mM sample 1 by increasing the saturation power up to B1 = 1500 Hz. As expected, the intensities of serotonin signals do grow, but falsepositives start to appear, especially from non-interacting analytes whose resonances lie closest to the saturating rf offset (Figure S14 and section 5 of SI). High-power saturation is also used in STD spectroscopy of biomacromolecules, where the larger excitation region resulting from the leaked rf field essentially provides a larger pool of saturated spins on the receptor.26 At difference with standard STD experiments, however, in water-STD experiments the opposing NOE from bulk water to non-interacting analytes contrasts the occurrence of false positives when the rf power is increased (Figure S15). Strong binding and concentration. In cases where the equilibrium between the free and the NP-bound analyte is markedly shifted towards the bound state, the apparent lineshape of the interacting analyte can experience a significant broadening, which is more evident in the presence of large NPs or NPs passivated with particularly “stiff”’ monolayers. This is because, in the fast exchange regime over two sites, both the position of the apparent signal and (to a first approximation) its apparent linewidth are averaged over the populations of the two sites.27-28 Albeit a sizeable line broadening does not necessarily imply a poor STD efficiency, it inevitably results in a lower S/N ratio, which can make the interacting analytes barely detectable in the final spectrum. To avoid this issue it proves convenient to alter the populations of the free and bound analytes by playing with the relative concentrations of the analytes and the NPs, based on the fact that decreasing the nanoparticle concentration will increase the fraction of unbound analyte. Figure 4 illustrates the effect of nanoparticles concentration in HP water-STD spectra when serotonin interacts with 1-AuNPs (weak binding, Kb ≈ 100 M-1) and 3-AuNPs (stronger binding to a rigid monolayer, Kb ≈ 1000 M-1). In the case of weak binding, a low concentration of 1-AuNPs results in no signal (Figure 4b,c), which suggests that the fraction of bound species is too low to provide a significant saturation transfer during the experiment. The signal is however recovered when the NPs concentration increases (Figure 4a). On the other hand, in the case of strong binding, a lower concentration of 3-AuNPs corresponds to a narrowing of the signals in water-STD spectra (Figure 4d-f). In this case the fraction of bound species, while low enough not to broaden the analytes’ signals, is still sufficient to provide an effective saturation transfer. A similar behaviour as that discussed for serotonin also applies to dopamine, a catecholamine whose elevated concentration in children’s urine (around 50 M) is frequently correlated to neuroblastoma.29 Being more hydrophilic than serotonin, dopamine association with the monolayer of 3-AuNPs is favoured by reducing the ionic

To broaden the scope of our investigation, the same water-LOGSY, STD, and HP water-STD experiments were repeated using 2-AuNPs (Chart 1) as nanoreceptors. These nanoparticles have also proved to bind phenethylamines in water, yet with a stronger affinity with respect to 1-AuNPs.10 In the case of serotonin, the estimated binding constant in phosphate buffer was Kb ≈ 6000 M-1 (see SI). Based on this evidence, a sample consisting of 0.5 mM 2-AuNPs with 50 M serotonin (4) as the binding candidate and 50 M phenylalanine (7) as the non-interacting species was prepared. The solvent medium was H2O:D2O = 90:10 buffered with 10 mM phosphate at pH = 7.0. The spectra resulting from this “50 M sample 2” closely parallel those obtained on 50 M sample 1, once again with an impressive enhancement of serotonin signals in the HP water-STD spectrum (Figure 3d). Interestingly, despite the different binding constants, samples 1 and 2 display similar S/N ratios on serotonin. This is because, in general, the STD signal depends not only on the populations of the free and bound ligands, but also on the efficiency of the magnetization transfer within the nanoparticle-analyte adduct.25

Figure 3. 1H NMR spectra of 50 M serotonin (4), 50 M phenylalanine (7) and 0.5 mM 2-AuNP. a. Plain 1H NMR spectrum with DPFG-PE (W5) solvent suppression. b standard STD spectrum with 2 s saturation at the frequency highlighted by the arrow. c water-LOGSY with 2 s mixing time. d HP waterSTD with 2 s saturation at the frequency of H2O. The total acquisition time for spectra b d is the same (60 min). Asterisks denote impurities.

While raising the saturation power may seem a straightforward pathway to improve the sensitivity of 5



strength of the medium. As an example, Figure S16 reports an HP water-STD spectrum obtained from a sample of 50 M dopamine and 50 M 3-AuNPs buffered with 0.5 mM phosphate (in place of the usual 10 mM). In summary, the experimental conditions can be optimized to provide meaningful results both in the case

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of weak and in the case of strong binding between nanoparticles and analytes. Indeed, the proper nanoparticle concentration can be quite easily estimated on the basis of its affinity for the specific analyte under investigation.

Figure 4. Left: HP water-STD spectra of 50 M serotonin (4), 50 M N-methyl-phenethylamine (5), 50 M phloretic acid (6) with (a) 500 M 1-AuNPs (b) 50 M 1-AuNPs, (c) 25 M 1-AuNPs. Right: HP water-STD spectra of 50 M serotonin (4) and 50 M phenylalanine (7) with (d) 500 M 3-AuNPs (e) 50 M 3-AuNPs, (f) 25 M 3-AuNPs. In all the samples the solvent medium is H2O:D2O = 90:10 with 10 mM phosphate buffer.

more intense signals, with an average S/N ratio of 8.64 in the same 6.5 7.5 ppm region.

Biselective water-monolayer saturation. Remarkably, the peculiar chemistry of AuNPs offers a general strategy to combine the benefits of standard STD and water-STD experiments. In fact, virtually all the thiols used to build AuNP monolayers feature an alkyl spacer whose protons resonate at low chemical shifts (typically between 1 and 2 ppm). If the saturating pulse is split into two sidebands, e.g. by cosine modulation of the parent selective pulse,30 a direct saturation of the NP monolayer can be obtained by placing one of the two sidebands close to the alkyl resonances. The advantages of such biselective water-monolayer saturation are presented in Figure 5: trace (a) reports a high-power STD spectrum obtained on the 50 M sample 2 upon saturation of the dimethylsilyl resonances, while trace (b) reports the same high-power STD spectrum obtained by saturating water (note that this is the same spectrum of Figure 3d). In both cases the saturating rf field has the same power and leads to an appreciable enhancement on serotonin, whose resonances in the region 6.5 7.5 ppm reach an average S/N ratio of 5.10 for trace 5a and 6.94 for trace 5b. Nonetheless, the simultaneous biselective saturation of water and dimethylsilyl resonances returns a spectrum with even

Figure 5. 1H NMR spectra of 50 M serotonin (4), 50 M phenylalanine (7) and 0.5 mM 2-AuNP. a and b: high power saturation (50 ms Gaussians at B1 = 750 Hz) at the frequencies

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indicated by the arrows. c simultaneous biselective saturation of water and alkyl resonances (50 ms Gaussians at B1 = 750 × 2 Hz) achieved by cosine modulation at 1144 Hz. Asterisks denote impurities.

the target analytes in matrices with interfering/competitive species. Finally, by showing how nanoparticle-assisted NMR can detect relevant species at low concentrations, the results here reported herald the inspiring challenge of rationally designing nanoreceptors with increased selectivity towards target molecules. All of these aspects are currently under investigation in our laboratories.

CONCLUSIONS

ASSOCIATED CONTENT

In conclusion we have demonstrated that, with respect to NOE pumping experiments proposed in early NMR chemosensing protocols, water spins bound to NPs monolayers can deliver a remarkable boost of sensitivity in conjunction with STD experiments. Furthermore, by carefully raising the power of the saturation pulses, a bonus sensitivity gain can be appreciated because of the concurrent partial saturation of the monolayer spins. More generally, thanks to the peculiar chemistry of AuNPs, water saturation can be complemented with the saturation of alkyl resonances, leading to a new class of biselective “water-monolayer” STD experiments. Thanks to this novel approach, the determination of analytes in the micromolar range becomes possible in reasonable acquisition times, even with standard instruments (a 500 MHz NMR spectrometer in our case) and with relatively simple nanoreceptors. Nonetheless, the proposed technique is amenable to several improvements. For example, lowering the temperature of the system will cause a decrease of the NPs correlation times, which enhances the saturation transfer mechanism by improving the efficiency of spin diffusion. Preliminary results indicate that this strategy does provide slightly better S/N ratios. Along the same line, another way to improve the efficiency of spin diffusion may consist in increasing the nanoparticle size. However, while the binding equilibria is only marginally affected by the size,31 the broadening of the analytes signals can be significantly enhanced. In this context, we had already verified that the NPs’ size adopted in this work (about 2 nm core) allows for optimal results in chemosensing experiments based on NOE-pumping. Finally, we expect that the use of higher magnetic fields should also bring significant benefits not only in terms of higher sensitivity, but also because it shifts the underlying spin dynamics towards the spin diffusion limit (where NOE is the most efficient). Indeed, when any of such strategies is complemented with cryogenic probes, an even larger sensitivity gain will result, opening the pathway to NMR chemosensing analysis of mixtures in the micromolar range. At these concentrations, among other examples, serum serotonin can be suitable for prognosis evaluation of urothelial carcinoma in the urinary bladder, adenocarcinoma of the prostate, and renal cell carcinoma.32 The future steps to undertake will be the implementation of the proposed approach in systems of analytical significance and the evaluation of its performance with regard to linearity, accuracy, limit of detection and quantitation. In this context, the major obstacles that still need to be addressed mainly concern the quantitation of

Supporting Information. Synthesis and characterization of nanoparticles, binding constants estimation, additional NMR experiments, saturation profiles and high-power effect. This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Author * [email protected]

Author Contributions All authors have given approval to the final version of the manuscript. ‡These authors contributed equally.

Funding Sources This work was partly supported by P-DiSC #09BIRD2017UNIPD granted to F.M. by Università degli Studi di Padova and by PRIN 2015 RNWJAM granted by MIUR.

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT F.D.B. acknowledges support from COST Action CA15209: “European Network on NMR Relaxometry” (EURELAX). X.S. thanks the China Scholarship Council for a PhD fellowship (no. 201506870019)

ABBREVIATIONS DPFG: double pulsed field gradient; ePHOGSY: enhanced Protein Hydration Observed through Gradient SpectroscopY;  =  HP: high-power; NP: nanoparticle; PE: perfect-echo; rf, radiofrequency; STD: saturation transfer difference; waterLOGSY: Water-Ligand Observed via Gradient SpectroscopY.

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(20) The conventional phase of the signals in water-LOGSY spectra has been reversed to facilitate the comparison with STD spectra. (21) Liu, M.; Mao, X.-a.; Ye, C.; Huang, H.; Nicholson, J. K.; Lindon, J. C., Improved WATERGATE Pulse Sequences for Solvent Suppression in NMR Spectroscopy. J. Magn. Reson. 1998, 132 (1), 125-129. (22) Aguilar, J. A.; Kenwright, S. J., Robust NMR water signal suppression for demanding analytical applications. Analyst 2016, 141 (1), 236-42. (23) See Figure S17 for a comparison between low-power and high-power saturation profiles. (24) Bengs, C.; Levitt, M. H., SpinDynamica: Symbolic and numerical magnetic resonance in a Mathematica environment. Magn. Reson. Chem. 2018, 56 (6), 374-414. (25) Neuhaus, D.; Williamson, M. P., The Nuclear Overhauser Effect in Structural and Conformational Analysis. 2 ed.; Wiley-VCH: New York, 2000; p 136-143. (26) Cutting, B.; Shelke, S. V.; Dragic, Z.; Wagner, B.; Gathje, H.; Kelm, S.; Ernst, B., Sensitivity enhancement in saturation transfer difference (STD) experiments through optimized excitation schemes. Magn. Reson. Chem. 2007, 45 (9), 720-4. (27) McLaughlin, A. C.; Leigh, J. S., Relaxation times in systems with chemical exchange: Approximate solutions for the nondilute case. J. Magn. Reson. (1969) 1973, 9 (2), 296-304. (28) Sandström, J., Dynamic NMR Spectroscopy. Academic Press: London, 1982. (29) Davidson, D. F., Elevated urinary dopamine in adults and children. Ann. Clin. Biochem. 2005, 42 (Pt 3), 200-7. (30) Emsley, L.; Burghardt, I.; Bodenhausen, G., Double selective inversion in NMR and multiple quantum effects in coupled spin systems. J. Magn. Reson. (1969) 1990, 90 (1), 214-220. (31) Lucarini, M.; Franchi, P.; Pedulli, G. F.; Gentilini, C.; Polizzi, S.; Pengo, P.; Scrimin, P.; Pasquato, L., Effect of core size on the partition of organic solutes in the monolayer of water-soluble nanoparticles: an ESR investigation. J. Am. Chem. Soc. 2005, 127 (47), 16384-5. (32) Jungwirth, N.; Haeberle, L.; Schrott, K. M.; Wullich, B.; Krause, F. S., Serotonin used as prognostic marker of urological tumors. World J Urol 2008, 26 (5), 499-504.

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