*******

Field of the invention

The invention relates to a method for detection of organic molecules via NMR spectroscopy by means of magnetization transfer from gold nanoparticles functionalized on the surface with a monolayer of organic molecules to the analyte. The method is based on the magnetization transfer sequence from the functionalized gold nanoparticles to the analyte to be detected and subsequent removal of the signal of the gold nanoparticles. The invention further relates to the use of the functionalized gold nanoparticles for the method itself.

The work leading to this invention has received funding from the European Research Council under the European Union's 7th Framework Programme, project ERC Starint Grant 2010 MOSAIC, grant agreement n °259014

State of the art

The analysis of mixtures of chemical compound mixtures aimed to identify their composition or the presence of a specific compound is one of the main problems faced by chemical sciences. Until now, this issue has not yet received a response of general validity.

The most common analysis methods provide for the separation of the mixture components by means of chromatographic techniques (in particular, HPLC - high performance liquid chromatography - instruments) and their identification by means of spectroscopic techniques. Generally, the detectors used identify the presence of compounds based on changes in the refractive index or UV-Vis absorption. In these cases, the assignment of the identity of the identified compound takes place solely on the basis of the retention time of the chromatographic column. However, many molecules may share the same retention time and in each case it is necessary to have authentic samples (called standards) of each compound to be detected. In the most advanced solutions, the HPLC instrument is connected to a mass spectrometer able to measure the molecular weight of separated compounds. However, also this approach provides only partial information about the identity of the separated compounds, since different molecules can share the same molecular weight. Moreover, complex sample preparation procedures are also generally required to eliminate or suppress potential interferents and matrix effects, which adversely affect the reliability of the measurement.

NMR (Nuclear Magnetic Resonance) spectroscopy has been known for several years as the potentially most suitable technique for the identification of organic compounds. This technique provides a large amount of information about the sample under consideration which, analyzed by an expert, allows determining the structure of a compound, even unknown, with relative ease. The wide variety of measurable parameters and of experiments available further allow obtaining a wide range of structural and chemical information. However, to the increase in the number of components present in a sample to be examined corresponds an extremely large amount of information produced, which becomes a limitation, since the information cannot be uniquely associated to the compound it belongs to.

There are some techniques that allow reducing the amount of information (e.g. decoupled spectra, two-dimensional homo- and heteronuclear techniques) or extracting information from the NMR analysis regarding one sample only (e.g. selective TOCSY, DOSY), but also such techniques are subject to heavy restrictions regarding the structure of the investigated molecule and the features of the mixture.

A solution proposed for this problem is coupling of NMR analysis with the chromatographic separation of the components of the mixture (J.W. Jaroszewski, Planta Medica, 71 , 691 , 2005; J. W. Jaroszewski, Planta Medica, 71 , 795, 2005; K. Patel et al., Pharmaceutical Methods, 2, 2, 2010). However, this approach has achieved limited success, largely because of the low sensitivity of NMR spectroscopy, that is ill-suited to the small amounts of sample provided by chromatographic separation. The method also requires a complex calibration and the use of multi-frequency techniques for the suppression of interference of the solvent, since it is not possible for cost reasons to use deuterated solvents in the chromatographic separation. Another strategy exploits the capability of NMR spectroscopy to separate from each other the spectral fingerprints of the mixture components on the basis of their diffusion rate (P. Stilbs, Analytical Chemistry, 53, 2135, 1981 ). These methods are known by the name of "Diffusion Ordered Spectroscopy (DOSY) (K. F. Morris et al., Journal of the American Chemical Society, 1 14, 3139, 1992). Normally, however, the difference in the diffusion rate of molecules of similar size is quite little and a conventional DOSY experiment does not reach sufficient resolution to distinguish the single components.

More recently, the method of chromatographic NMR has been proposed in scientific and patent literature. In this method, a suitable component ("stationary phase") is added to the sample to increase the difference in the diffusion rate of the compounds and thereby, the DOSY resolution: this component can be silica gel (S. Viel et al., Proceedings of the National Academy of Sciences, 100, 9696, 2003), a polymer (J. S. Kavakka et al., Organic Letters, 1 1 , 1349, 2009), micelles (M. E. Zielinski et al., Magnetic Resonance in Chemistry, 47, 53, 2009) or microemulsions (C. Pemberton et al., Langmuir, 27, 4497, 201 1 ). The various components of the mixture interact in a more or less strong manner with the stationary phase, altering the effective diffusion rate thereof proportionally to the residence time therein. However, the interactions involved are generally very weak and the differentiation of analytes is often ineffective. Moreover, the significant amount of stationary phase added causes the deterioration of the homogeneity of the magnetic susceptibility and of the spectrum obtained up to make it unintelligible (C. Pemberton et al., Journal of Magnetic Resonance, 208, 262, 201 1 ).

In order to solve this problem, costly high resolution solid state MRI techniques (HR-MAS) or solvents with magnetic susceptibility "uniformized" to that of the stationary phase have been used (J. A. Chin et al., Journal of Combinatorial Chemistry, 2, 293, 2000; B. Antalek, Concepts in Magnetic Resonance, 14, 225, 2002; C. Carrara et al., Journal of Magnetic Resonance, 194, 303, 2008; G. Pages et al., Analytical Chemistry, 78, 561 , 2006; S. Caldarelli, Magnetic Resonance in Chemistry, 45, S48, 2007). However, the improvements recorded have implied significant increases in the cost of the measurements. Recently, the focus has been on the use of solvent-soluble stationary phases such as microemulsions, micelles and polymers (C. Pemberton et al., Langmuir, 27, 4497, 201 1 ; R. E. Hoffman et al, Journal of Magnetic Resonance, 220, 18, 2012). While this method has led to narrower signals compared to the previous methods, these soluble stationary phases generate other signals that overlap with those of the mixture components, making the DOSY analysis impossible or requiring the use of perfluorinated, perdeuterated or more recently, silicone stationary phases (S. Huang, Jet a., Angewandte Chemie International Edition, 53, 1 1592, 2014) to prevent an accidental overlap with the signals of the analyte. However, these expedients are of limited applicability since the perfluorinated/perdeuterated reagents available on the market are few and the differentiation ability of silicone polymers is modest.

A last analysis method recently proposed involves the use of fluorinated supramolecular receptors (Y. Zhao et al., Journal of the American Chemical Society, 136, 10683, 2014; Y. Zhao et al., Journal of the American Chemical Society, 135, 18770, 2013). The receptor complexes a chemical compound or class of compounds, thereby producing a signal (fluorine resonant frequency shift) different for each product analyzed. If this method responds to the problem, typical of supramolecular and natural receptor-based sensors, of the inability to distinguish positive analysis from interferents having similar features, there remains the need for genuine standards of each compound and the possibility of obtaining similar signals from different compounds.

In an earlier patent application, the authors of the present invention described an NMR method for the analysis of complex mixtures based on the magnetization transfer from gold nanoparticles functionalized with a monolayer of organic molecules to the molecules to be detected (B. Perrone et al., Patent application PD2013A000153; B. Perrone et al., Journal of the American Chemical Society, 135, 1 1768, 2013; M.-V. Salvia et al., Journal of the American Chemical Society, 137, 886, 2015).

This method takes advantage of the possibilities offered by NMR spectroscopy to distinguish molecules based on the differences in the diffusion rate and to transfer the magnetization between two chemical species that are at a short distance. By applying the "NOE pumping" sequence to a sample to which gold nanoparticles, protected by a monolayer of organic molecules, have been added, it is possible to separate and highlight only the signals of the interacting species. In fact, the NOE pumping experiment involves the deletion of all signals relating to the species with a high diffusion rate (sample components) but not those of nanoparticles. Thereafter, the nanoparticles transfer the retained magnetization to the molecules able to establish an interaction with them. The signal of the interacting molecules reappears in the NMR spectrum of the sample and such molecules can therefore be identified (a different experiment, called STD, based on a similar principle, allows similar results in shorter time but is not of general applicability as the NOE pumping). If the interaction between the nanoparticle and the analyte is selective, it is possible to determine its presence in the sample with certainty. The NMR spectrum analysis highly reduces the possibility of false positives. The possibility of easily modifying the chemical features of the monolayer of organic molecules coating the surface of gold nanoparticles in principle allows identifying gold nanoparticles able to selectively interact with the analyte of interest. In the patent mentioned, the molecular recognition takes place by hydrophobic partition, and thus in aqueous medium, of the analytes in the monolayer. The key element of recognition therefore is the affinity of the substrate (i.e. the analyte) for lipophilic phases, expressed as isooctane/water partition coefficient. In a subsequent publication, the authors showed how the combination of hydrophobic interaction with electrostatic one increases the affinity and selectivity for anionic molecules and consequently lowers the detection limit. However, some limitations to this method remain (M.-V. Salvia et al., 2015, ref. cit).

Firstly, if the detection by NMR-NOE pumping based on the magnetization transfer alone, hereinafter referred to as "NMR chemosensing" for brevity, removes the signals of molecules not interacting with the gold nanoparticles from the spectrum, it does not remove those of the same nanoparticles that may overlap with those of the analyte, thereby interfering with the analysis. Secondly, since the interaction between analyte and nanoparticle is based mainly on the hydrophobic effect, the method is limited to the analysis of samples in aqueous solution, with clear restrictions as regards the amount and type of detectable analytes. Summary

A first object of the invention is to solve the drawbacks of the analysis technique of mixtures of organic compounds by NMR chemosensing which, while allowing the detection of predetermined analytes in complex mixtures of organic molecules through the use of gold nanoparticles functionalized on the surface, exhibits problems of interference from the signals of the same nanoparticles. A second object of the invention is to expand the field of application of the NMR chemosensing technique to analytes not soluble in water, these being the majority of organic compounds.

At the purpose to overcome the first drawback, namely the presence of signals of gold nanoparticles in the NMR spectra obtained with the magnetization transfer sequence, a novel method has been developed based on the detection of NMR spectra using the magnetization transfer sequence from the functionalized gold nanoparticles to the analyte to be detected and then removing the spectroscopic signal of the functionalized gold nanoparticles from the final spectrum. In this novel method, the signal sequence of the analysis by magnetization transfer (e.g. NOE pumping) is followed by the sequence of analysis using CPMGz, which removes the signals of species characterized by short relaxation times Tz from the spectrum. Since this feature is generally possessed by relatively large species, such as gold nanoparticles, this insertion allows removing the signals of the same nanoparticles from the final spectrum without affecting those of the analyte to be identified.

In order to overcome the second drawback, relating to the applicability of the NMR chemosensing method only in aqueous solutions, have been developed gold nanoparticles able to recognize organic molecules by means of non-covalent interactions, such as hydrogen bond or aromatic interaction, as well as the hydrophobic partition. Since these interactions are also effective in non-aqueous solutions, this allows the application of the method to solutions of organic solvents and to analytes not water-soluble.

In a first aspect the object of the present invention is a method for detection of organic molecules in a sample comprising one or more organic compounds, using for this detection a magnetization transfer nuclear magnetic resonance (NMR) spectroscopy, wherein:

- the chemical species acting as stationary phase for the detection of the organic molecules in the sample being examined consists of Au nanoparticles functionalized on the surface with a monolayer of thiol residues, and

comprising at least the steps of:

- magnetizing a sample comprising the Au nanoparticles functionalized and the sample to be examined by the application of nuclear magnetic resonance (NMR) pulses;

- removing the magnetization of one or more organic compounds of the sample by applying a diffusion filter;

- transferring the magnetization from the functionalized Au nanoparticles to one or more organic compounds of the sample to be examined;

- removing residual magnetization of functionalized Au nanoparticles by the application of a sequence of "Carr-Purcell-Meiboom-Gill modified with a filter- z" (CPMGz) acting as a filter for transverse relaxation times T2,

- recording of a spectrum of signals resulting from the residual magnetization of one or more organic compounds of the sample to be examined.

The purpose of the functionalization of the gold nanoparticles is to enable selective non covalent interaction between the same and the organic molecules to be detected both in aqueous medium and in an organic environment. Such functionalization of the gold nanoparticles is obtained by treatment of the same with organic molecules with a thiol functional group that, self-assembling on the gold core of the nanoparticle, forms a monolayer that gives to the Au nanoparticles solubility and stability in solution, but, above all, the capacity of capturing molecules that are able to form interactions complementary to those carried out by the monolayer.

In a second aspect, the object of the present invention is therefore the use of Au nanoparticles functionalized with a monolayer of thiol residues represented by the general formula (I)

-S-(A)n-(Y)ffl-(B)p-X (I)

wherein :

- A is selected from -CH2-, -CF2-, or a combination thereof and n is an integer comprised from 6 to 12;

- B is selected from -CH2-, -CF2-, an aryl residue or a combination thereof and p is an integer comprised from 0 to 4;

- Y is selected from an amide group -CONH-, urea -NHCONH-, thiourea - NHCSNH-, a dimethyl silane group -Si(CH3)2- an ether group -O- or a combination thereof and m is an integer equal to 0 or 1 ;

- X is selected from a macrocyclic compound of general formula -(Z\N)q- where Z is selected from -CH2CH2- or -C6H ₄ - or a combination thereof, W is selected from O, N, S and q is an integer comprised from 4 to 8, a carboxyl group, a phosphoric group, a phosphonic group, a sulfonic group, a sulfuric group, an amino group, an amide group, a guanidine group, a pyridinium group, an urea group or a thiourea group,

for the method for detection of organic molecules in a sample comprising one or more organic compounds, based on magnetization transfer nuclear magnetic resonance (NMR) spectroscopy object of the invention.

The molecules of interest for the method are organic molecules characterized by solubility of at least 0.1 mM in water or in organic solvent and diffusion coefficient higher than 1 x10 ^"10 m ² /sec (PM < 2000).

In a further aspect, an object of the invention relates to nanoparticles functionalized with specific thiol residues.

The method of the invention has proved to fulfill the purpose since, as will emerge from the following description, the presence of a single analyte could be detected in mixtures composed of many, also very similar, molecules.

The advantage described and other related ones will become apparent from the following detailed description of the invention and explanatory figures.

Brief description of the figures

Figure 1. The figure shows a schematic representation of the operating principle of the determination of organic molecules (A, B, and C in the Figure) using techniques based on the analysis of NMR spectra. Panel (a) schematizes the NMR signals recordable when a sample containing many organic molecules is analyzed via standard NMR spectroscopy; in this case, the signals for all the chemical species present are obtained and the identification of a single analyte is usually impossible. Panel (b) schematizes the NMR signals recordable with the introduction of Au nanoparticles coated with a monolayer consisting of organic molecules due to which they selectively interact with one of the organic species present in the sample; the application of the magnetization transfer method (such as NOE pumping) allows the identification of the analyte of interest (shown in the figure as A). In fact, the interaction between the analyte and the nanoparticle allows transferring the magnetization, created by the NMR pulse, from the functionalized Au nanoparticle to the interacting molecule. Since all species with non-zero magnetization are visible on NMR, both the signals of the searched analyte A and those of the nanoparticle appear in the spectrum, and are then detected. Panel (c) schematizes the NMR signals recordable with the application of the NOE pumping-CPMGz sequence object of the invention; this method allows removing the signals of the nanoparticles, thereby facilitating the analysis of the signal obtained from the searched analyte alone.

Figure 2. The figure shows a sequence of pulses for the NOE-pumping-CPMGz experiment. Phase cycle: φι = x -x; φ2 = x x y y -x -x -y -y; φ3 = x x y y -x -x -y -y -x -x -y -y x x y y; φ ₄ = x; φδ = y y y y -y -y -y -y; φ _Γ = x -x -x x x -x -x x -x x x -x -x x x -x. Δ = diffusion delay, Tmix = mixing time, τ = half-echo time (~1 ms). G1 and G3 are encoding-decoding gradients lasting 1000 με. The GPMGz filter is highlighted in grey.

Figure 3: The figure shows a schematic representation of the protocol used for the method of detecting an analyte according to the invention.

Figure 4: The figure shows a schematic representation of the functionalized gold nanoparticles used in the present invention and the chemical structure of some examples of molecules coating the surface of the Au nanoparticle.

Figure 5. The figure shows the ¹ H-NMR spectra, obtained by the method according to the invention, of a mixture (Mixture 1 , Table 2) containing tyramine, L- phenylalanine, phloretic acid, N-methylphenethylamine, sodium para- toluenesulfonate, arbutin, 3-chlorophenol in the presence of gold nanoparticles (Au MNP 1 ) coated with a monolayer of organic molecules described in example 1 . A) standard NMR spectrum, B) NMR spectrum recorded with NOE pumping sequence, C) NMR spectrum recorded with NOE pumping-CPMGz sequence, object of the invention. It is seen that in spectrum C, the nanoparticle signals are significantly reduced, thereby facilitating the identification and quantification of the analyte.

Figure 6. The figure shows the ¹ H-NMR spectra, obtained by the method according to the invention, of a solution of cadaverine in the presence of gold nanoparticles (Au MNP 1 ) coated with a monolayer of organic molecules described in example 1 . A) standard NMR spectrum, B) NMR spectrum recorded with NOE pumping sequence, C) NMR spectrum recorded with NOE pumping- CPMGz sequence, object of the invention. It is seen that in spectrum B, recorded with the NOE-pumping sequence, the nanoparticle signals overlap with those of the analyte, making the identification difficult, while the problem does not occur in spectrum C where the NOE pumping-CPMGz sequence has been used.

Figure 7. The figure shows an example of a calibration curve for the tyramine analyte in the presence of Au nanoparticles MPN 1 , in deuterated methanol buffer (·, signal at 3.1 ppm; O, 2.9 ppm; i, 6.8 ppm; 7.1 ppm). The curve was constructed by recording the NMR spectra of samples containing tyramine in varying concentrations by means of the method of the invention. The tyramine signals were integrated and shown in the graph according to the concentration of tyramine. The three calibration curves were obtained by data interpolation using equation 1 .

Figure 8. The figure shows the results obtained with the method object of the invention in the analysis of viologen by the addition of Au MPN 1 of example 1 or Au MNP 2 of example 2 using the NOE pumping-CPMGz magnetic resonance technique, in deuterated methanol/dichloromethane solvent. A) Standard proton spectrum. B) Proton spectrum recorded by the NOE pumping-CPMGz sequence by the addition of Au MNP 1 nanoparticles. C) Proton spectrum recorded by the NOE pumping-CPMGz sequence by the addition of Au MNP 2 nanoparticles. It is noted that the analyte signals are only recovered when the Au MNP 2 nanoparticles are added. Figure 9. The figure shows the ¹ H-NMR spectra, obtained by the method according to the invention, of a mixture containing N-methylphenethylamine, phenylalanine, glucose, HEPES in the presence of gold nanoparticles (Au MNP 3 of example 3 and Au MNP 4 of example 4) in aqueous solvent (deuterated water) at pH 7. A) Standard proton spectrum. B) Proton spectrum recorded by the NOE pumping-CPMGz sequence by the addition of Au MNP 3 nanoparticles. C) Proton spectrum recorded by the NOE pumping-CPMGz sequence by the addition of Au MNP 4 nanoparticles. It is noted that the analyte signals are not identifiable in spectrum A due the presence of the signals of the other molecules present in much higher concentration, whereas in spectra B and C, the analyte signals alone are recovered with both nanoparticles.

Figure 10. The figure shows the results obtained with the detection assay of phenethylamines in the presence of Au MNP 3 of example 3 and Au MNP 4 of example 4 in the detection of tyramine and 4-nitrophenethylamine as representative of possible new molecules. A) Spectrum recorded by NOE- pumping-CPMGz sequence of tyramine in the presence of Au nanoparticles MNP 3 in buffered deuterated water (HEPES 10 mM) at pD 7. B) Spectrum recorded by NOE-pumping-CPMGz sequence of 4-nitrophenethylamine in the presence of Au nanoparticles MNP 3 in buffered deuterated water (HEPES 10 mM) at pD 7. C) Spectrum recorded by NOE-pumping-CPMGz sequence of tyramine in the presence of Au nanoparticles MNP 4 in buffered deuterated water (HEPES 10 mM) at pD 7. D) Spectrum recorded by NOE-pumping-CPMGz sequence of 4- nitrophenethylamine in the presence of Au nanoparticles MNP 4 in buffered deuterated water (HEPES 10 mM) at pD 7. It is noted that the Au nanoparticles MNP 3 are able to reveal both the tyramine and the 4-nitrophenethylamine while the Au MNP 4 are not effective in the second case.

Figure 11. The figure shows the ¹ H-NMR spectra, obtained by the method according to the invention, of a mixture containing naproxen and sodium salicylate in the presence of gold nanoparticles (Au MNP 5 of example 5) coated with a monolayer of organic molecules described in example 5. A) Standard NMR spectrum, B) NMR spectrum recorded with the NOE pumping-CPMGz sequence, object of the invention. Detailed description of the invention

Definitions and abbreviations

Unless otherwise defined, all scientific and technical terms used in all parts of the description of the invention have the usual meanings that a man skilled in the art of the invention commonly understands.

The acronym Au MPN is used herein as abbreviation for "Au nanoparticles functionalized with a self-assembled monolayer of residues of organic molecules" or equivalent expressions, such as "Au nanoparticles protected (or coated) with a self-assembled monolayer of residues of organic molecules". Generally, the abbreviation is followed by the number indicating the coating molecule, for example Au MPN1 indicates gold nanoparticles functionalized with molecule 1 (shown in example 1 ). The nanoparticles can be prepared by chemical reduction of a gold salt (HAuCU) in the presence of stabilizing agents following methods reported in the literature (F. Manea, et al., Langmuir, 24, 4120, 2008).

"Analyte", "substrate" or "target molecule" are equivalent terms indicating any organic molecule to be determined in a sample to be analyzed. These expressions are to be considered, for the purposes of the description of the present invention, equivalent and in their extended meaning.

By "stationary phase" it is meant a chemical species, introduced into the tube for the NMR analysis, which can bind to an analyte and is provided with a lower diffusion coefficient than the analyte itself. In the present invention, this is an Au nanoparticle MPN functionalized with organic molecules of general formula (I). "NOE" is the abbreviation for "Nuclear Overhauser Effect", a physical phenomenon that consists in magnetization transfer between two spins via dipolar intra- or inter- molecular interactions, commonly known by one skilled in the art and described in the literature (G.M. Clore et al., Journal of Magnetic Resonance, 48, 402, 1982; G.M. Clore et al., Journal of Magnetic Resonance, 53, 423, 1983; B. Meyer et al., European Journal of Biochemistry, 246, 705, 1997). The preferred technique in this case is the so-called "NOE-pumping" (A. Chen et al., Journal of the American Chemical Society, 120, 10258, 1998) or modifications thereof (A. Chen et al., Journal of the American Chemical Society, 122, 414, 2000). "CPMGz" is the abbreviation for sequence of "Carr-Purcell-Meiboom-Gill modified with a filter-z", the purpose of which is to remove signals characterized by short relaxation times from the NMR spectrum, while avoiding the phase distortions typical of CPMG sequences (F. Rastrelli et al., Journal of the American Chemical Society, 131 , 14222, 2009).

The "relaxation time" T∑ is a parameter that describes the time required, in an NMR experiment, for the magnetization produced in the nanoparticles by the radio frequency pulses used, to return to its initial value. In particular, the relaxation time T∑ corresponds to the time in which the transverse magnetization of the sample decreased by 37% after its formation.

"NOE pumping-CPMGz" or "NOE pumping-CPMGz sequence" are equivalent and are also used here to briefly indicate the method object of the invention.

Description

The detection of organic chemical species by NMR is substantially based on the recording of spectrometric signals and since each chemical species has its own set of signals, this allows uniquely identifying the molecule itself. In principle, this technique therefore is a powerful analytical instrument. As schematically shown in Figure 1 , panel (a), however, by applying the standard NMR spectroscopy to mixtures of organic molecules (indicated in the figure as A, B and C), NMR signals for all the chemical species present are obtained and the identification of a single analyte is actually not feasible due to the complexity of the spectra recorded. A first overcoming of this limitation has been possible with the application of NMR techniques with magnetization transfer (also referred to as NMR chemosensing technique), whose operating scheme is shown in Figure 1 , panel (b). In this detection technique, the analyte is determined by means of the magnetization transfer from a functionalized gold nanoparticle (stationary phase) to the analyte itself. In the NMR chemosensing method, this stationary phase consists of gold nanoparticles protected with a monolayer of organic molecules capable of selectively interacting with the analyte in water by hydrophobic interactions (or partition). Using this NMR technique, the magnetization is accumulated on the functionalized Au nanoparticle and then transferred to the interacting molecule (or analyte). The analyte thus magnetized is then detected in the same NMR experiment. The magnetization transfer process is made selective by the selectivity of the interaction between the functionalized nanoparticles and the analyte and, since only the molecules with magnetization are detected by the NMR analysis, only the species interacting with the functionalized Au nanoparticles are detected. In practice, the method allows eliminating all the undesired signals from the NMR spectrum of the sample (except those of the nanoparticles themselves), allowing the unambiguous identification of the analyte alone, and the quantification thereof.

The NMR spectroscopic determinations usable for this purpose are based on "transferred Nuclear Overhauser Effect (tr-NOE)" or "Saturation Transfer Difference" (STD) processes. In particular, the most general determination, based on the determination for tr-NOE, is called "NOE-pumping".

As previously anticipated and schematically shown in Figure 1 , panel (b), the main limitation encountered when using this method based only on the magnetization transfer is the presence of nanoparticles signals in the final spectrum that interfere with the analyte signals. In fact, this method first involves the suppression of the analyte signals from the spectrum by means of a diffusion filter, the subsequent (partial) magnetization transfer, such as via dipolar interactions (NOE) from the functionalized gold nanoparticle to the analyte and the spectrum recording. Since at the time of detection the magnetization is thus present both on the nanoparticles and on the analyte, the signals of both species are present in the final spectrum. In the case of overlap (when the signals of the analyte and of the particles are at the same position in the spectrum), the presence of the functionalized gold nanoparticle signals can make difficult or even impossible to identify the analyte, especially if it is present in a small amount.

This limitation becomes even more relevant when using the STD method instead of the "NOE pumping" method. In this case, in fact, an isolated signal of the stationary phase (functionalized nanoparticle) is saturated selectively. Via "spin diffusion", the saturation is then propagated to the entire nanoparticle and, by intermolecular route, to any other analyte interacting with it. A first reference spectrum is recorded by irradiating an area that contains no signal and it is then subtracted ("difference") from a second spectrum obtained by recording a signal of the stationary phase. The subtraction allows highlighting the signals of the species that were affected by the saturation transfer, and then due to the analyte- functionalized Au nanoparticle interaction. However, all signals of the stationary phase, whose intensity is increased by the propagation of the magnetization, are highlighted and also amplified.

The object of the present invention is in the first place a new method, called "NOE pumping-CPMGz", of which Figure 2 shows a phase cycle, which allows solving the drawbacks mentioned above by removing the signals of the functionalized nanoparticles from the final spectrum.

According to the method object of the invention, in fact, a sample containing one or more organic chemical species to be analyzed and the functionalized nanoparticles is magnetized by NMR pulses; the magnetization is then removed by an appropriate series of pulses, called diffusion filter, and the residual magnetization remained in the monolayer of Au nanoparticles is transferred to the interacting molecules; the residual magnetization of the nanoparticles is then removed by a pulse sequence called CPMGz; finally, the signal produced by the magnetization remained only on the analyte is recorded, allowing to exclusively detect the latter by means of NMR spectroscopy.

This result is therefore obtained by inserting the CPMGz sequence, which operates as a "filter Ti at the end of the magnetization transfer sequence. This filter allows removing the signals characterized by a short relaxation time T∑ from the final spectrum. Since the relaxation time T∑ of a chemical species is inversely related to the speed of rotation of the same, and consequently to its dimensions, large sized species have short relaxation times. A filter of T∑ is therefore able to remove the nanoparticle signals from an NMR spectrum without altering those of the molecules present. If applied at the end of the magnetization transfer sequence, the filter therefore allows removing the signals of functionalized gold nanoparticles, leaving only those of the interacting species.

Also for this novel NMR chemosensing method, the magnetization transfer can be pursued both with "Transferred Nuclear Overhauser Effect (NOE-tr)" or "Saturation Transfer Difference" (STD) processes, but because of the limits of the magnetization transfer according to the STD technique, for the purposes of the present invention, the magnetization transfer method by means of "NOE-pumping" is to be preferred, since it is applicable in a more general manner and does not require any type of selective excitation of the functionalized gold nanoparticles. The novel "NOE pumping-CPMGz" method, moreover, is a further advance over the known NMR chemosensing method, since it can be also applied for the determination of analytes dissolved in non-aqueous environment. Most of the organic compounds is not, in fact, soluble in water and this strongly limits the field of use of the NMR chemosensing method based only on magnetization transfer. In fact, this provides that the selective recognition between functionalized gold nanoparticle and analyte is determined by hydrophobic interactions which can occur only in water and consequently, the use of a different solvent makes impossible to recognize the analytes. This problem is solved by the structure of the thiols coating the gold nanoparticles by conjugating the same with functional groups bearing a positive or negative charge or with supramolecular receptors capable of selectively interacting with compounds to be determined by means of ionic pair or specific recognition non-covalent bonds.

Therefore, the functionalized gold nanoparticles can be designed and selected according to the type of analyte or analytes to be detected.

The functionalized nanoparticles that can be used for the purposes of the present invention are characterized by relatively small size (hydrodynamic radius, to which both the gold core and the monolayer contributes, around 5-9 nm), and this is significant for the relaxation time T∑, and they are coated by a monolayer of molecules characterized by the presence of an alkyl, or fluoroalkyl chain that for the NMR technique is to be considered equivalent to the alkyl chain, terminated with one or more functional groups or with a supramolecular receptor. Particles of this type are partially already known and described (for example, in G. Guarino et.

Al., Journal of the American Chemical Society, 134, 7200, 2012 or in M.V. Salvia et al., 2015, cit. ref.).

In short, the features that the nanoparticles must have for the purposes of the present invention are:

a gold core of average size (diameter) comprised from 1 to 5 nm; the functionalization with organic molecules representable by the general formula (I):

HS-(A Y B)p-X

(I)

wherein A, B, Y, X, n, m and p have the meanings mentioned above for the residue of general formula (I) and wherein :

-SH is a thiol functional group, necessary to attach the molecule to the surface of the bare gold nanoparticle with consequent formation of the self-assembling protective monolayer;

- A represents a linear alkyl or fluoroalkyl chain necessary for creating a stable monolayer and, in some cases, a hydrophobic pseudo-phase in the self-assembled monolayer on the surface of the Au nanoparticle; Y, with m = 1, represents a functional linking group between the linear chain A and B;

- B, with p≠ 0, represents an alkyl or fluoroalkyl chain or an aryl residue linking X and Y;

X is a functional group able to confer solubility in an aqueous and/or organic environment to the gold nanoparticle and to determine non- covalent interactions between the functionalized Au nanoparticle and the analyte to be detected of electrostatic or hydrogen bonding type.

As schematically shown in Figure 4, each moiety of these molecules therefore has a substantial role for the recognition of the substrates. The alkyl (CH2) _n or fluoroalkyl -(CF2) _n moiety, represented in the general formula (I) with (A) _n , forms a hydrophobic psuedo-phase in the self-assembled monolayer capable of capturing organic molecules with similar characteristics, when operating in aqueous solution. The functional groups, represented in the general formula (I) with Y, B and X, in particular X, contribute both to ensure the solubility of the Au nanoparticle in the selected solvent but also to the recognition of the analyte. When this has a net charge, X groups with opposite charge provide an electrostatic interaction able to modulate the selectivity of the functionalized Au nanoparticle. When the substrate has an aromatic moiety, aromatic B or X groups can provide complementary aromatic interactions π-π. When the substrate has hydrogen bond donor and/or acceptor groups, complementary Y and X groups can provide the right interactions with the substrate in non-aqueous solvent.

The efficacy of the analyte recognition is, therefore, directly related to the selection of the coating of the Au core for the target.

In a preferred embodiment, meanings of A, Y, B and X of the thiol residue with general formula (I), which can be used for the functionalization of gold nanoparticles, are as follows:

- A is a linear alkyl chain;

- Y is an amide group -CONH-;

- B with p≠ 0 is a short linear alkyl chain or an aryl;

- X is a crown ether macrocycle, a pyridinium group or a sulfonic group. Most preferred functionalizations are with thiol residues in which:

- A is equal to -(CH2)7- Y is equal to -CONH-, B is equal to -CH2- and X is a heterocycle 18-crown-6 (-S-(CH ₂ )7-CONH-CH2-18-crown-6); - A is equal to -(CH2)7- Y is equal to -CONH-, and X is a heterocycle dibenzo-18-crown-6 (-S-(CH2)7-CONH-CH2-dibenzo-18-crown-6);

- A is equal to -(CH ₂ )n- and X is a -SOs ^" (-S-(CH ₂ )n- SOs ^" );

- A is equal to -(CH2)7-, Y is equal to -CONH-, B is equal to aryl and X is a -SO3- (-S-(CH2)7-CONH-C ₆ H -S03-);

- A is equal to -(CH2)s- and X is a pyridinium group (-S-(CH2)s-C5H ₄ N ⁺ ).

Most preferred functionalizations are with thiol residues in which:

- A is equal to -(CH2)7- Y is equal to -CONH-, B is equal to -CH2- and X is a heterocycle 18-crown-6 (-S-(CH2)7-CONH-CH ₂ -18-crown-6);

- A is equal to -(CH2)7- Y is equal to -CONH-, and X is a heterocycle dibenzo-18-crown-6 (-S-(CH ₂ )7-CONH-CH2-dibenzo-18-crown-6);

- A is equal to -(CH2)7-, Y is equal to -CONH-, B is equal to aryl and X is a -SO3- (-S-(CH2)7-CONH-C ₆ H -S03-);

which are also new and therefore part of the invention.

The technical features of the detectable molecules are the following:

- solubility in a solvent of choice of at least 0.1 mM, and in particular of

0.5 mM for the determination based on "NOE pumping" and 0.1 mM for the determination based on STD; diffusion coefficient higher than 1 x10 ^"10 m ² /sec (PM < 2000);

presence of a net charge (positive or negative), or of a group able to accept/donate hydrogen bonds (for example, primary ammonium, guanidinium, carbonyl, alcohol).

Moreover, the interaction between the analyte and the functionalized Au nanoparticles should necessarily take place in conditions of fast exchange. This condition is normally guaranteed by the functionalized Au nanoparticles for molecules which have the characteristics herein previously mentioned.

As a non limitative example, this class of molecules comprises: carboxylates, benzoates, benzenesulfonates, salicylates, organic phosphates and phosphonates, quaternary ammonium salts, protonated amines, alkylpyridinium derivatives.

For the purposes of the present invention the following are preferred:

functionalized nanoparticles with a thiol residue wherein A is equal to - (CH2)7- Y is equal to -CONH-, and X is a heterocycle dibenzo-18- crown-6 (-S-(CH2)7- YH-CH2-dibenzo-18-crown-6) for analytes consisting of aromatic quaternary ammonium salts;

functionalized nanoparticles with thiol residue, wherein A equal to - (CH2)7- Y equal to -CONH-, B equal to -CH2- and X is a heterocycle 18-crown-6 (-S-(CH2)7-CONH-CH2-18-crown-6) or wherein A is equal to -(CH2)7- Y is equal to -CONH-, and X is a heterocycle dibenzo-18- crown-6 (-S-(CH ₂ )7-CONH-CH2-dibenzo-18-crown-6) for analytes consisting of protonated primary amines;

functionalized nanoparticles with a thiol residue, wherein A equal to - (CH ₂ )i i- and X is a -SO3- (-S-(CH ₂ )n- SO3-) or wherein A is equal to

-(CH ₂ )7-, Y is equal to -CONH-, B is equal to aryl and X is a -SO3- (-S- (CH2)7-CONH-C6H ₄ -S03-) for methamphetamine analogues; functionalized nanoparticles with a thiol residue, wherein A is equal to CH2)s- and X is a pyridinium group (-S-(CH2)s-C5H ₄ N ⁺ ) for carboxylated compounds such as naproxen.

The detection of an organic molecule according to the method object of the invention comprises the following steps: 1 . optionally, dissolution of the sample to be studied in a suitable solvent (water or buffered solution at a pH suitable to ensure the complete dissolution of the analyte, organic solvent). This step can, in fact, be not necessary if the sample is already available as a solution;

2. optionally, centrifuging the sample for removing insoluble species or macromolecules;

3. adding to the sample a suitable amount of selected functionalized Au nanoparticles with residues of general formula (I);

4. recording the magnetization transfer NMR spectrum by means of "NOE pumping-CPMGz" or STD-CPMGz;

5. calculating the analyte concentration by integrating the analyte signals and comparing with a calibration curve obtained with a set of samples of known titre of the same.

In order to select the Au nanoparticles with the most suitable functionalization to interact with the analyte to be determined, prior to the preparation of the sample to be subjected to NMR or in any case, prior to the addition of the same Au nanoparticles, the sample being examined can be subjected to analysis for the determination of the diffusion coefficient, solubility, log P, and charge.

The protocol for the execution of the method is schematically shown in Figure 3 and comprises the steps of:

optionally, characterizing the compound to be detected according to its diffusion coefficient, solubility in aqueous or organic solvent, and charge;

selecting the functionalization of the nanoparticle suitable for interacting with the compound to be determined;

dissolving the sample being examined in a deuterated aqueous or organic solvent or adding a deuterated aqueous or organic solvent to the sample being examined;

optionally centrifuging the sample for removing insoluble species or macromolecules;

adding the selected functionalized Au nanoparticles with residues of general formula (I) to the sample; and calculating the concentration of the organic molecule to be detected by integrating the signals related to the same detected by the magnetic resonance spectrum recorded and comparison with a calibration curve obtained with a series of samples of known concentration of the same.

The determination of an organic molecule according to the invention provides the following experimental conditions.

The sample preparation can be different, depending on its origin and physical state:

1 a) sample not in solution (extract of vegetable or animal origin, raw reaction product, medicine or drug tablet): a sample amount of between 2 and 20 mg (preferably 10 mg) is dissolved in 1 ml_ of solvent or buffer (carbonate, 100 mM pD 10; 10 mM phosphate pD 5-9; 10 mM HEPES, pD 7; 10 mM acetate, pD 3.5) in deuterated water. The sample is subjected to centrifugation at 12,000 rpm to remove any unsolved material and 0.6 ml_ of the same are transferred to a tube for NMR;

1 b) sample in solution (environmental sample, biological fluid, process sample): 0.5 ml_ of sample are mixed with 0.1 ml_ of deuterated solvent (the corresponding perdeuterated of the same solvent in which the buffer is dissolved) or, if the sample is an aqueous solution, in phosphate buffer (100 mM, pD 7) in deuterated water (or another buffer prepared in deuterated water); the sample is subjected to centrifugation at 12,000 rpm and transferred to a tube for NMR.

The samples thus prepared are admixed with the Au nanoparticles MPN up to a final concentration of nanoparticles of from 5μΜ up to 150μΜ and preferably comprised from 15 to 50 μΜ.

The NMR spectra are, then, recorded and the integrals of the signals are measured. Finally, the analyte concentration is determined by the equation 1 :

(Equation 1 )

wherein Ci is the concentration of analyte in the sample (expressed as molar units); li is the integral of a signal / of the sample measured in the spectrum obtained according to the method described in the invention;

N is the concentration of Au nanoparticles expressed as molar units;

parameters Ki and Ai are determined, for each signal / ^' , from the calibration curve constructed using a series of samples with known titre of analyte interpolated according to equation 2:

/, = A- (Equation 2).

Generally, calibration can be performed as described below.

In order to quantify the analyte in solution it is necessary to construct a calibration curve obtained in the same matrix used for the analysis. After choosing an appropriate coating for the Au nanoparticle, an NMR spectrum is recorded with increasing concentrations of the analyte to be determined [preferably at concentrations of between 0.1 and 20 mM]. For each spectrum obtained, the integral (/) of each analyte signal is calculated using the application available in the NMR spectrum processing software used, then said integral values are reported in the graph as a function of the analyte concentration (C, expressed in molar units) and data are interpolated with equation 2. The interpolation can be performed independently for each signal, obtaining a Ki value for each, or simultaneously for all the signals, obtaining an average K value.

Parameters Ai and Ki resulting from the interpolation of the calibration curve allow determining the unknown concentration of analyte G knowing the integral // of one of its signals obtained through equation 1 .

As will be clear from the following experimental part, given by way of non-limiting example of the invention, this method has been applied to the detection of different chemical species, and in particular to the so-called "designer drugs". These are synthetic drugs based on the general structure of methamphetamine (N-methyl-1 - methyl-2-phenylamine). In these substances, the introduction of substituents on the aromatic ring allows the creation of new substances with the same pharmacological properties but not tabulated in the lists of prohibited substances and then formally legal. The analysis of these substances with the traditional methods can accordingly be problematic, since no methods and standards exist usable for unknown substances. The method of the invention therefore is optimal for this problem since it also allows determining unknown substances through the analysis of their NMR spectrum.

The molecular backbone of such drugs is linked to 2-phenylethylamine. In this molecule, a hydrophobic moiety (the phenylethyl residue) and a positively charged hydrophilic moiety (the amino group protonated under physiological conditions) are easily recognizable. For the determination of these molecules by the NOE pumping-CPMGz method, therefore, functionalized nanoparticles with thiols alkylsulfonic thiols were developed which can produce hydrophobic and electrostatic interactions with positively charged species. The need for identifying unknown molecules that may potentially have signals in each region of the NMR spectrum also makes the complete removal of the nanoparticles signals necessary. The use of alkylanionic nanoparticles and of the "NOE pumping- CPMGz" sequence allows determining the presence of analogues of synthetic drugs also in the presence of excipients and masking agents.

As regards the technical problem of using these analytical methods with chemical species in solvents other than the aqueous ones, most of the organic compounds are not in fact soluble in water, the structure of the thiols that coat the nanoparticles was conjugated with supramolecular receptors. In fact, any supramolecular receptor conjugated to a nanoparticle becomes an NMR sensor for organic molecules. In particular, hereinafter, it is shown that the conjugation of nanoparticles with a crown ether 18-crown-6 allows achieving a system capable of selectively recognizing the protonated primary amines in methanol. On the other hand, the functionalization of the same with a crown ether dibenzo-18-crown-6 allows determining the presence of species characterized by the presence of electron-poor aromatic compounds in solvents such as dichloromethane or acetonitrile. The interactions responsible for the recognition in this case are aromatic (π-π) or hydrogen bond interactions.

By the NMR chemosensing method "NOE pumping-CPMGz", the nanoparticle signals are attenuated to an extent of 95-100% compared to the signals of the functional ized Au nanoparticles and in most cases deleted: the identification of the sample is therefore much more precise and reliable.

EXAMPLES

For illustrative but non-limiting purposes of the present invention, below are examples of preparations of functionalized Au nanoparticles.

Example 1. Preparation of Au nanoparticles with a residue -S-(CH2)7-CONH-CH2- 18-crown-6 (Au MPN 1 )

The nanoparticles are prepared by chemical reduction of a gold salt (HAuCU) in the presence of the thiol residue

according to the method reported in the literature (F. Manea, et al., 2008, ref. cit). The average diameter of the gold core is approximately 2 nm, the overall average hydrodynamic diameter (including the stabilizing monolayer of organic molecules) is about 5 nanometers.

Example 2. Preparation of Au nanoparticles with a residue -S-(CH2)7-CONH-CH2- dibenzo-18-crown-6 (Au MPN 2)

The nanoparticles are prepared by chemical reduction of a gold salt (HAuCU) in the presence of the thiol residue

according to the method reported in the literature (F. Manea, et al., 2008, ref. cit). The average diameter of the gold core is approximately 2 nm, the overall average hydrodynamic diameter (including the stabilizing monolayer of organic molecules) is about 5 nanometers.

Example 3. Preparation of Au nanoparticles with a residue -S-(CH2)n- SO3 ^" (Au MPN 3) The nanoparticles are prepared by chemical reduction of a gold salt (HAuCU) in the presence of the thiol residue according to the method reported in the literature (F. Manea, et al., 2008, ref. cit). The average diameter of the gold core is approximately 2 nm, the overall average hydrodynamic diameter (including the stabilizing monolayer of organic molecules) is about 5 nanometers.

Example 4. Preparation of Au nanoparticles with a residue -S-(CH2)7-CONH- CeHU-SOs- (Au MPN 4)

The nanoparticles are prepared by chemical reduction of a gold salt (HAuCU) in the presence of the thiol residue

according to the method reported in the literature (F. Manea, et al., 2008, ref. cit). The average diameter of the gold core is approximately 2 nm, the overall average hydrodynamic diameter (including the stabilizing monolayer of organic molecules) is about 5 nanometers.

Example 5. Preparation of functionalized Au nanoparticles with a residue -S- (CH2)8-C ₅ H N ⁺ (Au MPN 5)

The nanoparticles are prepared by chemical reduction of a gold salt (HAuCU) in the presence of the thiol residue according to the method reported in the literature (F. Manea, et al., 2008, ref. cit). The average diameter of the gold core is approximately 2 nm, the overall average hydrodynamic diameter (including the stabilizing monolayer of organic molecules) is about 5 nanometers. The functionalized Au nanoparticles prepared were used for the determination of different analytes:

(a) protonated primary amines in a complex mixture of organic compounds of molecular weight and chemical structure similar to the analyte, in an organic solvent, by means of NOE pumping-CPMGz sequence in combination with functionalized nanoparticles with supramolecular receptors for the amines;

(b) a pesticide (viologen) in an organic solvent by NOE pumping-CPMGz sequence in combination with nanoparticles functionalized with aromatic functional groups;

(c) a designer drug (N-methylphenethylamine) analogue in a mixture of organic compounds selected to simulate a commercial formulation containing masking agents, in aqueous solvent, using NOE pumping- CPMGz sequence in combination with nanoparticles functionalized with anionic functional groups;

(d) an antiinflammatory drug (naproxen) in aqueous solvent, using NOE pumping-CPMGz sequence in combination with nanoparticles functionalized with cationic aromatic functional groups.

The organic compounds similar in structure are shown in Table 1 and Table 2 shows the compositions of the samples to be tested:

Table 1 : Chemical structure of the analytes used in the examples

Table 2. Composition of the mixtures of molecules analyzed in the experimental part and in the examples.

Mixture 7 Naproxen (N) 0.5 mM D ₂ O, pD 7

Salicylic acid (P) 0.5 mM

All NMR spectra shown in the Figures were recorded at room temperature (298 K) with a Bruker Avance III 500 spectrometer having a resonant frequency ¹ H of 500.13 MHz.

Example 6. Detection of primary amines in an organic solvent (tyramine and cadaverine)

For the detection of primary amines in an organic solvent, nanoparticles coated with the organic molecules Au MPN 1 (example 1 ) and Au MPN 2 (example 2) were used. In fact, it is known that crown ethers, and in particular 18-crown-6, are good supramolecular receptors for protonated primary amines (R. M. Izatt et al., Journal of the American Chemical Society, 1979, 101 , 6273).

Deuterated methanol was selected as solvent, able to easily solubilize most organic compounds with net charge. Only Au NMP 1 were found to be soluble in this solvent and were then tested.

Au nanoparticles MNP 1 were used according to the method described for the analysis of the mixture of compounds 1 in Table 2 (tyramine, L-phenylalanine, phloretic acid, N-methylphenethylamine, sodium p-toluensulfonate, arbutin, 3- chlorophenol) using the "NOE pumping-CPMGz" method, object of the present invention, and the "NOE-pumping" NMR method for comparison. The effectiveness of the Au nanoparticles MPN 1 is demonstrated in Figure 5, where the spectra ¹ H-NMR of the mixture 1 in Table 2 are shown. In the case of spectrum A), in which a standard NMR analysis was carried out, many signals are seen associated to all the molecules present in the sample; the target analyte signals (tyramine) may not be easily identified since in many cases they are overlapped to those of the other molecules present in the sample. In spectrum B), recorded with the "NOE pumping" sequence, only the tyramine signals are clearly and selectively seen; the nanoparticle signals, however, are also visible which could make difficult to identify the sample. In spectrum C), recorded by the "NOE pumping-CPMGz" sequence, the nanoparticle signals are attenuated to an extent of 95-100% compared to the signals of the nanoparticles of spectrum B and in most cases deleted: the identification of the sample is therefore much more precise and reliable.

The same Au nanoparticles MNP 1 were used (Figure 6) for the analysis of biogenic diamines such as cadaverine (mixture 2 table 2). In this case, one of the analyte signals is completely overlapped to those of the gold nanoparticles and the analysis with the NOE pumping sequence (spectrum B) does not allow unambiguously determining the presence of the analyte. The application of the "NOE pumping-CPMGz" sequence (spectrum C), according to the invention, allows removing all the gold nanoparticle signals, showing with certainty those attributable to the analyte.

Calibration

An example of calibration curve is shown in Figure 7, where the abscissa axis shows the values of the integrals of the tyramine signals and the ordinate axis shows the concentration of tyramine in solution.

The parameters obtained from the overall interpolation of the data in Figure 7 (where N = 2x10 ^"3 M) are: K = 160 ± 5 M ^" \ Ai = 590 ± 80 M ^" \ A ₂ = 410 ± 50 M ^" \ A3 = 310 ± 30 M ^"1 , A ₄ = 270 ± 40 M ^"1 and wherein Ai, A2, A3 and A ₄ are parameters Ai calculated for the signals at 2.9 ppm, 3.1 ppm, 6.8 ppm and 7.1 ppm, respectively.

Example 7. Detection of viologen in an organic solvent

Method

The viologen, also known as paraquat, is a non-selective herbicide. For its determination nanoparticles coated with thiols 1 and 2 of the examples 1 and 2 were tested. The solvent used is a mixture of deuterated methane and dichloromethane in the ratio 1 :3. The concentration of the substrate (mixture 3, table 2) is 10 mM.

The sample was prepared by dissolving the analyte in the mixture of solvents (about 0.5 ml_), adding a part of stock solution of Au MPN 1 nanoparticles of example 1 or Au MNP 2 nanoparticles of example 2 and transferring the whole in a tube for standard NMR of 5 mm diameter.

The NMR spectrum was acquired using the "NOE pumping-CPMGz" sequence. Results

In Figure 8 the spectra of viologen (H, Table 1 ) obtained with a conventional NMR experiment (A) and the NOE pumping-CPMGz method using Au MNP 1 nanoparticles (B) and Au MNP 2 nanoparticles (C) can be seen. Even the only visual comparison clearly shows that only the Au MNP 2 are able to detect the analyte while no signal appears in the spectrum NOE pumping-CPMGz registered with Au MNP 1 nanoparticles. The analyte viologen is not able in fact to interact with the residual 18-crown-6 present on Au MNP 1 nanoparticles, because, despite being a quaternary ammonium salt, it cannot form hydrogen bonds. It can instead interact with the Au MNP 2 nanoparticles through interaction with the aromatic electron-rich aryl groups of the dibenzo-18-crown-6 residue.

Example 8. Detection of "designer drugs" analogues in aqueous solution

Method

Designer drugs are modified psychoactive substances synthetic derivatives so which do not to fall in the lists of prohibited substances while maintaining psychotropic effects. This goal is obtained by inserting one or more substituents in the basic structure of a known psychoactive molecule, in particular methamphetamine, which substituents modify its chemical structure but not the interaction with the receptor. For example, in the case of amphetamine substituents of the aromatic residue are inserted. Therefore, the general problem in the analysis of these substances, which may be new and not known, can be the non-availability of a standard for a comparison.

For the determination of designer drugs in aqueous solvent nanoparticles coated by the organic molecules of examples 3 and 4 were prepared.

The structure of methamphetamine, selected as reference target, contains a positively charged site (the protonated amine group) and a lipophilic residue (aromatic portion). It can therefore be assumed that a good interaction occurs with a monolayer coating of a nanoparticle characterized by the presence of terminal groups of opposite charge (anions) and an inner hydrophobic portion.

In this example, the sample being analyzed is a mixture of N- methylphenethylamine, used as a model designer drug due to its chemical similarity with methamphetamine, phenylalanine, glucose (mixture 4 table 2). The sample was prepared by dissolving the mixture in 10 mM HEPES buffer with pH 7.0 in deuterated water (about 0.5 ml_), adding a part of stock solution of Au MPN 3 nanoparticles or Au MNP 4 nanoparticles and transferring the whole in a standard NMR tube of 5 mm diameter.

The comparison between the performance of the two nanoparticles was carried out by comparing their ability to detect derivatives of phenethylamine structure with different substituents. The nanoparticles were added to solutions of tyramine (mixture 5, table 2) and 4-nitrophenethylamine (mixture 6, Table 2) in deuterated water buffered with 10 mM HEPES with pH 7 and the NMR spectra were recorded with the sequence "NOE pumping-CPMGz".

NMR spectra were acquired using the pulse sequence "NOE pumping-CPMGz", of the invention, by using the following acquisition parameters: spectral width = 5 kHz, frequency of the transmitter = 2350.60 Hz, acquired points = 8 k, acquired scans = 3072, recycle delay = 2 s, mixing time = 400 ms, diffusion delay Δ= 50 ms, Gz(max) = 52 G/cm, duration δ = 5 ms. Before proceeding with the Fourier transform, to the FID (Free Induction Decay) a 64 k zero-filling and an exponential window function with "line-broadening" of 2 Hz were applied.

Results

The spectra of the mixture 4 obtained by a traditional method (A) and with the method of the invention by addition of Au MPN 3 nanoparticles (B) and Au MPN 4 nanoparticles (C) can be seen in Figure 9. As in the previous example, the simplification of the spectrum and the selection of the N-methylphenethylamine by the nanoparticles are evident.

In Figure 10 which shows the spectra obtained with the mixture 6, it can be seen in addition that, while the Au MNP 3 nanoparticles, which can recognize the substrate by using only hydrophobic and ion pair interactions, can be used for determination of phenethylamines with any substituent of the aromatic ring, the Au MNP 4 nanoparticles, which can also detect aromatic interactions, are not effective in the case of analytes substituted with electron-attracting residues.

Example 9. Detection of naproxen in a complex mixture

Method The sample being examined consists of a library of organic compounds soluble in water, the chemical structure of which is reported in Table 1 and the composition of which is reported in table 2 as in mixture 7. The concentration of each compound is 0.5 mM while the Au MPN 5 nanoparticles (example 5) have a concentration of about 15 μΜ. The solvent used was deuterated water containing a carbonate HEPES buffer with pD 7. The sample was prepared by dissolving the analyte mixture in the buffer (about 0.5 ml_), adding a part of stock solution of Au MPN 5 nanoparticles and transferring the whole in a standard NMR tube of 5 mm diameter.

NMR spectra were acquired using the pulse sequence "NOE pumping-CPMGz", by using the following acquisition parameters: spectral width = 5 kHz, frequency of the transmitter = 2350.60 Hz, acquired points = 8 k, acquired scans = 3072, recycle delay = 2 s, mixing time = 400 ms, diffusion delay Δ= 50 ms, Gz(max) = 52 G/cm, duration 5 = 5 ms. Before proceeding with the Fourier transform, a 64 k "zero-filling" and an exponential window function with "line-broadening" of 2 Hz was applied to the FID (Free Induction Decay).

Results

The spectra of the mixture 7 obtained by a traditional method (A) and with the method of the invention (B) can be seen in Figure 1 1 . The simplification of the spectrum is evident, the Au MNP 5 nanoparticles, which can provide π-π interactions in addition to the electrostatic interactions, select naproxen with respect to salicylate.

From these examples the ease, speed, quantitative capability and versatility of application of the method can be appreciated, both in an aqueous and in organic environment, as well as the advantages obtainable with the same compared to the known methods heretofore.

In particular, the method of the present invention showed a much higher sensitivity (up to 10 times) with respect to a method based only on the NOE-pumping method.