WO2016026916A2

WO2016026916A2 - Phase transfer based chemical sensing

Info

Publication number: WO2016026916A2
Application number: PCT/EP2015/069097
Authority: WO
Inventors: Stefan GULDIN; Yang Ye; Silke Krol; Francesco Stellacci
Original assignee: Ecole Polytechnique Federale De Lausanne (Epfl)
Priority date: 2014-08-19
Filing date: 2015-08-19
Publication date: 2016-02-25
Also published as: WO2016026916A3

Abstract

The present invention relates to a phase transfer based chemical sensing, specifically to a method for selective separation, detection and quantification of a polycyclic organic compound in a sample and a method for selective extraction of a polycyclic organic compound from a sample.

Description

PHASE TRANSFER BASED CHEMICAL SENSING

FIELD OF THE INVENTION

The present invention relates to a phase transfer based chemical sensing, specifically to a method for selective separation, detection and quantification of a polycyclic organic compound in a sample and a method for selective extraction of a polycyclic organic compound from a sample. BACKGROUND OF THE INVENTION

One classic probe for chemical and biological sensing are gold nanoparticles (AuNPs) that exhibit a distinct optical footprint based on their surface plasmon resonance and allow for surface functionalization, typically via thiol chemistry. The creation of molecular receptors in the ligand shell of AuNPs allows for selective interaction with ions, such as mercury, methyl mercury, cadmium or zinc, as well as polycyclic aromatic hydrocarbons, cocaine, glucose, oligonucleotides or certain proteins. Typically, specific binding leads to AuNP aggregation and a broadening of the light absorbance, resulting in a gradual colour shift of the solution from red to blue. This kind of colorimetric sensing has several disadvantages: 1) The dynamic range, i.e. the detectable concentration window of target molecules is limited by the fact that the observed colour is always a combination of AuNPs that have interacted with the target (and therefore aggregated), and excess probes; 2) The selectivity relies solely on the specificity of the probe- target interaction over probe interaction with other molecules in the incubation phase; 3) The detection is only possible, if the target-probe interaction triggers an aggregation induced colour shift.

SUMMARY OF THE INVENTION

In one aspect, the invention provides a method for selective separation, detection and quantification of a polycyclic organic compound in a sample comprising:

a) providing a two-layer solution consisting of the first layer which is an incubation phase and the second layer which is a read-out phase, wherein the incubation phase is immiscible with the read-out phase,

b) adding in the incubation phase a degradation agent or adding gold nanoparticles coated with ligands or with reactive surface specific to the polycyclic organic compound to be detected, wherein said gold nanoparticles are soluble in the incubation phase and insoluble in the read-out phase,

c) adding a sample containing the polycyclic organic compound insoluble in the readout phase in the incubation phase under conditions sufficient to allow interaction of the polycyclic organic compound with the degradation agent of step b), or the gold nanoparticles of step b), whereby said interaction either modifies the solubility of the polycyclic organic compound and allows transfer of said modified polycyclic organic compound into the read-out phase, or modifies the solubility of the gold nanoparticles in the read-out phase and allows transfer of said gold nanoparticles into the read-out phase,

d) detecting and quantifying the polycyclic organic compound in the sample by measuring the amount of modified polycyclic organic compound in the read-out phase, or by measuring the amount of the polycyclic organic compound bound to the gold nanoparticles in the read-out phase, or by measuring the amount of transferred gold nanoparticles in the readout phase and/or residual gold nanoparticles in the incubation phase.

In a further aspect, the invention provides a method for selective extraction of a polycyclic organic compound from a sample comprising:

a) providing a two-layer solution, wherein the first layer is an incubation phase immiscible with the second layer which is a read-out phase,

b) adding in the incubation phase gold nanoparticles coated with ligands specific to the polycyclic organic compound to be extracted, wherein said gold nanoparticles are soluble in the incubation phase and insoluble in the read-out phase,

c) adding a sample containing the polycyclic organic compound to be extracted in the incubation phase under conditions sufficient to allow binding of the polycyclic organic compound to the gold nanoparticles, wherein binding of the polycyclic organic compound to the gold nanoparticles modifies the solubility of said gold nanoparticles and allows transfer of said gold nanoparticles into the read-out phase,

d) releasing the polycyclic organic compound bound to the gold nanoparticles into the read-out phase,

e) recovering the polycyclic organic compound from the read-out phase.

In a further aspect, the invention provides a kit for detecting and quantifying doxorubicin in a sample, comprising

a) methanol, b) a cyanobiphenyl liquid crystal and/or an immiscible organic solvent,

c) gold nanoparticles coated with 1 1-mercapto-l-undecanol and 2-phenylethanethiol ligands specific to doxorubicin and/or its metabolites and/or a buffer agent for adjusting pH above pH 8.5, preferably between 9 and 10 and/or a degradation agent.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 shows AuNP phase transfer-based biochemical sensing. A) Scheme of AuNP probe with specific receptors (ligands) for target polycyclic organic compound. Binding of the target polycyclic organic compound to the AuNP shell leads to a change in solubility, e.g. from hydrophilic to hydrophobic. B) Alternative concept for phase-transfer based sensing where the target polycyclic organic compound cleaves the ligand surface or substitutes ligands on the AuNP surface, which similarly leads to a change in solubility. C) Two-phase system consisting of an AuNP containing incubation phase (here: top) and complementary, immiscible read-out phase (here: bottom). Upon addition of a polycyclic organic compound, mixing and phase separation, detection of the target polycyclic organic compound is possible through AuNP phase transfer into the read-out phase, which is the consequence of specific interaction of the target polycyclic organic compound with the AuNP and subsequent change in solubility. D) Alternative scenario where the presence of a degradation agent manipulates the chemical structure of the polycyclic organic compound and thus triggers a phase transfer of the polycyclic organic compound to the read-out phase. At an excess concentration of the degradation agent, the amount of phase-transferred molecules correspond to the initial concentration of the polycyclic organic compound, as illustrated two scenarios. A combination of A-C) and D) is also feasible, where the polycyclic organic compound is chemically modified by catalytic degradation, e.g. on the surface of gold nanoparticles.

Figure 2 shows biochemical sensing of doxorubicin (DOX). A) Two-phase system consisting of a methanolic AuNP solution and a liquid crystalline read-out in the isotropic phase. Depending on the target polycyclic organic compound to receptor ratio (here: 0.1 ; 0.2; 0.3; 0.75) a different amount of AuNP probes is transferred to the read-out phase, observable by the naked eye through a change in coloration. In contrast, when no AuNPs are present, the DOX is retained in the top phase as shown in the control samples. B) Two dimensional contour plot where the absorbance in the LC at the characteristic wavelength of 544 nm is shown as a function of AuNP and DOX concentration in the initial incubation phase.

Figure 3 shows doxorubicin (DOX) -induced AuNP phase transfer to heptane. Analogous phase transfer experiments t o Figure 2A, where heptane is used as an alternative read-out phase. Here, the incubation phase is in the bottom. In A-E), phase separated samples are shown with a target molecule to receptor ratio of 0; 0.3; 0.5; 0.75 and 1, respectively. Addition of DOX leads to a change in AuNP solubility in the incubation phase, A uNP prevalence at the 2-phase interface and subsequent complete removal from the incubation phase. To the right, control samples are shown without AuNPs but an identical amount of DOX, which is retained in the methanolic phase.

Figure 4 shows specificity of AuNP phase transfer. A) Example of AuNP probe with a design motif for the drug target doxorubicin (DOX), here through a binary ligand mixture of 11 -Mercapto-l -undecanol and 2-Phenylethanethiol. While binding to the supramolecular receptor is observed for DOX as well as its metabolites doxorubicinol (DOX-ol) and doxorubicinone (DOX-one), AuNP phase transfer is specific to the interaction of DOX to the ligand shell (B)).

Figure 5 shows doxorubicin (DOX) detection from human serum. Absorbance of read-out phase depending on the contents of the incubation phase. When DOX is extracted from human serum, a similar extent of phase transfer is observed as for the case when the DOX is directly added to the methanolic AuNP solution. In contrast, no phase transfer is observed in the absence of DOX.

Figure 6 illustrates the concept of Multidimensional sensing. In a) and b) phase transfer is shown for samples without and with nanoparticles (NPs) (Abs 0/0.5) and as a function of the relative concentration of DOX and DOX-ol at a constant total amount of both compounds. The variation of the alkyl-chain length for 4-Cyano-4'-pentylbiphenyl (5CB, a) and 4-Octyl- 4'-cyanobiphenyl (b, 8CB) with 5 vs. 8 carbons, respectively, results in characteristic trends that allow to a certain extend for the absolute determination of DOX and DOX-ol. c) Illustration of a binary sensor array consisting of three types of AuNPs and three different types of read-out phases that allows for 2D principal component analysis.

Figure 7 shows degradation agent (DA)-induced phase transfer. Chemical structure of a) doxorubicin (DOX) and b) 7,8-dehydro-9,10- desacetyldoxorubicinone (DOX-end). The addition of NaH or SBH resulted in the degradation of DOX into DOX-end and thus a phase transfer of the compound as evidenced by UV-Vis absorbance spectroscopy of the read-out phase (c) and photographs (d).

Figure 8 shows schematic diagram of phase-transfer based biochemical sensing.

Figure 9 shows (a) Absorption and (b) emission spectra of samples using LL extraction. Figure 10 shows absorption spectra of SP extraction samples of different washing volumes, (a) with Dox; (b) without Dox. Figure 11 shows absorption spectra of SP extraction samples washed by (a) solvents of different polarity and (b) MeOH-H20mixtures.

Figure 12 shows HPLC chromatogram of as-received Dox, D-ol and D-end mixed at equal molarities (500 nm).

Figure 13 shows integrated absorbance and linear fitting of Dox, D-ol and D-end at different concentrations.

Figure 14 shows (a) Absorption and (b) Emission spectra of incubated LL-extracted Dox solutions of different pH.

Figure 15 shows photograph of incubated LL-extracted Dox solutions of different pH after phase-transfer.

Figure 16 shows HPLC chromatogram of LL-extracted Dox solution with Tris buffer of pH 9.2 after incubation at 55°C for 24 h.

Figure 17 shows pixel plot of integrated absorbance of LL-extracted (a) Dox and (b) D-ol with Tris buffer of different pH after incubation at 55°C for 24 h.

Figure 18 shows pixel plot of integrated absorbance of (a) Dox and (b )D-ol in 90%MeOH- 10%H2O mixture with Tris buffer of different pH after incubation at 55°C for 24 h.

Figure 19 shows HPLC chromatogram of LL-extracted Dox solution with 600 μΜ SBH after incubation at 55°C for 24 h.

Figure 20 shows HPLC pixel plot of integrated absorbance of SP-extracted Dox and D-ol with pH 9.6 Tris buffer at different incubation intervals.

Figure 21 shows HPLC pixel plot of integrated absorbance of SP-extracted Dox and D-ol with pH 9.0 Tris buffer at different incubation intervals.

Figure 22 shows HPLC pixel plot of integrated absorbance of SP-extracted Dox and with 600μΜ SBH at different incubation intervals.

Figure 23 shows HPLC pixel plot of integrated absorbance of 3 and 30 μg ml SP extracted Dox with SBH of different concentration after 24 h incubation.

Figure 24 shows HPLC pixel plot of integrated absorbance of 5 μg ml SP-extracted Dox and D-ol with BB of different pH MW-incubated at di^ erent temperatures for 10 min.

Figure 25 shows HPLC pixel plot of integrated absorbance of 5 μg/ml SP-extracted (a) Dox and (b) D-ol with BB of di^erent pH thermal-incubated at different temperatures for 10 min. Figure 26 shows integrated absorbance and linear fitting of degradation products from Dox and D-ol of different concentration. Figure 27 shows absorption spectra: (a) MeOH phase of incubated Dox samples after incubation; (b) 5CB phase of incubated Dox samples after phase-transfer; (c) MeOH phase of D-ol samples after incubation; (b) 5CB phase of D-ol samples after phase transfer.

Figure 28 shows peak absorbance of (a) Dox and (b) D-ol samples at different concentrations after incubation.

Figure 29 shows peak absorbance of (a) Dox and (b) D-ol samples at different concentrations after phase transfer (5CB phase).

Figure 30 shows LWCC-Absorption spectra of 1 ng/ml D-end in MeOH.

Figure 31 shows LWCC-Absorption spectra of (a) 0.1 and 1 ng/ml D-end in 90%MeOH10%5CB mixture; (b) 1 ng/ml D-end in 90%MeOH-10%5CB mixture with background removed; (c) 1 ng/ml D-end in 90%MeOH-10%5CB mixture with allometric fitting; (d)l ng/ml D-end in 90%MeOH-10%5CB mixture with Lorentzian fitting.

DETAILED DESCRIPTION OF THE INVENTION

Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. The publications and applications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. In addition, the materials, methods, and examples are illustrative only and are not intended to be limiting.

In the case of conflict, the present specification, including definitions, will control. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of skill in art to which the subject matter herein belongs. As used herein, the following definitions are supplied in order to facilitate the understanding of the present invention. The term "comprise" is generally used in the sense of include, that is to say permitting the presence of one or more features or components. Also as used in the specification and claims, the language "comprising" can include analogous embodiments described in terms of "consisting of " and/or "consisting essentially of. As used in the specification and claims, the singular form "a", "an" and "the" include plural references unless the context clearly dictates otherwise.

As used in the specification and claims, the term "and/or" used in a phrase such as "A and/or B" herein is intended to include "A and B", "A or B", "A", and "B".

Herein is disclosed a novel route to biological and chemical sensing that builds up on previously reported probe-target interaction but allows to improve specificity as well as quantitative readout by the introduction of a stimuli-induced phase transfer process as illustrated in Figure 1. This route allows to greatly improve the detection at low concentrations since only probes (i.e. gold nanoparticles (AuNPs)) with occupied receptor sites (i.e. occupied ligands) will transfer from the incubation phase into the read-out phase. As demonstrated in Figure 2, the extent of AuNP phase transfer allows for the quantitative determination of the polycyclic organic compound, namely selective extraction, separation, detection and quantification. Alternatively and especially at very low concentrations, a gradual titration with AuNPs can also be used. The requirement of phase transfer further improves specificity in suitable material systems. Further the read-out signal is not a superimposition of polycyclic organic compound-free AuNPs and polycyclic organic compound-bound (loaded) AuNPs but, because of the extraction through phase transfer, a direct measure of the polycyclic organic compound.

Attaching the polycyclic organic compound to AuNPs results in AuNPs readily going from the incubation phase into the read-out phase. The transfer can be accelerated by either temporarily forming a homogeneous mixture of incubation and read-out phase, e.g. at elevated temperatures or through emulsification of the two immiscible phases, for example in the form of stirring or shaking of the mixture.

Other methods for biological or chemical sensing include fluorescence, currents or electrochemical signals, Raman scattering, adsorption on surfaces etc. and would equally benefit from the herein proposed extraction step to isolate the polycyclic organic compound from a crowded or parasitic environment and expose only polycyclic organic compound-bound (loaded) AuNPs to the above-mentioned detection systems.

Furthermore, phase transfer based chemical sensing does not necessarily rely on binding of the polycyclic organic compound to the ligand shell of gold nanoparticles and resulting nanoparticle transfer. A chemical modification of the polycyclic organic compound to make it soluble in the read-out phase represents another viable pathway. This can be induced by the presence of a degradation agent and/or reactive gold nanoparticle surface that modifies the polycyclic organic compound and thereby modifies the solubility of the polycyclic organic compound and allows transfer of said modified polycyclic organic compound into the read-out phase. At an excess concentration of the degradation agent, the amount of phase-transferred molecules correspond to the initial concentration of the polycyclic organic compound.

In some embodiments, in the method of the invention the pH of the incubation phase is above pH 8.5. In preferred embodiments, the pH of the incubation phase is between 9 and 10. The pH above 8.5, preferably pH 9 - 10, is adjusted by a buffering agent, such as Tris buffer. In other embodiments, in the method of the invention further comprises heating the incubation phase at temperature between 50°C and 110°. In preferred embodiments, the incubation phase is heated between 80° and 90°. In other embodiments, the method of the invention further comprises measuring the absorbance of the incubation phase before the step b) (i.e. before adding the degradation agent or the gold nanoparticles and the sample containing the polycyclic organic compound into the incubation phase) and/or measuring the absorbance of the incubation phase after the step c) (i.e. after adding the degradation agent or the gold nanoparticles and the sample containing the polycyclic organic compound into the incubation phase (i.e. after incubation)).

In other embodiments, the method of the invention further comprises measuring the absorbance of the read-out phase before the step b) (i.e. before adding the degradation agent or the gold nanoparticles and the sample containing the polycyclic organic compound into the incubation phase) and/or measuring the absorbance of the read-out phase after the step c) (i.e. after adding the degradation agent or the gold nanoparticles and the sample containing the polycyclic organic compound into the incubation phase (i.e. after the phase transfer of the step c)).

In other embodiments, the method of the invention further comprises the step of mixing the incubation phase with the read-out phase after the step c) (i.e. after adding the degradation agent or the gold nanoparticles and the sample containing the polycyclic organic compound into the incubation phase), with optional heating and subsequent cooling of the mixture. This mixing step can improve the phase transfer of the modified polycyclic organic compound into the readout phase, or the phase transfer of the polycyclic organic compound bound to the gold nanoparticles into the read-out phase, or the phase transfer of the gold nanoparticles in the readout phase.

Polycyclic organic compounds may contain two-, three-, four-, five-, six-, seven-, or more member rings, said rings may be aromatic rings, cycloalkane rings, cycloalkene rings, cycloalkyne rings or combination hereof. The rings may include heteroatoms such as N, O, and S. The rings may carry substituents. In some preferred embodiments, the polycyclic organic compound is an anthracycline antibiotic. The most preferably anthracycline antibiotic is doxorubicin. The present invention can be applied to at least gold nanoparticles in the size range from 1000 nanometers to 1 nanometer.

The ligands coating the surface of gold-nanoparticles are selected to be specific to the polycyclic organic compound to be detected. The choice of ligands is motivated by weak molecular interactions between the ligand and the polycyclic organic compound, such as hydrogen bonding, pi-pi interactions, electrostatic, or hydrophobic interactions. One type or a combination of ligands can be used to form supramolecular receptors for the polycyclic aromatic compound in the ligand shell. The ligands are polyfunctional, containing one functional group to interact with the gold surface (e.g. a thiol group) and other functional groups to interact with a specific polycyclic organic compound. Examples for ligands of different chemical structures include linear, branched, cyclic and/or aromatic hydrocarbon chains, charged (e.g. primary amine, sulfonate, carboxylate) or hydrophilic (e.g. alcohol) or combinations thereof separate by a linear hydrocarbon chain linker. For example branched hydrocarbon chain would prevent crystallisation and favour a random arrangement, cyclic/aromatic hydrocarbons may have pi-pi interaction with the polycyclic organic compound, alkyl straight chains can help for hydrophobic interaction.

The methods of the present invention use two phases that have to be immiscible or immiscible at least under certain conditions. Typically one phase that contains the degradation agent or gold nanoparticles, and a polycyclic organic compound, called incubation phase and then another phase that serves as target for phase transfer and polycyclic organic compound or gold nanoparticle quantification, called read-out phase. The incubation phase of the present invention can be organic or inorganic solvent, in particular aqueous solution, which is immiscible or at least immiscible under certain conditions with readout phase that can be organic or inorganic solution, in particular a liquid crystal solution, such as 4-cyano-4'-pentylbiphenyl (5CB), 4-octyl-4'-cyanobiphenyl (8CB), (4-Cyano-4'-n- heptylbiphenyl (7CB), trans-4-(4'-n-Pentylcyclohexyl)benzonitrile, 5-Butyl-2-(4- heptyloxyphenyl)-pyrimidine.

The interaction of the polycyclic organic compound with the gold nanoparticles and/or the degradation agent can be at least one of the following: - binding of the polycyclic organic compound to the gold nanoparticles, wherein binding of the polycyclic organic compound to the gold nanoparticles modifies the solubility of said gold nanoparticles in the read-out phase and allows transfer of said gold nanoparticles into the readout phase, or

- substituting ligands on the gold nanoparticles surface by the polycyclic organic compound, wherein substitution of ligands on the gold nanoparticles surface by the polycyclic organic compound modifies the solubility of said gold nanoparticles in the read-out phase and allows transfer of said gold nanoparticles into said read-out phase, or

- the degradation agent and/or reactive gold nanoparticle surface chemically modifies the polycyclic organic compound and thereby modifies the solubility of the polycyclic organic compound and allows transfer of said modified polycyclic organic compound into the read-out phase.

The degradation agent can be a reducing agent or an oxidising agent or a catalyst such as an inorganic catalyst or an enzyme, as well as a pH buffer > 8.5 for pH -induced degradation, such as Tris buffer. In some embodiments, the reducing agent can be for example sodium hydride or sodium borohydride or related species. In other embodiments, the degradation agent can be grafted or adsorbed on the surface of the gold nanoparticles from particle preparation or an enzyme can be bound on the surface of gold nanoparticles. One example are boron alkoxides that can be grafted on the alcohol function of 11 -mercaptoundecan- 1 -ol, 6-mercapto- 1 -hexanol or similar ligands, where the boron species contribute to the degradation of the polycyclic organic compound. In some embodiments for pH-induced degradation, the pH of the incubation phase is above pH 8.5 and in preferred embodiments, the pH of the incubation phase is between 9 and 10, which is adjusted by a buffering agent, such as Tris buffer.

Also, the reactive surface of gold nanoparticle can be the surface coated with functional ligands that react as catalysts, such as enzymes, and thereby modifies the chemical structure of the polycyclic organic compound. Absorbance of the incubation phase (before and after the incubation process) and of the readout phase (before and after the phase transfer process) is measured by optical means known in the art, such as UV-Vis or fluorescence spectroscopy. The measuring of the amount of the polycyclic organic compound in the read-out phase is preferably carried out by optical means, such as through UV-Vis or fluorescence spectroscopy. For ultralow concentrations detection can be improved by using a liquid waveguide capillary cell for enhanced optical path and advanced analysis techniques such as 2D component analysis or spectral fingerprint analysis.

The strength of the methods of the present invention lies in this two-fold specificity. Besides binding to a specific molecular receptor site another requirement has to be fulfilled for a positive read-out. The loaded AuNPs need to be soluble in the complementary read-out phase, allowing to differentiate with high precision between chemically very similar molecules. Along these lines, a 2D sensor array consisting of a number of different types of AuNPs and different types of read-out phases enables to greatly amplify the read-out accuracy and to extend this sensing principle to a wide range of molecules by 2D principal component analysis. For three different read-out phases with significant solvation orthogonality, a sensor array of six pixels, is sufficient to reach high redundancy. An example array consists of two sample solutions (extracted body fluid with/without NPs) and three LCs. Adequate processing and mathematical optimisation of a 2D projection plane then allows to identify the principal components. This approach is particularly important if an individual combination of incubation phase and readout phase does not exhibit sufficient target-specific response to reach the required resolution for the polycyclic organic compound of interest, in a case where a multitude of similar polycyclic organic compounds are present in the sample or a number of different target polycyclic organic compounds need to be determined.

In some embodiments, the AuNPs of the invention are used for extraction, detection and quantification of polycyclic organic compounds that do not have a distinct absorbance or fluorescence footprint themselves. Thus, binding of such polycyclic organic compounds to AuNPs provides an optically detectable complex due to absorbance properties of the AuNPs.

In some embodiments of the invention, boron-containing species, as well as pH buffers were considered as promising candidates to establish a phase-transfer based sensing route without the use of gold nanoparticles (AuNPs). The advantage of this embodiment would be that the detected phase-transfer could be directly dependent on the amount of DOX in solution and would not also be related to the concentration of AuNPs and thus DOX receptor sites. In the latter case, AuNP titration or characterisation at different AuNP concentrations would be necessary to achieve sufficient unambiguity.

In an embodiment of the method of the invention, the method for selective separation, detection and quantification of a polycyclic organic compound in a sample comprises:

b) adding in the incubation phase a degradation agent,

c) adding a sample containing the polycyclic organic compound insoluble in the readout phase in the incubation phase under conditions sufficient to allow interaction of the polycyclic organic compound with the degradation agent of step b), whereby said interaction modifies the solubility of the polycyclic organic compound and allows transfer of said modified polycyclic organic compound into the read-out phase,

d) detecting and quantifying the polycyclic organic compound in the sample by measuring the amount of modified polycyclic organic compound in the read-out phase.

In some embodiments, in the method the pH of the incubation phase is above pH 8.5. In preferred embodiments, the pH of the incubation phase is between 9 and 10. The pH above 8.5, preferably pH 9 - 10, is adjusted by a buffering agent, such as Tris buffer.

In other embodiments, the method further comprises one or more of following steps:

heating the incubation phase at temperature between 50°C and 110°. In preferred embodiments, the incubation phase is heated between 80° and 90°.

- measuring the absorbance of the incubation phase before the step b) (i.e. before adding the degradation agent and the sample containing the polycyclic organic compound into the incubation phase) and/or measuring the absorbance of the incubation phase after the step c) (i.e. after adding the degradation agent and the sample containing the polycyclic organic compound into the incubation phase (i.e. after incubation)).

- measuring the absorbance of the read-out phase before the step b) (i.e. before adding the degradation agent and the sample containing the polycyclic organic compound into the incubation phase) and/or measuring the absorbance of the read-out phase after the step c) (i.e. after adding the degradation agent and the sample containing the polycyclic organic compound into the incubation phase (i.e. after the phase transfer of the step c)). the step of mixing the incubation phase with the read-out phase after the step c) (i.e. after adding the degradation agent and the sample containing the polycyclic organic compound into the incubation phase), with optional heating and subsequent cooling of the mixture.

In other embodiment of the method of the invention, the method for selective separation, detection and quantification of a polycyclic organic compound in a sample comprises:

a) providing a two-layer solution consisting of the first layer which is an incubation phase and the second layer which is a read-out phase, wherein the incubation phase is immiscible with the read-out phase and wherein the pH of the incubation phase is above pH 8.5, preferably between 9 and 10.

b) optionally heating the incubation phase at temperature between 50°C and 110°, preferably between 80° and 90°.

c) adding in the incubation phase a degradation agent,

d) adding a sample containing the polycyclic organic compound insoluble in the readout phase in the incubation phase under conditions sufficient to allow interaction of the polycyclic organic compound with the degradation agent of step c), whereby said interaction modifies the solubility of the polycyclic organic compound and allows transfer of said modified polycyclic organic compound into the read-out phase,

e) detecting and quantifying the polycyclic organic compound in the sample by measuring the amount of modified polycyclic organic compound in the read-out phase.

measuring the absorbance of the incubation phase before the step c) (i.e. before adding the degradation agent and the sample containing the polycyclic organic compound into the incubation phase) and/or measuring the absorbance of the incubation phase after the step d) (i.e. after adding the degradation agent and the sample containing the polycyclic organic compound into the incubation phase (i.e. after incubation)).

measuring the absorbance of the read-out phase before the step c) (i.e. before adding the degradation agent and the sample containing the polycyclic organic compound into the incubation phase) and/or measuring the absorbance of the read-out phase after the step d) (i.e. after adding the degradation agent and the sample containing the polycyclic organic compound into the incubation phase (i.e. after the phase transfer of the step d)). the step of mixing the incubation phase with the read-out phase after the step d) (i.e. after adding the degradation agent and the sample containing the polycyclic organic compound into the incubation phase), with optional heating and subsequent cooling of the mixture. In a particular embodiment of the method of the invention, the method for selective separation, detection and quantification of a polycyclic organic compound in a sample, wherein the polycyclic organic compound is doxorubicin (DOX) and wherein said method comprises a) providing a two-layer solution, wherein the first layer is methanol immiscible at temperatures below 20°C with the second layer which is a cyanobiphenyl liquid crystal and/or an immiscible organic solvent,

b) adding to the methanol phase gold nanoparticles coated with 11-mercapto-l- undecanol and 2-phenylethanethiol ligands specific to doxorubicin to be detected, wherein said gold nanoparticles are soluble in the methanol phase and insoluble in the cyanobiphenyl liquid crystal and/or immiscible organic solvent phase,

c) adding a sample containing doxorubicin in the methanol phase under conditions sufficient to allow binding of doxorubicin to the ligands or the surface of the gold nanoparticles, wherein the interaction of doxorubicin with the gold nanoparticles modifies the solubility of said gold nanoparticles and allows transfer of said gold nanoparticles into the cyanobiphenyl liquid crystal and/or immiscible organic solvent phase,

d) detecting and quantifying doxorubicin in the sample by measuring the amount of transferred doxorubicin bound to the gold nanoparticles in the read-out phase of a cyanobiphenyl liquid crystal and/or an immiscible organic solvent.

measuring the absorbance of the incubation phase before the step b) (i.e. before adding the gold nanoparticles and the sample containing doxorubicin into the incubation phase) and/or measuring the absorbance of the incubation phase after the step c) (i.e. after adding the gold nanoparticles and the sample containing doxorubicin into the incubation phase (i.e. after incubation)).

measuring the absorbance of the read-out phase before the step b) (i.e. before adding the gold nanoparticles and the sample containing doxorubicin into the incubation phase) and/or measuring the absorbance of the read-out phase after the step c) (i.e. after adding the gold nanoparticles and the sample containing doxorubicin into the incubation phase (i.e. after the phase transfer of the step c)).

the step of mixing the incubation phase with the read-out phase after the step c) (i.e. after adding the gold nanoparticles and the sample containing doxorubicin into the incubation phase), with heating up to 30° and subsequent cooling below 20° of the mixture.

In a further particular embodiment of the method of the invention, the method for selective separation, detection and quantification of a polycyclic organic compound in a sample, wherein the polycyclic organic compound is doxorubicin (DOX) and wherein said method comprises a) providing a two-layer solution, wherein the first layer is methanol immiscible at temperatures below 20°C with the second layer which is a cyanobiphenyl liquid crystal and/or an immiscible organic solvent,

b) adding to the methanol phase Tris buffer or sodium borohydride,

c) adding a sample containing doxorubicin in the methanol phase under conditions sufficient to allow degradation of doxorubicin into Dox-end and to allow transfer of Dox-end into the cyanobiphenyl liquid crystal and/or immiscible organic solvent phase,

d) detecting and quantifying doxorubicin in the sample by measuring the amount of transferred Dox-end in the read-out phase of a cyanobiphenyl liquid crystal and/or an immiscible organic solvent.

measuring the absorbance of the methanol phase before the step b) (i.e. before adding Tris buffer or sodium borohydride and the sample containing doxorubicin into the methanol phase) and/or measuring the absorbance of the methanol phase after the step c) (i.e. after adding Tris buffer or sodium borohydride and the sample containing doxorubicin into the methanol phase (i.e. after incubation)). measuring the absorbance of the read-out phase of a cyanobiphenyl liquid crystal and/or an immiscible organic solvent before the step b) (i.e. before adding Tris buffer or sodium borohydride and the sample containing doxorubicin into the incubation phase) and/or measuring the absorbance of the read-out phase of a cyanobiphenyl liquid crystal and/or an immiscible organic solvent after the step c) (i.e. after adding Tris buffer or sodium borohydride and the sample containing doxorubicin into the incubation phase (i.e. after the phase transfer of the step c)).

the step of mixing the methanol phase with the read-out phase of a cyanobiphenyl liquid crystal and/or an immiscible organic solvent after the step c) (i.e. after adding Tris buffer or sodium borohydride and the sample containing doxorubicin into the methanol phase), with heating up to 30° and subsequent cooling below 20° of the mixture.

In a preferred embodiment, the sample containing doxorubicin (DOX) is a body fluid such as blood serum, saliva, urine or lacrimation. In a further preferred embodiment, the cyanobiphenyl liquid crystal is selected from the group comprising 4-cyano-4'-pentylbiphenyl (5CB), 4- Cyano-4'-n-heptylbiphenyl (7CB), 4-Cyano-4'-n-octylbiphenyl (8CB). In another preferred embodiment, the immiscible organic solvent is, but not limited to, heptane.

In other embodiments of the invention, taking advantage of the distinct optical properties of polycyclic organic compound related species, such as DOX-related species, a phase-transfer based sensing technique was established by recording the absorbance of the incubation phase, such as MeOH phase, before phase-transfer and the read-out phase, such as liquid crystal (LC) phase, after phase-transfer. A schematic of the proposed detection principle is shown in Figure 8. To establish redundancy and thus a more reliable read-out, the simultaneous use of two or more incubation protocols and two or more LCs is proposed as individual pixels in a potential 2D principal component analysis.

Given a limited optical path length of a maximum of 1 cm in conventional UV-Vis spectrometry, clinically relevant concentration (10-1000 ng/ml for Dox) could not be detected with the set-up in our lab. However, it was still of great interests to investigate this method at a detectable concentration of 1-25 μg/ml. To explore the feasibility of the phase-transfer based sensing technique, two incubation protocols were employed to test both Dox and D-ol (Dox-ol) samples. In another study, the purpose was to verify whether phase-transfer based colorimetric sensing was able to characterise the linearity and reproducibility of the degradation process found in degradation after solid phase (SP) extraction. In order to lower the detection limit of phase-transfer based sensing technique of the invention, LWCC spectrometry (LWCC-3100- TIDAS 1 spectrometer with D4H light source, World Precision Instruments Deutschland GmbH) was employed in a set of preliminary experiments. Its 100 cm optical path and ultra- high signal-to-noise may enable us to detect anthracycline drugs at a significant lower concentration.

In another aspect, the invention provides a method for selective extraction of a polycyclic organic compound from a sample, such as a body fluid. Namely plasma, serum, and urine are typical matrices used in in-vitro analysis. Analytical detection from these body fluids typically starts with the extraction of designated molecules. Given the complex composition of human blood, a clean and specific extraction is of particular importance. Unwanted residues may influence the chemistry during the degradation process and absorbing species may overshadow the weak optical signal of target molecules because of the very low clinical concentrations. In another perspective, it would be possible to overcome individual variability and improve detection limit with a more efficient extraction protocol.

During the first step of plasma extraction, blood cells are removed by centrifugation from plasma. Chemicals like anticoagulant are added to prevent blood from clotting. The collected plasma can then be kept in a low-temperature environment, typically at -18°C for storage or transportation. To extract anthracyclines from human plasma, the major strategies compose of deproteinisation, liquid-liquid (LL) extraction and solid phase (SP) extraction. Organic solvents, metal ion and acid have been used to induce protein precipitation, which is then followed by either LL extraction or SP extraction.

Organic solvents such as MeOH, acetone and acetonitrile, zinc sulphate and hydrochloric acid (HC1) were used in LL extraction of DOX and DOX-ol. On the other hand, it has been reported that SP extraction based on reversed-phase column is capable of extracting DOX and its metabolites selectively while maintaining a recovery as efficient as in the case of the LL extraction.

In an embodiment, the invention provides a method for selective extraction of a polycyclic organic compound from a sample comprising: a) providing a two-layer solution, wherein the first layer is an incubation phase immiscible with the second layer which is a read-out phase,

d) releasing the polycyclic organic compound from the gold nanopartciles to the readout phase,

e) recovering the polycyclic organic compound from the read-out phase. To enable the polycyclic organic compound to be released readily from the gold nanoparticles after phase transfer, it is proposed that a reversible bond between the ligands coated on the gold nanoparticle surface and the polycyclic organic compound is used. For the present purposes, a "reversible" bond means essentially a type of bond which is mainly due to van der Waals, hydrogen bonding, or electrostatic forces.

If the interaction is reversible, one may retrieve the gold nanoparticles and release the polycyclic organic compound by for example temperature or selective solubility (a solvent that is a good solvent for the polycyclic organic compound but a poor one for the gold nanoparticles. It is also possible to use ligand replacement by an excess of thiolated molecules (e.g. glutathione) which replace the ligands and therefore cleave the receptor or etching and solving the gold particles by iodine or cyanide.

In a further aspect, the invention provides kits for carrying out the methods of the present invention. Kits of present invention contain at least appropriate incubation phase and read-out phase. In addition, kits may contain gold nanoparticles coated with appropriate ligands and/or a degradation agent.

According to an embodiment, the present invention provides a kit for detecting and quantifying doxorubicin (DOX) in a sample, comprising a) methanol,

b) a cyanobiphenyl liquid crystal and/or an immiscible organic solvent,

c) gold nanoparticles coated with 11-mercapto-l-undecanol and 2-phenylethanethiol ligands specific to doxorubicin and/or its metabolites and/or a buffer agent for adjusting pH above pH 8.5, preferably between 9 and 10 and/or a degradation agent.

In some preferred embodiment, the cyanobiphenyl liquid crystal is selected from the group comprising 4-cyano-4'-pentylbiphenyl (5CB), 4-Cyano-4'-n-heptylbiphenyl (7CB), 4-Cyano- 4'-n-octylbiphenyl (8CB); the immiscible organic solvent is, but not limited to, heptane and the buffer agent is selected from the group consisting of Tris buffer (2-Amino-2-hydroxymethyl- propane-l,3-diol).

In addition to the above-mentioned components, the kit can include informational material. The informational material can be descriptive, instructional, marketing or other material that relates to the use of methods of the present invention. The informational material of the kits is not limited in its form. In many cases, the informational material, e.g., instructions, is provided in printed matter, e.g., a printed text, drawing, and/or photograph, e.g., a label or printed sheet. However, the informational material can also be provided in other formats, such as Braille, computer readable material, video recording, or audio recording. In another embodiment, the informational material of the kit is a link or contact information, e.g., a physical address, email address, hyperlink, website, or telephone number, where a user of the kit can obtain substantive information about the formulation and/or its use in the methods described herein. Of course, the informational material can also be provided in any combination of formats.

In some embodiments, the kit contains separate containers, dividers or compartments for each component and informational material. For example, each component can be contained in a bottle, vial, or syringe, and the informational material can be contained in a plastic sleeve or packet. In other embodiments, the separate elements of the kit are contained within a single, undivided container. For example, the formulation is contained in a bottle, vial or syringe that has attached thereto the informational material in the form of a label.

A sample amenable to the methods of the present invention can be a sample from any sources, for example, but not limited to biological samples, e.g., collected from organisms, animals or subjects, environmental samples, food, food by-product, soil, archaeological samples, extra- terrestrial samples, organic samples, inorganic samples or any combinations thereof. In alternative embodiments, a sample can comprise one or more cells, one or more tissues, one or more complex fluids, or any combinations thereof. In some embodiments, the sample can comprise a tissue sample. In some embodiments, the sample can comprise a fluid sample. In some embodiments, a sample can comprise blood, sputum, cerebrospinal fluid, urine, saliva, sperm, sweat, mucus, nasal discharge, vaginal fluids or any combinations thereof. In some embodiments, a sample can comprise a biopsy, a surgically removed tissue, a swap, or any combinations thereof. In some embodiments, the term "sample" refers to a biological sample. The term "biological sample" as used herein denotes a sample taken or isolated from a biological organism, e.g., tissue cell culture supernatant, cell lysate, a tissue sample (e.g., biopsy), a homogenate of a tissue sample from a subject, or a fluid sample from a subject. Exemplary biological samples include, but are not limited to body fluids, such as blood, sputum, urine, cerebrospinal fluid, urine, sweat, mucus, nasal discharge, vaginal fluids, spinal fluid, pleural fluid, nipple aspirates, lymph fluid, the external sections of the skin, respiratory, intestinal, and genitourinary tracts, tears, saliva, milk, faeces, sperm, cells or cell cultures, serum, leukocyte fractions, smears, tissue samples of all kinds, plants and parts of plants, microorganisms (such as bacteria), viruses (such as cytomegalovirus, HIV, hepatitis B, hepatitis C, hepatitis [delta] virus), yeasts, embryos, fungi, cell-free sample material, etc. The term also includes both a mixture of the above- mentioned samples such as fungus-infected plants or whole human blood containing mycobacteria as well as food samples that contain free or bound nucleic acids, or proteins, or cells containing nucleic acids or proteins, environmental samples which contain free or bound nucleic acids, or proteins, or cells containing nucleic acids or proteins. The term "biological sample" also includes untreated or pre -treated (or pre-processed) biological samples.

A "biological sample" can contain cells from subject, but the term can also refer to non-cellular biological material, such as non-cellular fractions of blood, saliva, or urine. In some embodiments, the sample is from a resection, bronchoscopic biopsy, or core needle biopsy of a primary or metastatic tumour, or a cell block from pleural fluid. In addition, fine needle aspirate samples can be used. Samples can be either paraffin-embedded or frozen tissue. In some embodiments, a biological sample can comprise a biopsy, a surgically removed tissue, a swap, or any combinations thereof. The sample can be obtained by removing a sample of cells from a subject, but can also be accomplished by using previously isolated cells (e.g. isolated by another person). In addition, the biological sample can be freshly collected or a previously collected sample. Furthermore, the biological sample can be utilized for the detection of the presence and/or quantitative level of a polycyclic organic compound of interest. Representative target polycyclic organic compound include, but are not limited to biomolecules, such as nucleic acids, proteins, and derivatives and fragments thereof, or drug and prodrug molecules, such as pharmaceutically active ingredients and metabolites thereof.

In some embodiments, the biological sample is an untreated biological sample. As used herein, the phrase "untreated biological sample" refers to a biological sample that has not had any prior sample pre-treatment except for dilution and/or suspension in a solution. Exemplary methods for treating a biological sample include, but are not limited to, centrifugation, filtration, sonication, homogenization, heating, freezing and thawing, and any combinations thereof.

In accordance with some embodiments of various aspects described herein, a biological sample can be pre-treated, as described above, before employing the methods of the present invention. In some embodiments, the biological sample can be treated with a chemical and/or biological reagent(s). Chemical and/or biological reagents can be employed to protect and/or maintain the stability of the sample or target polycyclic organic compounds during processing. In addition, or alternatively, chemical and/or biological reagents can be employed to release or expose target polycyclic organic compounds from other components of the sample. One exemplary reagent is a protease inhibitor, which is generally used to protect or maintain the stability of the target polycyclic organic compounds during processing.

In some embodiments, the term "sample" as used herein can refer to an environmental sample (including, but not limited to, air, agricultural (e.g., but not limited to hydrofarms or hydroponic samples), pond, water, wastewater, and soil samples); biological warfare agent samples; research samples including extracellular fluids. In some embodiments, an environmental sample can comprise a sample collected from a working surface of an equipment or machine (e.g., but not limited to, food or pharmaceutical product processing equipment or machine), a device (e.g., but not limited to, biomedical devices, implantation devices, fluid delivery devices such as a tubing, and/or a catheter), and/or a building or dwellings (e.g., but not limited to, food processing plants, pharmaceutical manufacturing plants, hospitals, and/or clinics).

In some embodiments, a sample can comprise food (e.g., solid and/or fluid food as well as processed food) and/or food by-product. For example, the methods of the present invention can be used to detect a polycyclic organic compound, e.g., a particular nutrient, in food and/or food by-product, e.g., but not limited to, meat, milk, yoghurt, bread, starch-based products, vegetables, and any combinations thereof. In some embodiments, the methods of the present invention can be used to detect a contaminant, e.g., bacteria, fungus, spores, moulds, viruses as well as inorganic contaminants, such as mercury, in food and/or food by-product.

In some embodiments, a sample can comprise a pharmaceutical product (e.g., but not limited to pills, tablets, gel capsules, syrups, vaccines, liquids, sprays, and any combinations thereof). For example, the methods of the present invention can be used to detect the presence and/or measure the level of a particular active agent present in a pharmaceutical product. In some embodiments, the methods of the present invention can be used to detect a contaminant, e.g., bacteria, fungus, spores, moulds, and/or viruses, in a pharmaceutical product.

In some embodiments, a sample can comprise an archaeological sample. In some embodiments, an archaeological sample can be obtained or collected from artefacts, architecture, biofacts (or ecofact, e.g., an object found at an archaeological site), cultural landscapes, and any combinations thereof.

In some embodiments, a sample can comprise an extra-terrestrial sample. For example, an extra-terrestrial sample can be any object or specimen (e.g., rock, meteorite, and/or environmental samples) obtained or collected from outer space or universe, and/or planets beyond the planet Earth, e.g., the moon, other planets (e.g., but not limited to, Mars, and/or Jupiter) and/or non- stellar objects. In the food and feed industry, some embodiments of various aspects described herein have a wide variety of applications. For example, it can be used for identification and characterization of production organisms such as yeast for production of beer, wine, cheese, yogurt, bread, etc. Another area of use is related to the quality control and certification of products and processes (e.g., livestock, pasteurization, and meat processing) for contaminants. Other uses include the characterization of plants, bulbs, and seeds for breeding purposes, identification of the presence of plant- specific pathogens, and detection and identification of veterinary infections.

Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the invention includes all such variations and modifications without departing from the spirit or essential characteristics thereof. The invention also includes all of the steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations or any two or more of said steps or features. The present disclosure is therefore to be considered as in all aspects illustrated and not restrictive, the scope of the invention being indicated by the appended Claims, and all changes which come within the meaning and range of equivalency are intended to be embraced therein.

Various references are cited throughout this Specification, each of which is incorporated herein by reference in its entirety.

The foregoing description will be more fully understood with reference to the following Examples. Such Examples, are, however, exemplary of methods of practicing the present invention and are not intended to limit the scope of the invention.

EXAMPLES

Example 1: PHASE TRANSFER BASED SENSING OF DOXORUBICIN

Doxorubicin (DOX) is an anthracycline antibiotic widely use in cancer chemotherapy. Its cumulative dose-dependent side effects, i.e. cardiotoxicity, compromise the anticancer efficacy. Due to the low therapeutic index, i.e. a narrow toxic-therapeutic dose range and effective dosage, dosing of the anticancer drugs should take into account the individual variability of metabolic activity and of bio-distribution of each patient. Yet, no procedure in clinical practice allows routine therapeutic drug monitoring of DOX and metabolites.

The exact cardiotoxicity mechanism is still unsettled. Among Dox and its metabolites, there are three main metabolic routes: one-electron reduction, two-electron reduction and deglycosidation. The secondary alcohol-doxorubicinol (D-ol or Dox-ol), is the major metabolite which reduces doxorubicin through a two-electron reduction, while doxorubicione (D-one or Dox-one), the products of deglycosidation, is a minor metabolite accounting for approximately 1-2 % of doxorubicin metabolism. Besides the increased formation of reactive oxygen species (ROS), the formation of D-ol has also been proposed to be associated with specific DNA mutations and thus responsible for the cardiotoxicity. The ROS hypothesis suggests the cardiotoxicity is induced during redox cycling of Dox, in which the unstable semiquinone compound is involved. ROS itself can also cause damage of healthy cells, e.g. through lipid peroxidation or oxidative stress. D-ol induces cytotoxicity by interfering with calcium and iron regulations.

The inventors have recently demonstrated that the assembly of binary ligand mixtures on gold nanoparticles (AuNPs) can create supramolecular receptor sites that bind specific classes of molecules by weak intermolecular forces such as pi-pi interactions, electrostatic interactions or hydrogen bonding. This was shown in a first study for AuNPs with an organic shell composed of a mixture of naphthalene-terminated thiol and hexanethiol ligands, which allowed the capturing of polycyclic aromatic hydrocarbons. Similarly, NPs with a shell composed of 11-mercapto-l-undecanol and 2-phenylethanethiol ligands are capable to bind DOX. When exposed to methano li c DOX-containing blood serum, AuNPs precipitate upon D O X binding due to the increase in hydrophobicity. Subsequent release of the target molecule by ligand exchange allows to reliably detect the binding of DOX by fluorescence spectroscopy.

While rational design of receptors by choosing suitable ligand mixtures allows reliable capturing of certain types of molecules even in crowded environment, this approach still lacks a reliable read-out mechanism for sensing, similarly to other approaches for colorimetric sensing explained above. T he inv ento rs have further extended the approach of nanoparticles-b ased colourimetric sensing by interfacing with an immiscible liquid that serves as read-out phase for AuNPs that have altered their solubility properties as a result of capturing target polycyclic organic compounds in their ligand shell. In this example, the inventors use cyanobiphenyl liquid crystals as complex liquid for AuNP read-out, in particular 4-cyano-4'-pentylbiphenyl (5CB), but also 4-Cyano-4'-n-octylbiphenyl (8CB), see Figure 6b. It is important to note that the inventors use 5CB and 8CB in the isotropic phase, thus, as a complex fluid with specific and temperature-dependent solubility properties. In fact, the methanolic incubation phase consisting of NPs and DOX mixes homogeneously with the read-out phase above room temperature and then phase separates below 20 ° C.

The conditions to allow reaction of DOX with ligands on the surface of gold nanoparticles are: 55°C (45-65°C), 4-24h, preferably overnight.

Results are shown in Figure 2. In Figure 2a, the phase transfer of AuNPs that were incubated with a varying amount of DOX to the 5 CB phase is compared to a control containing DOX without AuNPs. In case of AuNPs without DOX as well as DOX without AuNPs, no phase transfer was observed. In contrast, when AuNPs were previously incubated with DOX, phase transfer of loaded AuNPs occurred. This transfer was quantitative and depended on the amount of target polycyclic organic compound as well as available AuNP probes as shown in the two-dimensional plot in Figure 2b.

As previously discussed, the inv entors do not make use of the liquid crystalline character of 5CB but rather of its properties as a complex solvent. In fact a variety of immiscible solvents as exemplarily shown for heptane in Figure 3 show a comparable phase separation behaviour. Due to the density of heptane (0.6795 g/cm³) and methanol (0.7918 g/cm³), the position of read-out and incubation phase are reversed. In Figure 3a-e, phase separated samples are shown with a target molecule to receptor ratio of 0; 0.3; 0.5; 0.75 and 1, respectively. To the right, control samples are shown without AuNPs but an identical amount of DOX, which is retained in the methanolic phase. Again, the addition of DOX leads to a change in AuNP solubility in the incubation phase, AuNP prevalence at the 2-phase interface and subsequent complete removal from the incubation phase. Yet, the DOX-loaded NP probes are not well soluble in heptane, making this solvent somewhat less ideal than 5CB for quantitative read-out.

In Figure 4A a schematic of the supramolecular receptor on the AuNP surface is shown alongside with the chemical structure of DOX as well as its main metabolites, namely doxorubicinol (DOX-ol) and doxorubicinone (DOX-one). DOX-ol is metabolised by a two- electron reduction that results in a conversion of a ketone into a hydroxyl group, while DOX- one is formed by a deglycosidation. While the presence of DOX results in quantitative phase transfer of AuNPs into the LC read out phase, no phase transfer is observed for DOX-ol (Figure 4B). This is somewhat surprising since binding of all three molecules was confirmed by fluorescence experiments. The inventors rationalise that binding to the receptor occurs via π-π interaction and hydrogen bonding of the anthracycline, which is identical in all three cases. Accordingly, the subtle differences in the protruding moieties from the ligand shell results in a solubility contrast that allows differentiation by the phase transfer-based readout.

For therapeutic drug monitoring and relevance in clinical practise, DOX should be detected from human body fluids, such as blood, or non-invasively collected in saliva, urine or lacrimation. Compatibility of this sensing approach with biological samples is not self- evident. While extraction of DOX from biological fluids can be readily carried out by liquid- liquid or solid phase extraction, residual proteins rapidly bind to the surface of NPs and may impede a target-specific phase transfer. The inventors observed that DOX extracted from human serum showed a delayed binding to the NP receptors perhaps due to competition with unspecific protein binding, but overall loading remains comparable. In Figure 5 the extent of phase transfer is compared for three different samples: I) DOX is added directly to the AuNP-containing incubation phase, II) the same amount of DOX is added to human serum (spiked sample) and subsequently extracted by protein precipitation in methanol before mixing the incubation phase and further processing. As evidenced by UV-Vis spectroscopy of the read-out phase, phase transfer remains efficient even for biological samples. The slight decrease may also be related to loss of the absolute amount of DOX extraction from the serum. In the absence of DOX, no AuNP phase transfer is observed. These results underline the potential of the invention for clinical practise.

This approach is applicable to a wide range of drugs that need therapeutic drug monitoring. Depending rational design of the molecular receptors, similar results are foreseen for other widelyused chemotherapeutic drugs, such as epirubicin, irinotecan (SN-38) or methotrexate. It has previously been shown that specific enzymes triggered an alteration of AuNP solubility, enabling to efficiently detect protease, kinase, phosphatases, /^-lactamase by phase transfer. Example 2: SENSING OF DOXORUBICIN BY DEGRADATION AGENT-INDUCED PHASE TRANSFER

The phase transfer based (bio-)chemical sensing is also possible without AuNPs that act as polycyclic organic compound receptor. Phase transfer of the polycyclic organic compound for extraction and read-out is also feasible by the modification of its chemical structure, e.g. through degradation. The degradation pathways of DOX are well known. The presence of a degradation agent, such as a reducing agent such as sodium hydride (NaH) as well as boron- containing species, such as sodium borohydride (NaBfU; SBH), degrades DOX into its main aromatic backbone 7,8-dehydro-9,10-desacetyldoxorubicinone (D-end or DOX-end) as shown in Figure 7a,b. While the solubility of DOX alone in CB is very low, the solubility of DOX- end is significantly higher. The degradation of DOX to DOX-end therefore allows inducing a phase transfer of the reduced DOX into the read-out phase for quantitative detection by UV- Vis fluorescence or absorbance spectroscopy or merely for extraction and isolation of the compound. This is shown in Figure 7c,d. It is important to note that this degradation also occurs spontaneously in body fluids or samples that were extracted from body fluids with time or elevated temperatures.

D-end has been reported to be a stable degradation product of Dox in a basic aqueous environment while D-ol follows an another degradation pathway due to the lack of the ketone group on C- 13. It is important to note that D-end itself is soluble in the liquid crystal (LC) even without the gold nanoparticles (NP) shuttle.

Example 3: EXTRACTION OF ANTHRACYCLINES FROM HUMAN PLASMA 3.1 Liquid-liquid (LL) extraction

The extraction of Dox and D-ol (Dox-ol) started with mixing 60 μΐ 2mM Dox (doxorubicin, hydrochloride salt, >99%, LC Laboratories) or D-ol (doxorubicinol, hydrochloride salt, >85%, Chemtos) in 0.4 ml human plasma. After shaking for 5 min, 1.6 ml ice-cold MeOH was added. Then the sample was sonicated at room temperature (RT) for 2 min and vortexed for 10 min. The pellet and supernatant were separated after centrifugation at 14000rpm, 4°C for 15 min. The collected supernatant would be used without further purification. Control samples without adding Dox were prepared for absorbance and emission measurement. After this first measurement, 1 ml of the sample was mixed with 1 ml 5CB (CAS No. 40817-08-1, Synthon Chemicals) and heated in the oven (55 C) for 15 min. The sample was cooled in the fridge (4°C) before phase separation. 480 μΐ of the 5CB phase and 240 μΐ of the MeOH phase was used to measure the absorbance. Both phases were residing in the cuvette and only the lower phase accounted for the measured absorbance, which was carried out at 40°C with a scan speed of 200 nm/min. 970 μΐ of the 5CB phase was used to measure the emission with the excitation at 470 nm and as speed of 200 nm/min. 30 μΐ MeOH was added to obtain a clear LC phase at RT. Throughout the study, a JascoV670 spectrophotometer coupled with ETCR-762 water Peltier cooler was used for absorbance measurement and a Jasco FP-6 00 spectrofluorometer for emission measurement, unless otherwise denoted. Absorption and emission spectra of the LL extracted solution both before and after phase- transfer are shown in Figure 9. For background correction, absorbance at 700 nm was offset to 0 for all samples. Absorbance of pure MeOH and 5CB solution were measured as reference. From these dotted curves, 5CB has substantial parasitic absorption especially in the high-energy range (<450 nm), while influence of MeOH is negligible. However, the solid lines shows there are considerable amount of residual absorbing species from human plasma and some of them also transfer into the 5CB phase. To mimic single-beam absorbance detection used in many micro-device, the measurements were performed without the reference beam. This may have brought in uncertainty, which resulted in the negative absorbance of the pure MeOH sample. As for the fluorescence of the extracted solution, the emission intensity is rather weak and no pronounced peak has been observed.

3.2 Solid phase (SP) extraction

The SP extraction started with adding 20 μΐ of a 2 M Dox in MeOH stock solution into 0.5 ml RT-conditioned human plasma. The solution was then vortexed and briefly sonicated. After the cartridge was conditioned by 1.5 ml MeOH, 1.5 ml MeOH-H20 mixture (50%-50%) and 3 ml 0.05 M Na2HP04 aqueous solution (DSP), the plasma solution was pipetted. Another 1.5 ml 0.05 M DSP was used in the washing protocol. In the end, a Dox-extracted solution was then collected by eluding the cartridge with 1.5 ml MeOH. During these steps, the flow rate was controlled manually at 5 ml/min. Absorbance of the obtained samples was measured without further purification. Control samples without adding Dox stock solution were also prepared to study the residual absorption. Various samples were prepared using different washing protocols. In the first group, the cartridges were conditioned by 1.5 ml MeOH, 1.5 ml MeOH- H20 mixture (50%50%) and 3 ml 0.05 M DSP, while 0.05 M DSP of different volumes was used in the washing protocol. Organic solvents with different polarity, namely heptane, pentane, ether, cyclohexane and dichloromethane, were used to wash the samples in the second experiment. 3 ml of each was employed in every sample. MeOH-H20 mixtures of different ratios were also tested in another experiment. The cartridges were conditioned by 1.5 ml MeOH, 1.5 ml MeOH-H20 mixture (50%-50%) and 2.25 ml 0.05 M pH 7.0 phosphate buffer (PB) before the washing of the cartridge. 4.5 ml of 0.05 M pH 7.0 PB and 2.25 ml 10% MeOH - 90% H20 mixture were used in the first sample and 2.25 ml of 0.05 M pH 7.0 PB, 2.25 ml 10% MeOH - 90% H20 mixture and 2.25 ml ether were used in the other sample. In all experiments, Dox was collected by eluding the cartridge with 1.5 ml MeOH. To study the Dox recovery of SP extraction, a preliminary experiment was carried out to compare the absorbance of extracted Dox sample and corresponding reference prepared from stock solution. The efficient recovery of Dox using SP extraction is evidenced by the results shown in Figure 10. The recovery rate is still higher than 85%, even with extensive washing. When the volume of 0.05 M DSP used for washing was increased from 1.5 ml to 3 ml, the maximal absorbance decreased below 0.01 AU. The absorbance from residual species could not be further decreased with further washing. As for the effect of organic solvent of different polarities, the slightly polar diethyl ether was able to remove most of the absorbing species with the maximal absorbance around 0.005 AU (Fig 2.3(a)), while other candidates proved less efficient than 0.05 M DSP.

To maintain Dox, polar solvents were avoided and very apolar solvents were proposed when planning the experiment. However, the results of samples washed by heptane and cyclohexane in particular, proved the hydrophilicity of residues from human plasma. With the observation that the residual species were co-eluded with MeOH, the washing using MeOH-H20 mixture was assumed to be more efficient. The absorbance decreased when increasing MeOH ratio while the loss of Dox was also observed at the same time. Therefore 10% MeOH rather than more concentrated solution was used as a standard in the following experiments. To provide a neutral buffering environment, a minor adjustment was also made and DSP was replaced by PB. Comparing the results in Figure 11, the washing with 4.5ml of PB and 2.25ml 10%MeOH- 90%H2O was able to yield <0.005 AU of absorbing residues. No further improvement was achieved in the sample in which 10%MeOH-90%H2O and ether were combined in the washing. Considering the use of MeOH-H20 mixture would not introduce new chemical species to the system, the washing only with MeOH-H20 mixture has been chosen as the optimised protocol to extract Dox and D-ol from human plasma. In conclusion, an optimised SP extraction based on a commercially available C 18 cartridge was established as a more efficient procedure to selectively extract Dox than by LL extraction. SP extraction of D-ol was also carried out and similar results were obtained. The residing maximal absorbance decreased from 0.05 AU to 0.005, which makes it feasible to detect of Dox and D- ol at clinically relevant concentrations. It is also important to note that the SP extracted solution contains almost no water content while the number for LL extraction is >10%. This is beneficial for the consecutive phase-transfer process as water inhibits the mixing of MeOH and 5CB as found in a previous study. Consequently, SP extraction protocol described above was used to supply Dox and D-ol for the experiments in the present invention.

Example 4: pH BUFFER AND SODIUM BOROHYDRIDE (SBH)

Experiments were designed to explore viable HPLC protocols for the separation and quantification of Dox and D-ol. LL-extracted samples were then analysed to verify whether pH buffer and SBH could induce Dox degradation in a MeOH-based solution.

4.1 Experimental description

The analytical HPLC characterisations were performed with a Shimadzu LCMS-2020 Liquid chromatograph mass spectrometer, operated at 40°C. The mobile phase consisted of a mixture of H20 (A) and acetonitrile (B) and 0.05% trifluoroacetic acid (TFA). The flow rate was set to 1 ml/min. The eluding condition started with a solvent volume ratio of 95A/5B. Subsequently the fraction of B was linearly increased to 95% in the course of 30 min. Every measurement was followed by a 20-min column washing run where the amount of B in the mobile phase was linearly increased from 5% to 95%. The light absorption information was obtained by a photodiode array (PDA) with a spectral range from 210 nm to 600 nm and an optical path length of 1 cm. Considering that most Dox-related species absorb significantly around 500 nm[23], this wavelength was chosen for characterisation.

Samples with a serial number of 3.1 (S-3.1) were prepared to test HPLC protocols. Methanolic solutions with 12 μΜ Dox, D-ol and D-end (doxorubicin impurity, CAS No.

64845-67-6, Toronto Research Chemicals Inc.) each were prepared from stock solutions. 50 μΐ of each sample was then injected for HPLC analysis. To establish an absorbance-concentration relationship, S-3.2 were prepared similarly as S-3.1 but other concentrations of 2, 10 μΜ Dox, D-ol and D-end, 50 and 250 μΜ Dox and D-ol in MeOH were used for HPLC analysis. S-3.3 were prepared from LL-extracted Dox solutions with pH adjusted by citric acid and sodium hydroxide (NaOH). Samples of pH values: 4.2, 7.3, 9.1 and 10.7 were obtained before incubation in an oven (55 C) for 24 h. The absorption and emission spectra of each sample was recorded after the incubation. 0.5 ml of each incubated sample was then mixed with 0.5 ml 5CB for investigations of phase-transfer. S-3.4 were prepared for HPLC analysis, similarly as S-3.3 but the pH was adjusted with Tris buffers of pH 8.2, 9.2 and 9.7. As a complement, S-3.5 in a mixture of 90%MeOH10%H2O were prepared directly from methanolic stock solution. 600 μΜ SBH was used to replace Tris buffer for the preparation of S-3.6 before they were incubated under same conditions as S-3.4.

4.2 Results and discussion

Dox and D-ol were successfully separated with the linear 5B/95B eluding protocol in the HPLC analysis. During the 30-min span, D-ol was eluded at around 16.2 min and Dox slightly later, at around 17.1 min, confirmed by chromatograms of samples using both single compound and compound mixtures. The proximity of Dox and D-ol peaks can be explained by the very small difference in their chemical structure. It also suggests longer column and high-precision injection may be required to reliably separate Dox and D-ol by liquid chromatography. The hydrophobic degradation product of Dox, Dox-end (D-end) was eluded with the mixture containing 70% acetonitrile (26.3 min peak). This hydrophobicity confirmed the previous observations in phase-transfer experiments. The peak positions of these three standard compounds are shown in Figure 12, which will serve as reference in the following experiments. The absorbance at 500 nm of Dox and D-ol at four different concentrations and D-end at two different concentration are shown in Figure 13 (Sample S-3.2). Dox and D-ol data were linearly fitted using Origin Pro 8. Good linearities were obtained, proven by the adjusted R2 value of 0.9997 and 0.9998 for Dox and D-ol, respectively. The absorbance concentration relationship of Dox and D-ol was thus established for our specific HPLC protocol. At 500 nm, 1 μΜ Dox yields 44000 mAU in the PDA detector while 1 μΜ Dox results in 22000 mAU. In contrast, D- end has a limited solubility in MeOH and only two samples were prepared considering the detection limit of the HPLC system. Calculated according to the slope value, the absorbance- concentration relationship of D-end was determined to be 60000 mAU/μΜ. The first experiment of pH-induced Dox degradation using S-3.3 confirmed that Dox underwent pH specific degradation routes. After an incubation at 55 C for 24 h, solutions with a pH ranging from 4 to 11 showed different absorption and emission properties (Figure 14). In acidic and neutral environment, the Dox-like peak structure was conserved and the evaporation of MeOH during the incubation attributed to the increase of optical densities. It is also possible that Dox underwent deglycosidation to result in D-one. However, this process cannot be confirmed by UV-Vis spectroscopy as Dox and D-one have somewhat identical optical footprints. In a slightly basic solution, a small amount of D-end was produced, evidenced by the emergence of the triple absorption peak structure and the greatly enhanced emission intensity at 553 nm. The production of D-end accelerated when the pH of the solution increased from 7.3 to 9.1, while no optically identical compound was obtained in a very basic solution, e.g. at pH 10.7. The phase-transfer behaviours of these samples also supported the spectrometric results considering D-end had a purple-pink colour while Dox was orange. D-end was assumed to transfer into the bottom 5CB phase because of its hydrophobic nature. This is consistent with the observations shown in the photograph of Figure 15, where the solution of pH 9.1 exhibited the highest amount of phase transfer. Tris buffer at pH 9 provided a somewhat ideal basic media and enabled a rather efficient degradation of Dox into D-end. The chromatogram in Figure 16 is typical for Dox degradation with Tris buffer. The consumption of Dox is represented by the peak decrease at 17.1 min and the production of D-end is evidenced by the emergence of the peak at 26.3 min. Two reasons can explain the numerous secondary peaks appearing in the chromatogram. First, LL extraction did not prove to provide a clean supernatant and the residues from human plasma may interact with the multiple reaction sites of Dox. Second, Tris-induced Dox degradation is a multi-step and slow process. The reaction stayed at the intermediate stage after the incubation for 24 h, evidenced by the pronounced intensity from the Dox peak. The chromatography results using different Tris buffer are summarised in a pixel plot. The horizontal axis represents selected retention time where specific peaks were observed. Please note that this axis is therefore not to scale. For Dox samples, the characteristic peaks were: 17.1 (Dox), 20.1, 22.9, 26.3 (D-end) and 28.7 min while for D-ol samples characteristic peaks were identified at: 16.2 (D-ol), 18.3, 18.5 and 22.1 (D-ol-end) min. The colour of each pixel represents the integrated absorbance of the peak at a certain retention time. Results shown in Figure 17 confirm the trend observed from previous experiments: the Dox degradation is more pronounced in a more basic environment, e.g. the sample of pH 9.7 saw the complete disappearance of the Dox peak and the strongest D-end peak. It is important to point out that the degradation of D-ol is also more pronounced in a solution of higher pH. Most of the products of D-ol degradation are at 18.3 and 18.5 min and they have a similar absorption spectra to that of Dox and D-ol. When comparing Figure 17 and Figure 18, it is evident that incubated samples in a solvent mixture of 90%MeOH-10%H2O showed similar trends to that of LL-extracted supernatant originating from spiked human plasma. However, much less secondary peaks were observed in the chromatogram, suggesting that the reaction in neat solvents was much cleaner and generated less side-products. This observation further supported our assumption that the LL-extracted supernatant was not clean and plasma residues induced unexpected reactions. Results obtained for the degradation induced by SBH were significantly different to the one with Tris buffer. Comparing Figure 19 to Figure 16, firstly, the results from S-3.6 show that the SBH-induced Dox degradation was faster, also evidenced by the complete disappearance of Dox peak at 17.1 min. Second, given the degradation rate is higher, much less intermediate or side products were observed in the case of SBH. This is advantageous because a clean and complete reaction is of great importance for the consecutive effective quantification. Lastly, a second major degradation product appeared at 28.8 min (D-end). The chromatographic property suggests this product is even more hydrophobic than D-end and thus it is very likely to transfer to 5CB phase from MeOH.

4.3 Degradation after SP extraction

SP extraction proved to be a more efficient procedure to extract anthracyc lines than by LL extraction in the aspect of plasma residues. The optimised SP extraction established in 2.4 was therefore used in following systematic degradation study. It is also important to point out that the LL-extracted supernatant was assumed to be a mixture of MeOH and H20 with the percentage of H20 content above 10% because 0.4 ml human plasma was added with 1.6 ml MeOH as mentioned in 2.2. In contrast, the H20 content was negligible in SP extraction because neat MeOH was used in the eluding step. This difference in water content was anticipated to have an impact on the effectiveness of water-based pH buffer system, which is discussed later herein. 4.3.1 Experimental description

4.3.1.1 pH-induced degradation

To study the effect of Tris buffer at elevated temperature, different Dox and D-ol samples were added to Tris buffers of pH 9.6 (S-3.7) and 9.6 (S-3.8) and incubated at 90°C using high pressure tubes (Ace). Samples at different incubation intervals ranging from 2 h to 48 h were collected for HPLC analysis.

4.3.1.2 Degradation induced by boron compound

600 μΜ SBH was used to prepare S-3.9 before they were incubated in an oven (55°C) at different intervals ranging from 1 h to 168 h. S-3.10 were treated similarly as S-3.4 for HPLC analysis, but samples containing 3 and 30 μg/ml Dox were exposed to 6, 60 and 600 μΜ SBH, respectively. Borate buffer (BB) was used as a replacement for SBH in preparing S-3.11. The pH of BB solutions was found to be easily adjustable with sodium borate and NaOH in a range between 8.5 and 10.5. A concentration of 3 μg/ml Dox before SP extraction was used to be incubated with pH 9.5 BB. A control sample with both BB and 60 μΜ SBH was also added. After the same incubation treatment as for S-3.10, both Dox and reference samples with D-ol were analysed by HPLC.

To study the effect of microwave (MW) and thermal heating systematically, samples with 5 μ^ηιΐ Dox were: heated either under MW irradiation (S-3.12) or in an oil bath (S-3.13). At a constant incubation time of 10 min, the pH of BB (9.5 and 10.0) and the temperature (90, 100 and 110°C) were varied. Reference samples with D-ol were also analysed.

Sample series S-3.14 which contained both Dox and D-ol samples were prepared similarly as S-3.13 but BB of pH 9.5 and 90 C was chosen at an incubation time of 1 h. The stock Dox solution was prepared by SP extraction of 25 μg/ml Dox methanolic solution. Samples containing Dox at 1, 3, 5, 15, and 25 μg/ml were then prepared from the stock solution by dilussion with MeOH. This set of experiments was performed twice. 4.3.2 Results and discussion

4.3.2.1 pH-induced degradation

The degradation under a Tris buffer was further affected when following the SP extraction protocol for samples preparation. As indicated above, this seems to be related to the water content in the solution. After the eluding step with MeOH, the solution obtained by SP extraction contained almost no water. This difference resulted in a much slower degradation when we incubated the samples at the same temperature as for the LL-extracted samples. Therefore, an elevated temperature (90°C) was employed to study the degradation using SP- extracted solutions. It is important to mention here that the pH scale in a mixture of MeOH and H20 was found to be deviated from the one in H20. After the verification with a pH meter that was calibrated in aqueous solution, negligible deviations were obtained in Tris buffer and BB used in the experiment. The results obtained from S-3.7 and S-3.8 are shown in Figures 20 and 21. Both, the disappearance of Dox and the emergence of Dox-end with time are clearly depicted by the gradual colour change of the pixels. When comparing the two environment of different pH, similar time-dependent degradation behaviour was obtained while both the Dox and Dox-ol degradation rate were higher in pH 9.6. Despite of long incubation time, this protocol has major advantages. First, Dox-end was the only dominant product of Dox degradation, which is important to simplify the quantification process at a later stage. On the other hand, D-ol was well preserved under such conditions, evidenced by the fact that more than 50% of D-ol left after 24 h incubation. The major products at 18.3 and 18.5 min were observed to have similar hydrophilic and optical properties to D-ol.

4.3.2.2 Degradation induced by boron compound

S-3.9 were prepared to study the kinetics of SBH-induced degradation. The results shown in Figure 22 were in line with the finding of SBH-induced degradation in S-3.6, suggesting SBH had similar effect on both LL-extracted and SP extracted samples. It is also evident that the degradation rate was higher with SBH than with Tris buffer. The long-term incubation data (24 and 168 h) suggests that the degradation was close to completion after 24h. A possible concentration dependence of SBH on the degradation activity would challenge its usage in practice, when unknown concentrations of Dox and D-ol are present. This concentration dependence is supported by the results from S-3.10 shown Figure 23. At identical concentration of Dox, degradation rate and ratio of products varied greatly from sample to sample and was dependent on the concentration of SBH. The situation was even worse where SBH was over- dosed in the 3 μg/ml-600 μΜ SBH sample, neither Dox nor degradation products were observed suggesting, D-end could be further modified in a SBH-rich solution. Dosage dependence of SBH will definitely pose a challenge in clinical practice where a concentration within at least one order of magnitude would need to be reliably determined. It is important to note that the SBH concentration also had an impact on the pH of the solution. 600 μΜ SBH provided a solution of pH 9.5 while the pH of solution with 6 and 60 μΜ SBH was no higher than 8.5. These results suggest that the prerequisite of SBH-induced degradation is a basic environment with pH around 9.5. BB serves as an ideal replacement for SBH and induces Dox degradation independent of its concentration due to the pH buffering. S-3.11 with 3 μg/ml Dox were degraded with pH 9.5 BB and the products were similar to the control samples in which 60 μΜ SBH was also added. As for the samples with D-ol, both BB and SBH induced more intensive D-ol degradation than induced by Tris buffer. After the incubation at 55 C for 24 h, the conversion of D-ol was >80%, mostly to products with similar polarity, evidenced by a retention time of 18.3 and 18.5 min. Besides, a certain amount of product D-ol-end (22.1 min) were produced while this slightly hydrophobic compound was negligible in the Tris case. S-3.12 and S-13 were analysed in an effort to speed up the degradation kinetics. The results suggest that BB-induced Dox degradation can be accelerated at elevated temperature, evidenced by the results of MW (Figure 24) and oil bath (Figure 25) heating at 90, 100 and 110°C for 10 min. When comparing MW and thermal heating, MW appeared more aggressive and less D-ol was maintained after the incubation. Two common trends regarding Dox degradation can be observed from these two systematic studies: both the production rate of D-end (26.3 min) and D-end2 (28.7 min) increased with incubation temperature and pH. As for D-ol degradation, both the conversion of D-ol (16.2 min) and the production of D-ol-end (22.1 min) were higher at higher temperatures and pH 10.0. After a simple phase-transfer test, D-ol-end was also found in the 5CB phase, suggesting that the production of D-ol-end may make the quantification based on the read-out from 5CB phase difficult because it has similar optical footprint to D-end. In the following, S-3.14 were prepared to check the linearity of the degradation process as well as the reproducibility. A protocol consisting of a 1 h incubation at 90°C in a BB at pH 9.5 was chosen for further investigation. Samples of 1, 3, 5, 15, and 25 μg ml SP-extracted Dox and D- ol are summarised in Figure 26 with the error bars obtained from two repeated experiment. The negligible difference from both experiment proved the high reproducibility of the BB-induced Dox degradation protocol. As for linearity, the adjusted R2 value of linear fitting for D-end, D- end and D-end2 and D-ol-end were 0.99135, 0.99997 and 0.94880 respectively, suggesting a good linearity in the range of 1 25 μg/ml. It is also evident that the absorbance of D-ol-end was much less intense than the one of Dox degradation products, evidenced by the contrast spun from <1 to >4. The problem of the degradation of Dox-ol could thus be resolved by data treatment even under harsh incubation conditions.

To summarise this section, different degradation protocols of Dox and Dox-ol have been systematically studied, mainly by HPLC. Both Tris buffer and BB were able to induce Dox degradation despite the different outcomes. The advantage of using Tris buffer was the selective degradation of Dox into D-end while leaving D-ol relatively unaffected. Considering the fact that only one degradation product was produced, the quantification procedure would be much more simplified. In contrast, BB-induced degradation provided the advantages of being an accelerated reaction. The time span of a whole detection could be greatly reduced to lh, which would be of great interests in real clinical practice.

Example 5: SPECTROMETRY

5.1 Detection by UV-Vis spectrometry Two samples with 5 μg/ml SP-extracted Dox were exposed to a pH 9.5 BB before incubation in an oil bath. The first sample was incubated at 90°C for 10 min and the second one was at 100°C for 30 min. Absorbance measurements were carried out before an equal volume of 5CB was added to each sample. The samples were then briefly heated for mixing before cooling to 4°C in a fridge for phase separation. The absorbance of the 5CB phase was then measured at 40°C. Two samples with SP-extracted D-ol were also processed with same treatment. Dox and D-ol samples (S-3.14) prepared as above disclosed at concentrations of 1, 3, 5, 15 and μg/ml were also used for the separation experiments. The absorption spectra of 0.5 ml of the methanolic phase and 5CB was recorded before and after the phase transfer. A volume of 0.5ml was used for both components.

Absorption spectra of phase-transfer samples recorded by conventional UV-Vis spectrometer are shown in Figure 27. It is evident that the absorbance of samples that underwent different incubation protocols varied from each other. It is important to note that the same degradation agent was used in incubation and only time and temperature of the incubation was different. As discussed in Section 3.3, Dox degradation at pH 9.5 and elevated temperature results in a mixture of Dox-related species that do depend on the actual temperature and incubation time. When comparing the absorbance in the MeOH phase before and in the 5CB phase after phase- transfer, the absorbance in 5CB phase was always lower in every sample, suggesting some species were retained in the MeOH phase. The fading of MeOH phase was also observed by eye after phase transfer. This supports the selectivity of phase-transfer from MeOH to 5CB. It is important to note that in these experiments a somewhat harsh protocol was used, which typically led efficient Dox degradation in less than one hour. This protocol also significantly affects D-ol and results in the, at least partial, generation of the more hydrophobic compounds denoted as D-ol-end. It is therefore not surprising that phase-transfer was also observed for D- ol (Figure 27(d)), although to a much lower extend. It is important to note that the partial phase- transfer of D-ol related compounds does not impede the use of this method for quantitative sensing. In the case where no complementary LC could be found that selectively extracted Dox over D-ol (or vice versa), more sensing channels (e.g. with different degradation agents or temperatures) and a comprehensive data treatment by mathematical algorithms taking advantage of the absorbance contrast would required to yield the necessary reliability.

The selectivity of phase-transfer was further confirmed by the results in the experiment using samples at various concentrations. After incubation in a pH 9.5 BB at 90°C for 10 min, the absorption information at peak wavelengths was extracted for comparison. For Dox samples in MeOH, the three peaks were at 535, 501 and 472 nm, respectively while the peaks were identified at 531 , 496 and 472 nm for D-ol samples in MeOH. A red-shift of absorption spectra was observed in all samples because the higher refractive index of 5CB than MeOH, which led to a shift of the Dox peaks shift to 547, 511 and 482 nm and D-ol peaks at 541, 506 and 482 nm. When comparing Figure 28 and Figure 29, it is evident that both the linearity and reproducibility improved after phase-transfer. The improved linearity (adjusted R2 >0.9995) not only supports our assumption that only specific degradation products, i.e. D-end, D-end2 and D-ol-end would phase transfer from MeOH to 5CB, but also suggests the independence of concentration of degradation process. This was previously not observed in the case of SBH and is an important prerequisite for sensing applications.

5.2 Detection by LWCC spectrometry

The absorption spectra of 1, 10, 100 ng/ml D-end in MeOH, 10%MeOH-90%5CB and 90%MeOH-10%5CB were measured. As above outlined, conventional UV-Vis spectroscopy in 1 cm cuvettes would not allow to reach clinically relevant detection limits. The use of a LWCC was identified as particularly promising due to its greatly enhanced optical path length as well as an improved read-out as a result of a more stable light source and a dedicated detector with a superior signal-to-noise ratio. The absorption spectra of 1 ng ml D-end in MeOH solutionis shown in Figure 30. The peak structures were well resolved with negligible noise, suggesting the detection limit of our sensing technique could go down to 1 ng/ml and below when using this technique. However, the measurement of ultra-low concentrated D-end in solution may be overshadowed by parasitic absorption, namely from 5CB as well as residues of the human plasma. As shown in Figure 31, the strong absorption signal from 5CB overshadowed the one from D-end, especially in the spectra range below 550 nm. This phenomenon was not apparent in previous experiments where the absorbance was dominated by the anthracyclines due to a much higher concentration. In order to resolve a D-end related footprint, the 5CB absorption baseline, shown in Figure 31(b), was subtracted, which resulted in regaining the peak structures, though somewhat less pronounced. In order to understand whether the loss in transmittance was due to 5CB scattering or absorbance, the data was fitted by an allometric model (according to Rayleigh scattering) and a Lorentzian model (band absorption), respectively. The good fit with the latter suggests that the LC absorption is very likely to be contributed by parasitic absorption rather than scattering. The possibility to detect Dox and D-ol by phase-transfer based colorimetric sensing was investigated by both conventional UV-Vis spectrometry and LWCC spectrometry. Conventional UV-Vis results showed a good linearity and reproducibility of the degradation process. In the investigated case where the compounds were incubated for less than one hour under harsh conditions, 5CB did not provide orthogonal solubility, i.e. an ideal contrast in solvation between the Dox and D-ol related compounds. Thus 5CB should be replaced by a more apolar LC to prevent D-ol related products from transferring. The preliminary experiments using LWCC indicate that the technique of the invention may be capable of working at clinically relevant concentration levels.

Claims

1. A method for selective separation, detection and quantification of a polycyclic organic compound in a sample comprising:

2. The method of claim 1, wherein the pH of the incubation phase is above pH 8.5, preferably between 9 and 10.

3. The method of claim 1 or 2, further comprising heating the incubation phase at temperature between 50°C and 110°, preferably between 80° and 90°.

4. The method of any one of claims 1 to 3, further comprising measuring the absorbance of the incubation phase before the step b) and/or measuring the absorbance of the incubation phase after the step c).

5. The method of any one of claims 1 to 4, further comprising measuring the absorbance of the read-out phase before the step b) and/or measuring the absorbance of the read-out phase after the step c).

6. The method of any one of claims 1 to 5, further comprising the step of mixing the incubation phase with the read-out phase after the step c).

7. The method of any one of claims 1 to 6, wherein the polycyclic organic compound is an anthracycline antibiotic.

8. The method of claim 7, wherein the anthracycline antibiotic is doxorubicin.

9. The method of any one of claims 1 to 8, wherein the degradation agent is selected from the group consisting of a reducing agent, an oxidising agent, a catalyst and pH buffer for pH- induced degradation.

10. The method of any one of claims 1 to 9, wherein the degradation agent is on the surface of the gold nanoparticles.

11. The method of any one of claims 1 to 10, wherein the measuring of the amount of the polycyclic organic compound in the read-out phase is carried out by optical means, such as through uv-vis or fluorescence spectroscopy.

12. The method of claim 1, wherein the polycyclic organic compound is doxorubicin and wherein said method comprises

a) providing a two-layer solution, wherein the first layer is methanol immiscible at temperatures below 20°C with the second layer which is a cyanobiphenyl liquid crystal and/or an immiscible organic solvent,

b) adding to the methanol phase Tris buffer or sodium borohydride,

c) adding a sample containing doxorubicin in the methanol phase under conditions sufficient to allow degradation of doxorubicin into Dox-end and to allow transfer of Dox-end into the cyanobiphenyl liquid crystal and/or immiscible organic solvent phase, d) detecting and quantifying doxorubicin in the sample by measuring the amount of transferred Dox-end in the read-out phase of a cyanobiphenyl liquid crystal and/or an immiscible organic solvent.

13. The method of claim 1 , wherein the polycyclic organic compound is doxorubicin and wherein said method comprises

c) adding a sample containing doxorubicin in the methanol phase under conditions sufficient to allow binding of doxorubicin to the ligands or the surface of the gold nanoparticles, wherein interaction of doxorubicin with the gold nanoparticles modifies the solubility of said gold nanoparticles and allows transfer of said gold nanoparticles into the cyanobiphenyl liquid crystal and/or immiscible organic solvent phase,

d) detecting and quantifying doxorubicin in the sample by measuring the amount of transferred doxorubicin bound to the gold nanoparticles in the read-out phase of cyanobiphenyl liquid crystals and/or immiscible organic solvent.

14. The method of claims 12 and 13, wherein the sample containing doxorubicin is a body fluid such as blood, saliva, urine or lacrimation.

15. The method of any one of claims 12 to 14, wherein the cyanobiphenyl liquid crystal is selected from the group comprising 4-cyano-4'-pentylbiphenyl (5CB), 4-Cyano-4'-n- heptylbiphenyl (7CB), 4-Cyano-4'-n-octylbiphenyl (8CB) and wherein the immiscible organic solvent is heptane.

16. A method for selective extraction of a polycyclic organic compound from a sample comprising:

a) providing a two-layer solution, wherein the first layer is an incubation phase immiscible with the second layer which is a read-out phase, b) adding in the incubation phase gold nanoparticles coated with ligands specific to the polycyclic organic compound to be extracted, wherein said gold nanoparticles are soluble in the incubation phase and insoluble in the read-out phase,

e) recovering the polycyclic organic compound from the read-out phase.

17. The method of claim 16, wherein the sample is a body fluid such as blood, saliva, urine or lacrimation.

18. The method of claim 1, wherein the sample is obtained by the method of claim 16.

A kit for detecting and quantifying doxorubicin in a sample, comprising

a) methanol,

b) a cyanobiphenyl liquid crystal and/or an immiscible organic solvent,

20. The kit of claim 19, wherein the cyanobiphenyl liquid crystal is selected from the group comprising 4-cyano-4'-pentylbiphenyl (5CB), 4-Cyano-4'-n-heptylbiphenyl (7CB), 4-Cyano- 4'-n-octylbiphenyl (8CB); wherein the immiscible organic solvent is heptane and wherein the buffer agent is selected from the group consisting of Tris buffer (2-Amino-2-hydroxymethyl- propane-l,3-diol).