Measure of a quinolinate and tryptophan concentration

Abstract

The present invention relates to a disposable sensor (1) for measuring at least one of the quinolinate concentration and the tryptophan concentration in real time in human urine: - a molecularly imprinted polymer, MIP, support (11) comprising a polymer matrix (110), at least one of a first set of cavities (112) adapted to measure the quinolinate concentration and a second set of cavities (113) adapted to measure the tryptophan concentration within the polymer matrix (110); and - an optical fibre (12) coated with the MIP support (11). It also relates to a system (3) comprising same, a method for making same, and a method for measuring at least one of the quinolinate concentration and the tryptophan concentration using same.

Description

[0001] Measure of a quinolinate and tryptophan concentration

[0002] Technical Field

[0003] [1] This disclosure pertains to the field of measurement of quinolinate and tryptophan concentration. Such measurement may be applied, but is not limited, to the diagnostic and treatment of acute kidney injury.

[0004] Background Art

[0005] [2] Acute kidney injury (AKI) is a frequent and potentially lethal disease. It can evolve into a chronic kidney disease. AKI represents more than 10 % of all hospitalisations and the figure can rise up to 50% in intensive care units. Clinical and biological diagnostics are reached only hours or days after AKI. Established AKI generates additional insults that can aggravate it, such as heart or respiratory failure, dialysis and drug accumulation, with a risk of evolution towards chronic kidney disease. In addition to mortality and long-term morbidity, AKI is an enormous Public Health problem. It causes tremendous additional cost and strain on available Health resources. The current monitoring of AKI relies on quantification of haemodynamic parameters such as serum creatinine, which rises only 24 to 48 hours after kidney injury. Urinary quinolinate / tryptophan ratio has recently emerged as a non- invasive biomarker for AKI. However, the reference technique used for quinolinate and tryptophan measurements is liquid chromatography coupled with tandem mass spectrometry (LC / MS-MS), which is, nowadays, the gold standard.

[0006] [3] However, such a technique has the disadvantage of being carried out on an ad hoc basis, at given times, in the laboratory. It is an expensive technique that only allows a one-off assessment. It is not possible to carry out such measurements at the patient's bedside or a continuous monitoring.

[0007] Summary

[0008] [4] This disclosure improves the situation.

[0009] [5] A disposable sensor of at least one of a quinolinate concentration and a tryptophan concentration in real time in human urine is provided, which comprises:

[0010] - a molecularly imprinted polymer, MIP, support comprising a polymer matrix and at least one of a first set of cavities adapted to measure the quinolinate concentration and a second set of cavities adapted to measure the tryptophan concentration within the polymer matrix,

[0011] - an optical fibre coated with the MIP support.

[0012] [6] By combining the MIP support with an optical fibre, the concentration of quinolinate and / or tryptophan can be continuously measured and monitored. By allowing measurements of quinolinate or tryptophan concentrations, this disposable sensor provides rapid measurements of a marker of renal impairment in urine. This disposable sensor enables rapid off-site measurements, at hospitals, close to the patient during intensive care conditions rather than in the laboratory. Furthermore, this disposable sensor offers low production and operating costs. [7] This type of disposable sensors allows to better diagnose kidney disease. This disposable sensor may be used to predict acute renal failure, diagnose preclinical acute renal failure, and assess the renal toxicity of treatments. By measuring quinolinate and tryptophan concentrations continuously and in real time, it is possible to tailor nephroprotective treatment to the patient's needs during intensive care. This makes it easier to assess the effectiveness of nephroprotective treatments.

[0013] [8] This disposable real-time sensor allows to enslave treatment delivery in an automatic feedback loop, modulating doses of treatments (including, but not limited to metabolic treatments like nicotinamide and vasoactive agents like epinephrin or norepinephrin) in a goal-directed strategy.

[0014] [9] Further optional features of the disposable sensor are as follows:

[0015]

[0010] The MIP support may comprise MIP nanoparticles or a MIP film, or a combination of both.

[0016]

[0011] The MIP support may further comprise a fluorophore within the polymer matrix.

[0017]

[0012] The fluorophore may be chosen from the group consisting of N-allyl-4-(4'-methyl-piperazinyl)- 1 ,8-naphthalimide, N-allyl-4-(N,N'-(methyl) ethylene diamine)-1 ,8-naphthalimide, trans-4-[p-(N,N- dimethylamino)styryl]-N-vinyl benzyl pyridinium chloride and their mixtures.

[0018]

[0013] The optical fibre may be an evanescent-wave optical fibre.

[0019]

[0014] The disposable sensor may further comprise a lens at a proximal end of the optical fibre and a mirror at a distal end of the optical fibre opposite de proximal end.

[0020]

[0015] A measurement box is also provided and comprises:

[0021] - the disposable sensor described above and having an optical axis;

[0022] - a casing defining an inner chamber;

[0023] - a sample receiving hub within the inner chamber adapted to receive a sample holder; wherein the casing comprises a fibre probe connector adapted to receive a fibre probe with an optical axis; the fibre probe connector and the sample receiving hub are arranged so that, when the sample holder with the disposable sensor is received in the sample receiving hub, the optical axis of the optical fibre is aligned with the optical axis of the fibre probe.

[0024]

[0016] A system is also provided and comprises the disposable sensor as described above and a spectrometer coupled to the disposable sensor; the spectrometer being configured to emit an excitation wavelength corresponding to the polymer matrix of the disposable sensor and measure at least one of a first fluorescence proportional to the quinolinate concentration and a second fluorescence proportional to the tryptophan concentration from a secondary signal received from the disposable sensor.

[0025]

[0017] The spectrometer may be a spectrofluorometer.

[0026]

[0018] The system may further comprise the above measurement box and the spectrometer is coupled to the disposable sensor through the measurement box.

[0019] A method of making a sensor as described above is also provided and comprises:

[0027] - forming an MIP support on an optical fibre, from at least one template and matrix precursors, wherein the template is a quinolinate, a tryptophan template or both.

[0028]

[0020] Further optional features are the following:

[0029]

[0021] Forming the MIP support may further be from at least one fluorophore.

[0030]

[0022] The fluorophore may be chosen from the group consisting of N-allyl-4-(4'-methyl-piperazinyl)- 1 ,8-naphthalimide, N-allyl-4-(N,N'-(methyl) ethylene diamine)-1 ,8-naphthalimide, trans-4-[p-(N,N- dimethylamino)styryl]-N-vinyl benzyl pyridinium chloride and their mixture.

[0031]

[0023] The matrix precursors may be at least one monomer and a polymerization initiator.

[0032]

[0024] The monomer may be chosen from the group consisting of 4-vinylpyridine, ethylene glycol diacrylate, trimethylolpropane triacrylate, pentaerythritol triacrylate, polyethylene glycol acrylate, polyethylene glycol diacrylate, hydroxyethyl acrylate, ethylene glycol dimethacrylate, trimethylolpropane trimethacrylate, pentaerythritol trimethacrylate, polyethylene glycol methacrylate, polyethylene glycol dimethacrylate, hydroxyethyl methacrylate, acrylamide, methacrylamide, methylene bisacrylamide, ethylene bisacrylamide, piperazine bisacrylamide, N-isopropylacrylamide, N-tert-butylacrylamide, N-propylacrylamide, phenylacrylamide, 4-acrylamidobenzamidine, aminopropylacrylamide, diethylaminopropylacrylamide, and their mixtures.

[0033]

[0025] The polymerization initiator may be adapted for thermal polymerization, redox polymerization, photopolymerization, free radical polymerization, or controlled / living radical polymerization, such as, 2,2’-azobis(2,4-dimethyl)valeronitrile), 2,2’-azobis(2-methylpropionitrile), 2,2-dimethoxy-2- phenylacetophenone, benzyl N,N-diethyldithiocarbamate, phenylbis(2,4,6- trimethylbenzoyl)phosphine oxide, and their mixtures.

[0034]

[0026] Forming the MIP support may comprise:

[0035] - polymerizing the matrix precursors into a polymer matrix around the template; and

[0036] - removing the template from the polymer matrix, thereby leaving cavities behind.

[0037]

[0027] When fluorescence generating component is used, the polymer matrix encapsulates the fluorescence generating component upon polymerization.

[0038]

[0028] Polymerizing may be carried out in the absence of the optical fibre and carried out by precipitation polymerization, high-dilution polymerization, or controlled polymerization, to form the MIP support in the form of MIP nanoparticles, after removing the template, forming the MIP support further may thus further comprise coating the MIP nanoparticles onto the optical fibre.

[0039]

[0029] Alternatively, forming the MIP support may comprise polymerizing the MIP support in the form of an MIP film in situ on the optical fibre.

[0040]

[0030] A method of measuring at least one of a quinolinate concentration and a tryptophan concentration in real time is also provided and comprises:

[0041] - contacting the disposable sensor as described above with human urine, - injecting light inside the optical fibre of the disposable sensor,

[0042] - receiving fluorescence emission, FE, radiation back from the disposable sensor,

[0043] - determining the at least one of the quinolinate concentration and the tryptophan concentration based on the received FE radiation.

[0044]

[0031] This real time method of measurement of a quinolinate and tryptophan concentration is key for improving the AKI outcomes with personalized treatments. Ultimately, this method enables the personalised management of acute renal failure in intensive care units according to anatomical lesions and early response to standardised treatments.

[0045]

[0032] Further optionally features are as follows:

[0046]

[0033] The quinolinate concentration and the tryptophan concentration may be both measured, and the method may further comprise calculating a quinolinate / tryptophan ratio based on the determined quinolinate and tryptophan ratios.

[0047]

[0034] The method may further comprise indicating whether the quinolinate / tryptophan ratio is above a threshold.

[0048] Brief Description of Drawings

[0049]

[0035] Other features, details and advantages will be shown in the following detailed description and on the figures, on which:

[0050]

[0036] Fig. 1 is a schematic illustration of an exemplary disposable sensor according to the present invention.

[0051]

[0037] Fig. 2A to 2D are schematic illustrations of MIP supports in particulate form usable in the disposable sensor of Fig. 1 .

[0052]

[0038] Fig. 3A to 3C are schematic illustrations of MIP supports in film form usable in the disposable sensor of Fig. 1 .

[0053]

[0039] Fig. 4A is a schematic illustration of an exemplary disposable sensor according to the present invention.

[0054]

[0040] Fig. 4B is a schematic illustration of another exemplary disposable sensor according to the present invention.

[0055]

[0041] Fig. 5 is a graph representing a fluorescence intensity in count per second (CPS) as a function of emission wavelength (from 420 to 650 nm) for MIP nanoparticles with cavities corresponding to quinolinate, coated on an optical fibre and in the presence of various concentrations of quinolinate from 0 to 100 pM. The excitation wavelength was 410 nm and the detection was carried out with a 3 nm slit width.

[0056]

[0042] Fig. 6 is a graph representing a fluorescence intensity in CPS as a function of emission wavelength (from 420 to 650 nm) for MIP nanoparticles with cavities corresponding to L-tryptophan, coated on an optical fibre and in the presence of various concentrations of tryptophan from 0 to 100 pM. The excitation wavelength was 410 nm and the detection was carried out with a 3 nm slit width.

[0057]

[0043] Fig. 7 is a graph representing a fluorescence intensity in CPS as a function of emission wavelength (from 410 to 650 nm) for MIP nanoparticles with cavities corresponding to quinolinate in suspension with a concentration of MIP nanoparticles of 50 pg / mL and in presence of various concentration of quinolinate from 0 to 100 pM. The excitation wavelength was 400 nm and the detection was carried out with a 3 nm slit width.

[0058]

[0044] Fig. 8 is a graph representing a fluorescence intensity in CPS as a function of emission wavelength (from 410 to 650 nm) for MIP nanoparticles with cavities corresponding to L-tryptophan in suspensions with a concentration of MIP nanoparticles of 50 pg / mL and in presence of various concentrations of tryptophan from 0 to 100 pM. The excitation wavelength was 400 nm and the detection was carried out with a 3 nm slit width.

[0059]

[0045] Fig. 9 schematically illustrates a system comprising the disposable detector of Fig. 4A, the system notably comprising a spectrometer coupled to the disposable sensor.

[0060]

[0046] Fig. 10 schematically illustrates the interaction between an MIP support (in the form of an MIP film) having both type of cavities and quinolinate and tryptophan.

[0061]

[0047] Fig. 1 1 schematically illustrates an exemplary measuring box for use with the disposable sensor of Fig. 4A within the system of Fig. 9.

[0062]

[0048] Fig. 12 schematically illustrates an exemplary method for making the disposable sensor according to the present invention.

[0063]

[0049] Fig. 13 to Fig. 16 schematically illustrate steps of an exemplary embodiment of the method of Fig. 12, wherein the MIP support is in particulate form.

[0064]

[0050] Fig. 17 schematically illustrates steps of another exemplary embodiment of the method of Fig. 12, wherein the MIP support is in film form.

[0065]

[0051] Fig. 18 and Fig. 19 schematically illustrate step of a method for measuring a quinolinate concentration and / or tryptophan concentration.

[0066]

[0052] Fig. 20 (A) Images of the optical fiber waveguide before and during polymerization (XEX = 470 nm, 10 nm slit width) and before and after washing (XEX = 400 nm, 1 nm slit width), in comparison to a bare fiber. These images are for illustrative purposes only; fluorescence measurements are strictly done inside a dedicated measurement box. (B) Image of the photopolymerization and sensing setup using a bifurcated optical probe from the spectrofluorometer inserted into a dedicated measurement box wherein the 4 mL glass vial housing the fiber is located. (C) Image of the QA MIP- coated optical fiber by in situ photopolymerization, in comparison to a bare fiber.

[0067]

[0053] Fig. 21 Fluorescence spectra (AEX / EM = 400 / 490 nm) of QA MIP-coated optical fiber after incubation with increasing concentrations of QA-spiked 1 :9 urine:H2O (left); Corresponding fluorescence enhancement responses of QA MIP (dots) with BLT control MIP (crosses)-coated optical fibers (n = 3, mean ± s.e.m.) (right). Inset: Linear dynamic range of QA MIP.

[0068]

[0054] Fig. 22 Fluorescence spectra (AEX / EM = 400 / 490 nm) of BLT MIP-coated optical fiber after incubation with increasing concentrations of L-Trp-spiked 1 :9 urine:H2O (left); Corresponding fluorescence enhancement responses of BLT MIP (crosses) with QA control MIP (dots)-coated optical fibers (n = 3, mean ± s.e.m.).

[0069]

[0055] Fig. 23 Recognition of QA and BLT MIP-coated optical fibers towards target and competing urinary molecules, spiked in 1 :9 urine:H2O.

[0070]

[0056] Fig. 24 Images of scanning electron microscopy (SEM) analysis of MIP-QA on the waveguides

[0071] Description of Embodiments

[0072] Disposable sensor 1

[0073]

[0057] A disposable sensor according to the present invention will be described hereafter with reference to Fig. 1 to 4.

[0074]

[0058] Such sensor 1 is adapted for measuring at least one of the quinolinate concentration and the tryptophan concentration in real time in human urine.

[0075]

[0059] The sensor 1 comprises:

[0076] - a molecularly imprinted polymer (MIP) support 11 comprising a polymer matrix 110 and at least one of a first set of cavities 112 adapted to measure the quinolinate concentration and a second set of cavities 113 adapted to measure the tryptophan concentration also within the polymer matrix 110; and

[0077] - an optical fibre 12 coated with the MIP support 11.

[0078]

[0060] Quinolinate and tryptophan are markers of acute renal failure in human urine. More in particular, the ratio of quinolinate to tryptophan in human urine may be advantageously used to diagnose such failure.

[0079]

[0061] MIPs are tailor-made synthetic materials possessing specific cavities designed for a target molecule, in the present case: quinolinate and tryptophan. They are typically synthesized by copolymerization of functional and cross-linking monomers in the presence of a molecular template, in a solvent. The template can be the target molecule (quinolinate or tryptophan) or a derivative thereof. The most common approach to prepare MIPs is by the self-assembly method, due to the large variety of commercially available acrylic, methacrylic, vinylic and styrenic functional and cross-linking monomers. The functional monomers initially form a complex with the template, followed by the polymerization step. Thus, after polymerization, the monomer-template assembly is held in position by the highly cross-linked three-dimensional structure. Subsequent removal of the template leaves cavities with a size, shape and chemical functionality complementary to those of the template. The resulting binding sites can bind the target molecule with a very high specificity and affinity, comparable to that of biological receptors such as antibodies and enzymes.

[0062] The MIP support 11 may be MIP nanoparticles 11P or a MIP film 11 F, or even a combination of both. The MIP film 11 F is typically a continuous film.

[0080]

[0063] The MIP nanoparticles 11P may have an equivalent diameter of 20 to 400 nm, preferably 40 to 200 nm, for example 100 nm In the context of the present disclosure, “equivalent diameter” refers to the diameter of a sphere of same volume as the MIP nanoparticles 11P.

[0081]

[0064] Fig. 2A and 2B illustrate the case of MIP nanoparticles 11P having only one of the first and second sets of cavities 112, 113.

[0082]

[0065] Fig. 3A and 3B illustrate the case of MIP films 11 F having only one of the first and second sets of cavities 112, 113.

[0083]

[0066] When it is said that the MIP support 11 comprises a first set of cavities 112 and a second set of cavities 113, this does not necessarily mean that both types of cavities 112, 113 are provided on the same nanoparticle 11 P or film 11 F, but the following combinations may be contemplated:

[0084] - each MIP nanoparticle 11P comprises only one type of cavities 112 or 113 (hereafter “single-type nanoparticle”, 1 C);

[0085] - each MIP nanoparticle 11P comprises both type of cavities 112 and 113 (hereafter “double-type nanoparticles”, see Fig. 1 D);

[0086] - a mixture of single-type nanoparticles and double-type nanoparticles;

[0087] - each MIP film 11 F comprises only one type of cavities 112 or 113 (hereafter “single-type film”);

[0088] - each MIP film 11 F comprises both types of cavities 112 and 113 (hereafter “double-type film”, see Fig. 2C);

[0089] - a mixture of one or more single-type films and one or more double-type films;

[0090] - a combination of the above.

[0091]

[0067] For example, if the sensor 1 only measures the quinolinate concentration, it only has the first set of cavities 112. If the sensor 1 only measures the tryptophan concentration, it only has the second set of cavities 113. If the sensor 1 measures both the quinolinate concentration and tryptophan concentration, it has both sets of cavities 112 and 113.

[0092]

[0068] Thus, it is possible to use a combination of a first sensor 1 with the first set of cavities 112 and a second sensor 1 with the second set of cavities 113. However, it is advantageous to have both measurements made available in the same sensor 1. Therefore, in such case, the disposable sensor

[0093] I has both sets of cavities 112 and 113. In such case, the disposable sensor 1 may have two optical fibres 12, each with an MIP support 11 having one or the other set of cavities 112. The MIP supports

[0094] II of this type of disposable sensor 1 may be made of the same material or different materials. Alternatively or in addition, they may be of the same shape or different shapes.

[0095]

[0069] The MIP support 11 may coat the circumference surface of the optical fibre 12 (Fig. 4A) or a distal end 122 thereof when this latter is sheathed as further described below (Fig. 4B).

[0096]

[0070] The MIP support 11 on the optical fibre 12 may have a thickness of 2 to 400 nm, preferably 2 to 100 nm, for example 50 nm. The thickness may not be homogeneous over the whole of the optical fibre 12. In such case, the mentioned ranges correspond to average thickness, with a standard deviation of 10%. In general, the thinner, the better. Thus, when the MIP support 11 is in the form of MIP nanoparticles I I P, the thickness thereof preferably corresponds to once, twice or thrice the diameter of the MIP nanoparticles 11 P.

[0097]

[0071] The MIP support 11 may further comprise a fluorescence generating component such as a fluorophore 111.

[0098]

[0072] Depending on the nature of the fluorophore 111 , it can polymerize with the matrix precursors or be physically entrapped inside the polymer matrix 110 during polymerization thereof.

[0099]

[0073] The fluorophore 111 may be chosen from the group consisting of N-allyl-4-(4'-methyl- piperazinyl)-1 ,8-naphthalimide, N-allyl-4-(N,N'-(methyl) ethylene diamine)-1 ,8-naphthalimide, trans- 4-[p-(N,N-dimethylamino)styryl]-N-vinyl benzyl pyridinium chloride and their mixtures. These fluorophores have the advantage of fluorescence enhancement upon analyte binding, which is more specific than fluorescence quenching.

[0100]

[0074] However, other organic or inorganic fluorophores may be used, working either with fluorescence enhancement, with fluorescence quenching, with fluorescence wavelength shift, or with a change in fluorescence lifetime. For example, fluorescent nanoparticles may be used such as quantum dots, carbon dots, fluorescence upconverting nanoparticles. The advantage of upconverting nanoparticles is that a higher wavelength can be used for excitation, thereby not triggering the autofluorescence of the biological samples (urine).

[0101]

[0075] The disposable sensor 1 may comprise two different fluorophores, notably when it comprises cavities 112, 113 for both quinolinate and tryptophan. These two different fluorophores preferably have different emission wavelengths.

[0102]

[0076] Alternatively to the fluorophore 111 , the MIP support 11 may comprise optical cavities inside the polymer matrix 110 (such as Fabry-Perot cavities), photonic crystals or holograms as fluorescent generating component.

[0103]

[0077] The polymer matrix 110 may be obtained through polymerizing matrix precursors. The matrix precursors comprise a monomer and a polymerization initiator.

[0104]

[0078] The monomer may be chosen from the group consisting of 4-vinylpyridine, ethylene glycol diacrylate, trimethylolpropane triacrylate, pentaerythritol triacrylate, polyethylene glycol acrylate, polyethylene glycol diacrylate, hydroxyethyl acrylate, ethylene glycol dimethacrylate, trimethylolpropane trimethacrylate, pentaerythritol trimethacrylate, polyethylene glycol methacrylate, polyethylene glycol dimethacrylate, hydroxyethyl methacrylate, acrylamide, methacrylamide, methylene bisacrylamide, ethylene bisacrylamide, piperazine bisacrylamide, N-isopropylacrylamide, N-tert-butylacrylamide, N-propylacrylamide, phenylacrylamide, 4-acrylamidobenzamidine, aminopropylacrylamide, diethylaminopropylacrylamide, and their mixtures.

[0105]

[0079] The polymerization initiator may be adapted for thermal polymerization, redox polymerization, photopolymerization, free radical polymerization, or controlled / living radical polymerization, such as, 2,2’-azobis(2,4-dimethyl)valeronitrile), 2,2’-azobis(2-methylpropionitrile), 2,2-dimethoxy-2- phenylacetophenone, benzyl N,N-diethyldithiocarbamate, phenylbis(2,4,6- trimethylbenzoyl)phosphine oxide, and their mixtures.

[0106]

[0080] The optical fibre 12 may be made of glass, quartz, or a transparent polymer such as polystyrene and polymethylmethacrylate.

[0107]

[0081] The optical fibre 12 may have a core diameter comprised between 8 pm and 1 mm.

[0108]

[0082] The optical fibre 12 may be sheathed. Thus, the disposable sensor 1 may further comprise a sheath surrounding the circumference of the optical fibre 12.

[0109]

[0083] The disposable sensor 1 may have two optical fibres 12, notably one with an MIP support 11 with cavities 112 for the detection of quinolinate and another one with an MIP support 11 with cavities for the detection of tryptophan. In such case, both MIP supports 11 may be made of the same material or different materials. Alternatively or in addition, both MIP supports 11 may be of the same shape or different shapes.

[0110]

[0084] Fig. 5 and Fig. 6 are experimental graphic results representing fluorescence intensity in count per second (CPS) as a function of emission wavelength in nm for MIP nanoparticles 11 P coated on an optical fibre 12 and in the presence of various concentrations of quinolinate. In Fig. 5, the MIP nanoparticles 11 P have the first set of cavities 112 corresponding to quinolinate. In Fig. 6 the MIP nanoparticles 11 P have the second set of cavities 113 corresponding to tryptophan. Experimentally, MIP nanoparticles 11 P fluoresce increasingly in the presence of growing concentrations of their respective ligands: quinolinate 13 in Fig. 5, and tryptophan 14 in Fig. 6. The peak fluorescence intensity was found at 494 nm for quinolinate and 493 nm for tryptophan. These experimental tests validate the effectiveness of such disposable sensors 1 in vitro. The concentration of quinolinate can be determined through the fluorescence emission at 494 nm and that of tryptophan through the fluorescence emission at 493 nm.

[0111]

[0085] Fig. 7 and Fig. 8 illustrate the case where the MIP nanoparticles 11 P are in suspension. The ordinate and abscissa of these Fig. 7 and 8 are the same as those of Fig. 5 and 6. In Fig. 7, the MIP nanoparticles 11 P have the first set of cavities 112 corresponding to quinolinate. In Fig. 7 the MIP nanoparticles 11 P have the second set of cavities 113 corresponding to tryptophan. Similarly, MIP nanoparticles 11 P fluoresce increasingly in the presence of growing concentrations of their respective ligands: quinolinate 13 in Fig. 7, and tryptophan 14 in Fig. 8. The peak fluorescence intensity was found at 510 nm for both quinolinate and tryptophan.

[0112]

[0086] As illustrated in Fig. 4A and Fig. 4B, the disposable sensor 1 may further comprise a lens 13 at a proximal end 121 of the optical fibre 12 and a reflector 14 at a distal end 122 of the optical fibre 12 opposite the proximal end 121 thereof. The optical fibre 12 defines an overall optical axis 12A along which light travels although not in a straight manner within the optical fibre 12. This optical axis 12A is aligned with the optical axis of the lens 13.

[0113]

[0087] The lens 13 helps to inject a laser light into the optical fibre 12. The lens 13 may be made of glass, quartz, or a transparent polymer such as polystyrene and polymethylmethacrylate.

[0088] The reflector 14 may be a surface such as a planar mirror, or a metal layer.

[0114]

[0089] As illustrated in Fig. 9, the disposable sensor 1 as described above may be used in a system 3 further comprising a spectrometer 31 (such as a spectrofluorometer) and a fibre probe 32. The fibre probe 32 is connectable to the spectrometer 31 and comprises a bifurcated optical fibre 321. The spectrometer 31 typically comprises a light source 311 and a detector 312. The light source 311 is configured to emit an excitation wavelength corresponding to excitation wavelength of the fluorophore 111 in the disposable sensor 1 , and to measure at least one of a first fluorescence proportional to the quinolinate concentration and a second fluorescence proportional to the tryptophan concentration emitted by the MIP support 11.

[0115]

[0090] The excitation wavelength depends on the fluorophore used in the disposable sensor 1 and the state of the MIP support 11. The person skilled in the art knows how to adapt the excitation wavelength to the fluorophore.

[0116]

[0091] Likewise, the detection wavelength depends on the nature of the ligands, in the present case, quinolinate and tryptophan; and the state of the MIP support 11.

[0117]

[0092] The detector 312 may be a photomultiplier tube (PMT) or a diode array detector. The lens 13 enables a first signal S1 from light source 311 to converge into the optical fibre 12 and a second signal S2 to reach the fibre probe 32 and eventually the detector 312.

[0118]

[0093] In operation, excitation light 41 (e.g. 410 nm) is emitted by the light source 311 and guided up to the optical fibre 12 through the fibre probe 32 and the lens 13. This excitation light 41 undergoes total internal reflection inside the optical fibre 12 and generates an evanescent field that is perpendicular to the fibre surface and excites the fluorophore 111 inside the MIP support 11. Consequently, a secondary signal 25 is emitted from the MIP support 11 into the optical fibre 12 and travels back up to the detector 312. The MIP support 11 is adapted to fluoresce in presence of quinolinate or tryptophan thanks to the cavities 112, 113 thereof receiving specifically quinolinate or tryptophan. When the fluorophore 111 is excited, the MIP support 11 emits a first emission wavelength (e.g. centred at 493 nm) indicating the presence of tryptophan and the amplitude thereof being indicative of the tryptophan concentration, or a second emission wavelength (e.g. centred at 494 nm) indicating the presence of quinolinate and the amplitude thereof being indicative of the quinolinate concentration.

[0119]

[0094] As illustrated in Fig. 11 , the invention also provides a measurement box 2 for use in combination with the disposable sensor 1 described above, for example within the system 3 illustrated in Fig. 9. In one embodiment, such measurement box 2 comprises a casing 21 defining an inner chamber 22. Within the inner chamber 22, a sample receiving hub 23 is provided to house a sample holder 24 coupled with the disposable sensor 1. The casing 21 comprises a fibre probe connector 211 adapted to receive a fibre probe. The fibre probe connector 211 and the sample receiving hub 23 are arranged so that, when the sample holder 24 with the disposable sensor 1 is received in the sample receiving hub 23, the optical axis 12A of the optical fibre 12 is aligned with the optical axis of the fibre probe. Method for making the disposable sensor 1

[0120]

[0095] The present invention also provides a method for making a sensor 1 as described above. The method comprises forming an MIP support 11 on an optical fibre 12, from at least one template and matrix precursors. The template is a quinolinate template or a tryptophan template.

[0121]

[0096] Quinolinate template and tryptophan template refer to a shape around which polymerization of the monomers into the polymer matrix occurs and thus forming cavities inside the polymer matrix once the template is washed off. The shape of the cavities left by the quinolinate template molecule is specific to quinolinate and enables a molecule of quinolinate to be received therein. Similarly, the shape of the cavities left by the tryptophan template molecule is specific to tryptophan and enables a molecule of tryptophan to be received therein.

[0122]

[0097] A fluorescence generating component such as a fluorophore may also be used to form the MIP support.

[0123]

[0098] The fluorophore may be chosen from the group consisting of N-allyl-4-(4'-methyl-piperazinyl)- 1 ,8-naphthalimide, N-allyl-4-(N,N'-(methyl) ethylene diamine)-1 ,8-naphthalimide, trans-4-[p-(N,N- dimethylamino)styryl]-N-vinyl benzyl pyridinium chloride and their mixtures.

[0124]

[0099] Alternatively, other fluorescence generating component may be used as already mentioned above.

[0125]

[0100] Matrix precursors are typically monomers and a polymerization initiator.

[0126]

[0101] The monomer may be chosen from the group consisting of 4-vinylpyridine, ethylene glycol diacrylate, trimethylolpropane triacrylate, pentaerythritol triacrylate, polyethylene glycol acrylate, polyethylene glycol diacrylate, hydroxyethyl acrylate, ethylene glycol dimethacrylate, trimethylolpropane trimethacrylate, pentaerythritol trimethacrylate, polyethylene glycol methacrylate, polyethylene glycol dimethacrylate, hydroxyethyl methacrylate, acrylamide, methacrylamide, methylene bisacrylamide, ethylene bisacrylamide, piperazine bisacrylamide, N-isopropylacrylamide, N-tert-butylacrylamide, N-propylacrylamide, phenylacrylamide, 4-acrylamidobenzamidine, aminopropylacrylamide, diethylaminopropylacrylamide, and their mixtures.

[0127]

[0102] The polymerization initiator may be adapted for thermal polymerization, redox polymerization, photopolymerization, free radical polymerization, or controlled / living radical polymerization, such as, 2,2’-azobis(2,4-dimethyl)valeronitrile), 2,2’-azobis(2-methylpropionitrile), 2,2-dimethoxy-2- phenylacetophenone, benzyl N,N-diethyldithiocarbamate, phenylbis(2,4,6- trimethylbenzoyl)phosphine oxide, and their mixtures.

[0128]

[0103] Forming an MIP support 11 may comprise:

[0129] - polymerizing S110 the matrix precursors into a polymer matrix 110 around the template; and

[0130] - removing S120 the template from the polymer matrix 110, thereby leaving cavities 112, 113 behind.

[0131] When fluorescence generating component is used, the polymer matrix encapsulates the fluorescence generating component upon polymerization. MIP nanoparticles 11P

[0132]

[0104] Fig. 13 to 16 illustrate one embodiment of forming a MIP support 11. In this embodiment, the MIP support 11 is in the form of MIP nanoparticles-and polymerizing is performed in the absence of the optical fibre 12 and leads to formation of the MIP nanoparticles 11 P. Polymerizing is typically carried out by precipitation polymerization, high-dilution polymerization, or controlled polymerization. Also, after removing S120 the template, forming the MIP support 11 further comprises coating S130 the MIP nanoparticles 112 onto the optical fibre 12.

[0133]

[0105] Polymerizing may be performed at a temperature comprised between 40 and 50 °C, for example in a water bath.

[0134]

[0106] Before polymerizing S120 the matrix precursors, forming the MIP nanoparticles 11 P may comprise mixing S105 the matrix precursors, the fluorophore 111 and the template.

[0135]

[0107] Mixing may be performed in dimethyl siloxane (DMSO). The MIP nanoparticles 11 P and the cavities are better formed with DMSO. There is a stronger interaction between quinolinate or tryptophan and the fluorophore.

[0136]

[0108] In some casing, notably when the matrix precursors are susceptible to interaction with oxygen, forming the MIP nanoparticles 11 P may further comprise, after mixing S105, depleting S106 the reacting medium of oxygen. Depleting S106 may comprise sonicating S1061 the mixture obtained after mixing S105 and / or purging S1062. Sonicating S1061 may be performed at room temperature, i.e. between 20 and 25 °C. Sonicating S1061 may be carried out during 1 to 10 min, preferably 2.5 to 7.5 min, for example 5 min. Purging S1062 may be carried out using an inert gas, such as nitrogen, preferably on ice. Purging S1062 may be performing during 1 to 5 min, preferably 1 .5 to 2.5 min for example 2 min.

[0137]

[0109] Removing S120 the template may comprise:

[0138] - washing S121 away the template from the MIP nanoparticles 11 P leaving cavities 112, 113 in the MIP nanoparticles 11 P; and

[0139] - drying S122 the MIP nanoparticles I I P, notably under vacuum.

[0140]

[0110] Washing S121 may comprise one or more rounds of washing with one or more solvents. The following solvents may be used: methanol, acetic acid, ammonia, water and their mixture such as methanol / acetic acid (for example 9 / 1 ) and methanol / ammonia (for example 100 mM ammonia). The methanol / acetic acid ratio may preferably be 7 / 1 to 1 1 / 1 , preferably 8 / 1 to 10 / 1 , for example 9 / 1 . The methanol / ammonia washing mixture preferably comprises 90 to 1 10 mM ammonia, still preferably 95 to 105 mM, for example 100 mM. In one embodiment, washing may comprise a round with methanol / acetic acid (preferably three rounds), a round of methanol / ammonia (preferably two rounds), a round of ultrapure water (preferably two rounds) and a round with methanol (preferably three rounds).

[0141]

[0111] Coating S130 the MIP nanoparticles 11 P may comprise:

[0142] - preparing S131 a solution of MIP nanoparticles 11 P and a binder; - binding S132 the MIP nanoparticles 11P onto an optical fibre 12;

[0143] - drying S133 the coated optical fibre.

[0144]

[0112] The binder may be polyvinyl alcohol (PVA), preferably in an aqueous solution at up to 1 wt% PVA, still preferably up to 0.75 wt%, for example at 0.5 wt%.

[0145]

[0113] The concentration of MIP nanoparticles 11 P in the solution may be 4 to 6 mg / mL, preferably 4,5 to 5,5 mg / mL, for instance 5 mg / mL.

[0146]

[0114] Binding S132 may comprise immersing S1321 the optical fibre 12 into the solution and heating S1322 under agitation. Heating S1322 may be performed at a temperature of 40 to 100 °C, preferably of 75 to 95 °C, still preferably of 80 to 90 °C, for example 85 °C. Heating can be performed for preferably 10 to 120 min, still preferably 20 to 50 min, for example 30 min.

[0147]

[0115] Drying S133 may be carried out on air.

[0148] MIP film 11F

[0149]

[0116] Forming an MIP support 11 may alternatively lead to the formation of an MIP film 11 F which is illustrated by Fig. 17 to 18. In such case, polymerizing S110 the MIP film 11 F is made in situ on the optical fibre 12 and before that, forming an MIP film 11F may comprise:

[0150] - mixing S105 the matrix precursors, the template and optionally the fluorescence generating component; and

[0151] - immersing S106 the optical fibre 12 into the obtained mixture.

[0152]

[0117] Mixing S105 can be performed in a solvent mixture comprising methanol and water, or in a pure solvent such as DMSO. The methanol / water ratio may be 4:1 .

[0153]

[0118] Polymerizing S110 may a controlled polymerization notably from the surface of the optical fibre 12. It may be performed by injecting light through the optical fibre 12, notably through the lens 13. In one case, polymerization S110 may be obtained using the evanescent field generated over a short distance from the surface of the optical fibre 12 by the light travelling by total internal reflectance through the optical fibre 12. In another case, a sheath may be present around the optical fibre 12. In such case, the light injected into the optical fibre 12 will leak out at the distal end 122 of the optical fibre 12 and polymerize a rod as a continuation thereof. Such polymerization S110 may be controlled to obtain the desired length of the rod, for example by placing a stop surface facing the distal end 122 of the optical fibre 12 at a desired distance and fill the space between the distal end 122 of the optical fibre 12 and the stop surface with the monomer solution. In other cases, when there is no total internal reflectance within the optical fibre 12, polymerization S110 may be obtained at the surface of the optical fibre 12 thanks to the light leaking out therefrom. In such case, polymerization S110 may be controlled, i.e. stopped when the desired thickness of the polymer matrix 110 is obtained.

[0154]

[0119] Alternatively, the light may come from outside of the optical fibre 12 and polymerization S110 may be controlled.

[0120] Polymerizing S110 may be performed at room temperature, i.e. from 20 to 25 °C. Polymerizing may be performed under protection from outside light.

[0155]

[0121] Following polymerizing S110, removing S120 the template may comprise washing away the template. Washing may comprise one or more rounds of washing with one or more solvents. The following solvents may be used: ethanol, acetic acid, ammonia, water and their mixture such as ethanol / acetic acid (for example 4 / 1 ). In one embodiment, washing may comprise a round with ethanol / acetic acid (preferably three rounds) and a round of methanol (preferably three rounds).

[0156] Method for measuring at least one of a quinolinate concentration FQ1 and a tryptophan concentration FT] in real-time

[0157]

[0122] The present invention also provides a method of measuring at least one of a quinolinate concentration and a tryptophan concentration in real-time. Such method comprises:

[0158] - contacting S210 the disposable sensor 1 described above with human urine,

[0159] - illuminating S220 the MIP support 11 of the disposable sensor 1 with an excitation light 31 ,

[0160] - receiving S230 fluorescence emission (FE) radiation 32 back from the disposable sensor 1 ,

[0161] - determining S240 the at least one of the quinolinate concentration and the tryptophan concentration based on the received FE radiation 42.

[0162]

[0123] During contacting S210, when quinolinate Q and / or tryptophan T are present in the human urine, they will bound with their corresponding cavities 112, 113 as shown in Fig. 10. This will change the overall FR radiation 32 of the disposable sensor 1.

[0163]

[0124] Contacting S210 the disposable sensor 1 with human urine may be performed through immersion. Preferably immersion lasts 1 to 10 min.

[0164]

[0125] Contacting S210 may be terminated before illuminating S220 the MIP support 11 and receiving S230 FE radiation 42 or performed at the same time.

[0165]

[0126] Illuminating S220 may comprise injecting the excitation light 41 into the optical fibre 12 of the disposable sensor 1 , notably through the lens 13. The injected excitation light 41 is thus transmitted along the optical fibre 12 through total (or almost total) reflection. The light 41 excites the fluorescence generating component such as the fluorophore 111 comprised in the MIP support 11 and the FE radiation 42 is emitted and travels back.

[0166]

[0127] Illuminating S220 may be performed using a light source such as an LED, a laser, a lamp with a monochromator. Receiving S230 the FR radiation may be performed using a spectrofluorometer. The wavelength used for this step depends on the nature of the polymer matrix used in the disposable sensor 1 . When there is a fluorescence generating component in the polymer matrix of the disposable sensor 1 , the wavelength also depends on the fluorescence generating component. The skilled person would know how to determine the correct wavelength to use. For example, when the fluorophore is N-ally l-4-(4'-methyl-piperazinyl)-1 ,8-naphthalimide, the wavelength used in 410 nm.

[0128] Determining S240 the at least one of the quinolinate concentration [Q] and the tryptophan concentration [T] comprises detecting a peak at a first emission wavelength indicating the presence of quinolinate or a peak at a second emission wavelength indicating the presence of tryptophan.

[0167]

[0129] The wavelength(s) to consider when determining the at least one of the quinolinate concentration and the tryptophan concentration depends on the molecule to be detected and the nature of the polymer matrix. The skilled person would know how to determine the correct wavelength(s) to consider.

[0168]

[0130] The method may comprise both measurements S241 , S242 of the quinolinate concentration [Q] and the tryptophan concentration [T] and calculating S243 a quinolinate / tryptophan ratio [Q] / [T] based on the measured quinolinate and tryptophan concentrations. The method may comprise providing S245 the quinolinate / tryptophan ratio.

[0169]

[0131] Alternatively, the method, instead of providing the ratio, may be adapted to indicate S246 whether the ratio is above one or more thresholds Th1 . Alternatively, or additionally, the method may comprise indicating S246 whether the ratio is below one or more thresholds Th2. Thus, the method may comprise comparing S244 the quinolinate / tryptophan ratio [Q] / [T] with one or more thresholds.

[0170]

[0132] In other embodiments, the method may comprise both providing the quinolinate / tryptophan ratio [Q] / [T] and indicating S246 whether the ratio is above one or more thresholds Th1 or indicating S246 whether the ratio is below one or more thresholds Th2.

[0171] Examples

[0172]

[0133] Example 1 (MIP nanoparticles I I P)

[0173]

[0134] In this example, the MIP support 11 is in the form of MIP nanoparticles 11 P.

[0174]

[0135] 0.1 mmol quinolinate, or respectively tryptophan, are mixed with 0.1 mmol N-allyl-4-(4'-methyl- piperazinyl)-1 ,8-naphthalimide as fluorophore, 0.3 mmol 4-vinylpyridine and 2 mmol ethylene glycol dimethacrylate as polymer monomers and 0.044 mmol 2,2’-azobis(2,4-dimethyl)valeronitrile) as polymerization initiator, in 8 mL of DMSO, in a glass vial fitted with an airtight septum. The mixture is sonicated at room temperature for 5 min and purged with nitrogen for 2 min on ice. Polymerization is then performed overnight at 45 °C in a water-bath. Washing is performed with 3 rounds of methanol / acetic acid (9 / 1 ), 2 rounds of 100 mM ammonia in methanol, 2 rounds of ultrapure water, and 3 rounds of methanol. Then drying is performed overnight under vacuum.

[0175]

[0136] A polystyrene optical fibre 12 is then immersed in a solution of 5 mg / mL MIP nanoparticles 11 P in water containing 0.5 wt% polyvinyl alcohol (PVA) as binder in a 4 mL amber glass vial. The mixture is then heated to 85 °C in an oil bath under magnetic agitation. After 30 min, the optical fibre 12 is removed from the vial and dried on air resulting in an immobilization of MIP nanoparticles 11 P on the surface of the optical fibre 12. PVA acts as a glue.

[0137] Example 2 (MIP film 11 F)

[0176]

[0138] A polystyrene optical fibre 12 is immersed into a solution of 0,1 mmol quinolinate (or tryptophan), 0.2 mmol N-allyl-4-(4'-methyl-piperazinyl)-1 ,8-naphthalimide as fluorophore, 0.2 mmol 4-vinylpyridine and 2 mmol ethylene glycol dimethacrylate as polymer monomers, 1 .5 mol% phenylbis(2,4,6-trimethylbenzoyl)phosphine oxide as polymerization initiator, and 1 .5 mL methanol / water (4 / 1 ). Light (410 nm) is injected through the lens 13 of the sensor 1 during 20 min at room temperature and under protection from outside light resulting in an optical fibre 12 coated with a MIP film 11 F. The coated optical fibre 12 is then washed by immersion with 3 rounds of ethanol / acetic acid (4 / 1 ) during 15 min, 3 rounds of methanol during 15 min.

[0177]

[0139] Example 3: In s / tu photopolymerization and post-polymerization modification of MIPs on fiber optic waveguides and sensing studies in urine

[0178]

[0140] Experimental part

[0179]

[0141] The polystyrene waveguide was immersed into a 1 mL thin glass vial, which was placed inside a 4 mL amber glass vial, containing the template-monomer mixture of quinolinic acid (QA) or Boc-L- tryptohan (BLT), N-allyl-4-(N-methylpiperazinyl)-1 ,8-naphthalimide (AMPN), 4-vinylpyridine (4-VP), ethylene glycol dimethacrylate (EGDMA), and PI-RAFT agent 2-cyano-2-propyl dodecyl trithiocarbonate (CPDTC, 1 .5% relative to polymerizable double bonds) in DMSO (Table 1 ). The mixture was then purged with nitrogen for 5 min. Photopolymerization was performed by introducing light (AEX = 470 nm, 10 nm slit width, 0.88 mW / cm2) through the lens of the polystyrene fiber, using the bifurcated fiber optic probe of the spectrofluorometer for 2 min at room temperature and protected from outside light, resulting in a MIP-coated optical fiber. After polymerization, the MIP-coated fiber was washed 2 times by immersion for 15 min in a solution of methanol :acetic acid (9:1 ), and rinsed 2 times by immersion for 15 min in methanol. The extraction of the template was verified by performing an absorbance scan (AEX = 264 nm for QA; 280 nm for BLT) on the washings with UV- vis spectroscopy.

[0180]

[0142] Afterwards, the MIP-coated fiber underwent post-polymerization modification to improve the compatibility of the MIPs towards urine samples by introducing a hydrophilic polyethylene glycol (PEG) monomer. The MIP-coated fibers were immersed into another 1 mL thin glass vial placed inside a 4 mL amber glass vial containing a solution of PEG methyl ether methacrylate (PEGMEMA; average MN 500) with CPDTC (10% relative to polymerizable double bonds) in DMSO, followed by introducing light (AEX = 470 nm, 10 nm slit width, 0.88 mW / cm2) through the lens of the fiber for another 2 min at room temperature. The MIP-coated fiber was then rinsed 2 times by immersion for 15 min in methanol.

[0181]

[0143] Table 1 presents molar amounts of template, fluorescent and functional monomers, crosslinker, and photoiniferter used for the preparation of MIPs targeting QA and BLT by in situ photopolymerization, followed by post-polymerization.

[0144] [Table 1 ]

[0182] A. Photopolymerization

[0183] Amount (mmol) Total Monomer

[0184] Poly

[0185] 7mer _ . . mnT / - Solvent Concentration Template AMPN 4-VP EGDMA CPDTC ,o / ,

[0186] ( / o)

[0187] QA MIP 2 0.05 0.05 0.15 1 0.033 18.7

[0188] BLT MIP 2 0.05 0.05 0.15 1 0.033 18.7

[0189] _ (1 mL) _

[0190] B. Post-i

[0191] Polymer Amount

[0192] PEGMEMA Solvent

[0193] CPDTC

[0194] QA MIP 2 0.1 0.01 DMSO (1 mL)

[0195] BLT MIP 2 0.1 0.01 DMSO (1 mL)

[0196] * % monomer concentration: [(Total mass of monomers and crosslinker) / (Total mass of monomers and crosslinker + mass of solvent) x 100]

[0197]

[0145] For sensing studies, the MIP-coated optical fiber was inserted into 1 mL thin glass vials placed inside 4 mL amber glass vials containing 1 mL of 1 -100 pM QA or L-Trp-spiked 1 :9 urine:H2O for 5 min for each concentration. After incubation, the bifurcated fiber optic probe of the spectrofluorometer was used to introduce excitation light (AEX = 400 nm, 1 nm slit width) through the lens of the MIP- coated fiber. Fluorescence measurements were performed in the dark, with the fiber remaining immersed in the analyte solution through the same fiber optic probe.

[0198]

[0146] Results

[0199]

[0147] 1 . Sensing of QA in urine

[0200]

[0148] In situ photopolymerization of the MIPs targeting QA was performed on polystyrene waveguides as described above. Polymerization was initiated at 470 nm with an irradiation time of 2 min using the light source originating from the bifurcated optical fiber probe of the spectrofluorometer through the lens of the optical fiber waveguide (Fig. 20). As shown in Fig. 20C, a visible homogeneous polymer coating for QA MIP was obtained, as judged by the naked eye. After washing, the MIP-coated fibers were tested in 1-100 pM QA-spiked 1 :9 urine:H2O. A maximum fluorescence enhancement of -60% was achieved for the QA MIP-coated fiber (Fig. 21 ). The linear dynamic range for sensing spans from 1 to 10 pM of QA. Since acute kidney injury (AKI) and healthy persons have respectively mean QA values of 50 and 20 pM, their urine should be diluted before measurement, so as to fall within this dynamic range. It is worth noting that the QA MIPs do not bind L-Trp even at concentrations as high as 100 pM (Fig. 22, right). Moreover, this binding was specific as no enhancements were observed for the corresponding Boc-L-tryptophan (BLT) control MIP-coated fiber.

[0201]

[0149] 2. Sensing of L-tryptophan (L-Trp) in urine

[0202]

[0150] The in situ photopolymerization of the MIPs targeting BLT were performed on polystyrene waveguides, following the protocol described above for QA. After washing, the MIP-coated fiber was tested in 1-100 pM L-Trp-spiked 1 :9 urine:H2O, which yielded a maximum fluorescence enhancement of -45% for BLT MIP-coated fiber (Fig. 22, right). The linear dynamic range for sensing spans from 1 to 50 pM of L-Trp with MIP. This binding was specific as no enhancements were observed for the corresponding QA control MIP-coated fibers. Moreover, these BLT MIPs do not bind QA (Fig. 21 , right).

[0151] 3. Selectivity of MIP-coated optical fibers in urine

[0203]

[0152] Figure 23 shows how the MIP waveguides are specific for their corresponding target molecules as they do not recognize other molecules, even at high concentrations, present in urine. Note that there is some cross-reactivity with dipicolinic acid (DPA) which is structurally very similar to QA but DPA does not normally exist in urine, neither does D-Trp.

[0153] Figure 24 shows images of scanning electron microscopy (SEM) analysis of MIP-QA on the waveguides. Only QA MIP is shown because BLT MIP is similar. Upon close inspection of the QA MIP-coated fiber, a slightly rugged surface morphology is observed for the MIP fiber in comparison to the smooth surface aspect of a bare fiber. The cross-section shows that the polymer coating is porous, with a thickness of -10 pm.

Claims

Claims1 . A disposable sensor (1 ) for measuring at least one of the quinolinate concentration and the tryptophan concentration in real time in human urine:- a molecularly imprinted polymer, MIP, support (1 1 ) comprising a polymer matrix (1 10), at least one of a first set of cavities (1 12) adapted to measure the quinolinate concentration and a second set of cavities (1 13) adapted to measure the tryptophan concentration within the polymer matrix (1 10); and- an optical fibre (12) coated with the MIP support (1 1 ).

2. The disposable sensor of claim 1 , wherein the MIP support (1 1 ) comprises MIP nanoparticles (1 1 P) or a MIP film (1 1 F), or a combination of both.

3. The disposable sensor of claim 1 or claim 2, wherein the MIP support (1 1 ) further comprises a fluorophore (1 1 1 ) within the polymer matrix (1 10).

4. The disposable sensor of claim 3, wherein the fluorophore is chosen from the group consisting of N-allyl-4-(4'-methyl-piperazinyl)-1 ,8-naphthalimide, N-allyl-4-(N,N'-(methyl) ethylene diamine)-1 ,8-naphthalimide, trans-4-[p-(N,N-dimethylamino)styryl]-N-vinyl benzyl pyridinium chloride and their mixtures.

5. The disposable sensor of any of claims 1 to 4, further comprising a lens (13) at a proximal end (121 ) of the optical fibre (12) and a reflector (14) at a distal end (122) of the optical fibre (12) opposite the proximal end (121 ).

6. A system (3) comprising the disposable sensor (1 ) of any of claims 1 to 5, a light source and a spectrometer (21 ) coupled to the disposable sensor (1 ) through the measurement box (5); wherein the light source is configured to emit a light with a wavelength corresponding to the polymer matrix (1 10) of the disposable sensor (1 ) and measure at least one of a first fluorescence proportional to the quinolinate concentration and a second fluorescence proportional to the tryptophan concentration from a secondary signal received from the disposable sensor (1 ).

7. A method for making the disposable sensor of any claim 1 to 4, the method comprising: - forming an MIP support on an optical fibre, from at least one template and matrix precursors, wherein the template is a quinolinate, a tryptophan template or both.

8. The method of claim 7, wherein a fluorophore is also used to form the MIP support (1 1 ), the fluorophore being chosen from the group consisting of N-allyl-4-(4'-methyl-piperazinyl)-1 ,8- naphthalimide, N-allyl-4-(N,N'-(methyl) ethylene diamine)-1 ,8-naphthalimide, trans-4-[p-(N,N- dimethylamino)styryl]-N-vinyl benzyl pyridinium chloride and their mixture.

9. The method of claim 7 or claim 8, wherein the matrix precursors are at least one monomer and a polymerization initiator; preferably, the monomer is chosen from the group consisting of 4-vinylpyridine, ethylene glycol diacrylate, trimethylolpropane triacrylate, pentaerythritol triacrylate, polyethylene glycol acrylate, polyethylene glycol diacrylate, hydroxyethyl acrylate, ethylene glycol dimethacrylate, trimethylolpropane trimethacrylate, pentaerythritol trimethacrylate, polyethylene glycol methacrylate,polyethylene glycol dimethacrylate, hydroxyethyl methacrylate, acrylamide, methacrylamide, methylene bisacrylamide, ethylene bisacrylamide, piperazine bisacrylamide, N-isopropylacrylamide, N-tert-butylacrylamide, N-propylacrylamide, phenylacrylamide, 4-acrylamidobenzamidine, aminopropylacrylamide, diethylaminopropylacrylamide, and their mixtures; and / or preferably, the polymerization initiator is adapted for thermal polymerization, redox polymerization, or photopolymerization, such as, 2,2’-azobis(2,4-dimethyl)valeronitrile), 2,2’-azobis(2- methylpropionitrile), 2,2-dimethoxy-2-phenylacetophenone, benzyl N,N-diethyldithiocarbamate, phenylbis(2,4,6-trimethylbenzoyl)phosphine oxide, and their mixtures.

10. The method of one of claims 7 to 9, wherein forming the MIP support comprises:- polymerizing the matrix precursors into a polymer matrix around the template; and- removing the template from the polymer matrix, thereby leaving cavities behind.1 1 . The method of claim 10, wherein polymerizing is carried out in the absence of the optical fibre and is carried out by precipitation polymerization to form the MIP support in the form of MIP nanoparticles, after removing the template, forming the MIP support further comprises coating the MIP nanoparticles onto the optical fibre.

12. The method of claim 10, wherein forming the MIP support comprises polymerizing the MIP support in the form of an MIP film in situ on the optical fibre.

13. A method for measuring at least one of a quinolinate concentration and a tryptophan concentration in real time comprising:- contacting the disposable sensor of one of claims 1 to 5 with human urine,- injecting light inside the optical fibre of the disposable sensor,- receiving fluorescence emission, FE, radiation back from the disposable sensor,- determining the at least one of the quinolinate concentration and the tryptophan concentration based on the received FE radiation.

14. The method of claim 13, wherein the quinolinate concentration and the tryptophan concentration are measured, further comprising calculating a quinolinate / tryptophan ratio based on the determined quinolinate and tryptophan ratios.

15. The method of claim 14, further comprising indicating whether the quinolinate / tryptophan ratio is above a threshold.

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