Patent Description:
The document "<NPL>" is a Review article summarizing different L-Asparaginase-based biosensors for the quantification of L-asparagine. The reported articles in the review article use as detection methodologies the following techniques or reagents: Potentiometric ammonia gas detector, Ammonia gas electrode, Polypyrrole probe, Ammonium-selective glass electrode and the reagents: Nessler reagent (read in a spectrophotometer) and Phenol red (pH changes). Therefore, this document is silent on portable sensors capable of changing their color in the presence of different concentrations of L-asparagine, without the need of using any further lab equipment, electrodes, detectors or phenol red reagent.

The document "<NPL>. " describes the use of modified solid silica gel materials (Commercial silica gel <NUM> (<NUM>-<NUM>), spherical particles) with ionic liquids for the immobilization of the enzyme L-asparaginase and its quantification through biological activity tests with Nessler reagent (output read in a spectrophotometer). Regardless, this document does not describe a visual biosensor based on modified silica fabric with ionic liquids for the visual quantification of the amino acid L-asparagine in aqueous solutions, wherein the physical support for the enzyme changes its color depending on the L-asparagine concentration range.

The document "<NPL>" describes the immobilization of Leu71lle mutant L-asparaginase by crosslinking with glutaraldehyde on the side of a transparent plastic cuvette for the quantification of L-asparagine using the Nessler reagent (output read in a spectrophotometer). This document thus discloses a technology that requires a spectrophotometer to quantify the presence of L-asparagine.

The document "<NPL>" describes an assay for the determination of L-asparagine with a microplate-based biosensor using L-asparaginase immobilized by crosslinking with glutaraldehyde and deposited into the well of a microplate in <NUM>-well format and Nessler reagent (output read in a microplate equipment). Similarly, the detection of L-asparagine disclosed in this document is dependent on laboratory equipment, in this case a microplate reader.

The document <CIT> describes a colorimetric assay for the determination of organic substituents with primary or secondary amines or with thiol groups immobilized on or in insoluble materials. The visual detection or quantification of the solid-phase bound primary and secondary amines and thiol groups, comprises the addition of a fluid (acetone, acetonitrile, DMF (dimethylformamide), DMSO (dimethylsulfoxide), methanol, water or mixtures) to a substrate (animal or plant tissue, array surfaces, bone, dipsticks, macro-beads, membranes, microplates, nanoparticles, polymer, resin, silica gel, among others), and the further addition of DESC reagent (<NUM>-methyl-<NUM>-(<NUM>'-nitrophenyl)-imidazo[<NUM>,<NUM>-a]pyrimidinium perchlorate, doi: <NUM>/cc800031y). The color change intensity is proportional to the total amino groups and/or thiol groups concentration, detected via visual observation, optical microscope, or spectroscopically following <NUM> - <NUM> of DESC reagent addition. The document is however silent on a portable visual biosensor for the rapid detection of L-asparagine levels in aqueous solutions.

The document <CIT> describes a method and assay kit for the determination of the enzymatic activity of asparagine-β-amido hydrolase enzymes (glycosylasparaginases (EC <NUM>. <NUM>) and asparaginases (EC <NUM>. <NUM>)) in biological samples. In this method it is necessary the contact/incubation of the samples with a compound (that acts as a substrate (L-asparagine, L-aspartic acid derivatives with a free α-amino and α-carboxyl group of known fluorogenic compounds (<NUM>-amino-<NUM>-methylcoumarin, β-methylumbelliferone, fluorescein, β-naphtylamide, resorufin, <NUM>-aminoquinoline or resorcinol), derivatives of known coloured reagents (β-naphtylamide) or compounds used in chemiluminescence (D-luciferine or adamantyl-<NUM>,<NUM>-dioxethane) in a suitable buffer solution) containing a luminescent (fluorescent, phosphorescent or chemiluminescent) or a colored moiety attached to the β-amino group of L-asparagine or β-carboxyl group of L-aspartic acid. This label compound is cleaved off via the catalysis of asparagine-β-amido hydrolase enzymes present in the biological samples. The amount of released moiety is determined through luminometry (fluorometry or chemiluminometry) or spectrophotometry, quantitatively measuring the amount of asparagine-β-amido hydrolase enzymes present in the biological samples. The document is however silent on a portable visual biosensor for the rapid detection of L-asparagine levels in aqueous solutions, which does not require any further lab equipment such as luminometer, fluorometer, chemiluminometer or a spectrophotometer.

The document <CIT> describes a method for direct detection and quantitation of asparagine synthetase, an enzyme responsible for the cellular asparagine production, whose expression has been correlated with chemoresistance to certain cancer types, in biological samples. The method comprises substantial purification of asparagine synthetase protein from a sample (through SDS-PAGE separation, size exclusion chromatography, cation or anion exchange chromatography or antibody-mediated enrichment), adding standards (stable isotope peptide standards) to the protein and determining the amount of protein in the sample using mass spectrometry. The document is however silent on a portable visual biosensor for the rapid detection of L-asparagine levels in aqueous solutions, that does not require a previous purification of the compound to be detected, i.e., the L-asparagine amino acid.

The present disclosure relates to a biosensor based on silica fabric functionalized with ionic liquids for the attachment of the enzyme L-asparaginase and further detection of the L-asparagine amino acid. The functionalization of the silica fabric with ionic liquids enhances the binding/adsorption of the L-asparaginase enzyme (ASNase) through simple experimental procedures, thus allowing the detection of L-asparagine amino acid by visual observation. In an embodiment, upon addition of a solution into the modified fabric, and if L-asparagine is present in said solution, it interacts with the immobilized enzyme L-asparaginase, and is converted in L-aspartic acid and ammonium ions. By adding a revealing agent, such as Nessler's solution that changes color in the presence of ammonium ions, the color of the fabric changes as a consequence of the presence of ammonium ions, and consequently the presence of L-asparagine amino acid. Surprisingly, the color change of the functionalized fabric is proportional to the concentration of L-asparagine. Thus, the disclosed biosensor is a simple device that can be used for rapid detection of L-asparagine levels in aqueous solutions, by simple observation of change of color in a fabric, and without the need of special equipment such as microplate readers or spectrophotometers.

In an embodiment, the disclosed biosensor is a portable biosensor, which changes its color with the changes in the L-asparagine concentration levels. In a further embodiment, the biosensor is a visual biosensor based on modified silica fabric with ionic liquids for the visual quantification of the amino acid L-asparagine in aqueous solutions, wherein the silica fabric changes the color depending on the L-asparagine concentration range. The disclosed biosensor does not require the use of a spectrophotometer, microplate readers, or any lab equipment for the quantification of the L-asparagine levels, allowing its fast and simple determination.

In an embodiment, the visual detection of the L-asparagine consists on the change of the color of the modified silica fabric material where each concentration range of L-asparagine corresponds to a specific color.

In an embodiment, for the modification of the silica fabric, firstly a silane compatibilizing agent/anion source, namely (<NUM>-Chloropropyl)trimethoxysilane (C<NUM>H<NUM>ClO<NUM>Si), is added to the silica fabric. Then, after washing with organic solvents, such as toluene, ethanol, methanol, or mixtures thereof, and water, the cation (N,N-dimethylbutylammonium ([N<NUM>]+) or triethylammonium ([N<NUM>]+) is added and then washed with organic solvents, such as toluene, methanol or mixtures thereof, and water.

In an embodiment, high concentration ranges of L-asparagine (<NUM>-<NUM> M and <NUM>-<NUM> M) in aqueous solutions are detected by visual means, due to the change of the color of the modified silica fabrics ([Si][N<NUM>]Cl and [Si][N<NUM>]Cl). The visual detection includes a sequential addition of reduced volumes of aqueous solutions of L-asparaginase, sample (containing L-asparagine) and revealing solution (Nessler's reagent). Depending on the L-asparagine concentration levels, different colors of the modified silica fabric are obtained ranging from white-grey to brownish red.

The present disclosure relates to a fabric for immobilization of L-asparaginase enzyme, the fabric comprising: a plurality of silica fibers; and an ionic liquid supported on the silica fibers, wherein the ionic liquid comprises a quaternary ammonium cation and chloride as counterion; wherein the L-asparaginase enzyme is immobilized on the plurality of silica fibers through the ionic liquid.

The invention relates to a fabric for detecting a L-asparagine amino acid in a sample as defined in claim <NUM>, wherein the fabric comprises: a plurality of silica fibers; an ionic liquid linked to the plurality of silica fibers by a spacer arm, wherein the ionic liquid comprises a quaternary ammonium cation and a chloride as counterion; and a L-asparaginase enzyme immobilized in the plurality of silica fibers though the ionic liquid; wherein the spacer arm comprises a compound with formula CxHyClO<NUM>Si, wherein X is an entire number ranging from <NUM> to <NUM> and wherein Y is an entire number ranging from <NUM> to <NUM>; and wherein the presence of L-asparagine in the sample is detectable by a change of color of the fabric after addition of a revealing solution.

The invention relates to a fabric for detecting a L-asparagine amino acid in a sample as defined in claim <NUM>, wherein the fabric comprises: a plurality of silica fibers; an ionic liquid linked to the plurality of silica fibers by a spacer arm, wherein the ionic liquid comprises a quaternary ammonium cation and a chloride as counterion; and wherein the spacer arm comprises a compound with formula CxHyClO<NUM>Si, wherein X is an entire number ranging from <NUM> to <NUM> and wherein Y is an entire number ranging from <NUM> to <NUM>; and wherein the presence of L-asparagine in the sample is detectable by a change of color of the fabric after addition of a L-asparaginase enzyme solution to the fabric, addition of the sample, and addition of a revealing solution.

In the invention, the spacer arm (CxHyClO<NUM>Si) is selected from a list comprising (<NUM>-Chloropropyl)trimethoxysilane (C<NUM>H<NUM>ClO<NUM>Si), (<NUM>-Chloropropyl)triethoxysilane (C<NUM>H<NUM>ClO<NUM>Si), (Chloromethyl)trimethoxysilane (C<NUM>H<NUM>ClO<NUM>Si), (Chloromethyl)triethoxysilane (C<NUM>H<NUM>ClO<NUM>Si), (<NUM>-Chloroethyl)trimethoxysilane (C<NUM>H<NUM>ClO<NUM>Si), (<NUM>-Chloroethyl)triethoxysilane (C<NUM>H<NUM>ClO<NUM>Si), (<NUM>-Chlorobutyl)trimethoxysilane (C<NUM>H<NUM>ClO<NUM>Si), (<NUM>-Chlorobutyl)triethoxysilane (C<NUM>H<NUM>ClO<NUM>Si), or combinations thereof; preferably wherein the spacer arm is a (<NUM>-Chloropropyl)trimethoxysilane.

In an embodiment, the revealing solution is Nessler's reagent or phenol red, preferably Nessler's reagent.

In an embodiment, the silica fibers are woven.

In an embodiment, the quaternary ammonium cation comprised in the ionic liquid is selected from N,N-dimethylbutylammonium or triethylammonium.

In an embodiment, the mass of ionic liquid per mass of fabric ranges from <NUM>. g-<NUM> to <NUM>. g-<NUM>, preferably from <NUM>. g-<NUM> to <NUM>.

In an embodiment, the diameter of the silica fibers ranges from <NUM> to <NUM>, preferably from <NUM> to <NUM>, measured by scanning electron microscopy.

In an embodiment, the sample is a liquid biological sample, or a liquid food-derived sample. In a further embodiment, the liquid biological sample is a blood serum sample.

In an embodiment, the concentration of L-asparaginase enzyme, preferably the immobilized enzyme, in the fabric ranges from <NUM>/cm<NUM> to <NUM>/cm<NUM>, preferably from <NUM>/cm<NUM> to <NUM>/cm<NUM>.

An aspect of the present disclosure relates to the use of a fabric as defined in claim <NUM> in a sensor for detecting L-asparagine amino acid in an aqueous solution.

In an embodiment, the aqueous solution is a serum sample, or a food-derived sample.

The present disclosure also relates to a portable sensor for detecting L-asparagine amino acid in an aqueous solution comprising a fabric as defined in claim <NUM> for detecting a L-asparagine amino acid, preferably a sensor for monitoring and/or diagnosing a disease that causes an increase in the concentration of L-asparagine amino acid in blood, more preferably a sensor for monitoring and/or diagnosing leukemia disease.

In an embodiment, the immobilized L-asparaginase enzyme is available to catalyze the hydrolysis of the L-asparagine amino acid present in the aqueous solution into ammonia and L-aspartic acid. The produced ammonia, resultant from the presence of L-asparagine, is then detectable after addition of the revealing solution.

In an embodiment, the sensor is arranged to detect L-asparagine amino acid in a concentration ranging from <NUM>-<NUM> M to <NUM>, preferably <NUM>-<NUM> M to <NUM>-<NUM> M.

An aspect of the present disclosure relates to a method for detecting L-asparagine amino acid in an aqueous solution using the fabric as defined in claim <NUM> or the portable sensor defined in claim <NUM>, the method comprising the following steps: adding the aqueous solution to the portable sensor or fabric; adding a revealing solution to the portable sensor or fabric, preferably wherein the revealing solution is Nessler's reagent; analyzing the fabric or portable sensor for a change of color, wherein a change of color indicates the presence of L-asparagine amino acid in the aqueous solution.

An aspect of the present disclosure relates to a kit for detecting L-asparagine amino acid comprising the fabric as defined in claim <NUM> or the disclosed portable sensor as defined in claim <NUM> and a revealing agent. In a further embodiment, the kit also comprises a color scale to correlate the color of the disclosed fabric with the concentration of L-asparagine amino acid in the aqueous solution.

In an embodiment, the revealing agent is Nessler's reagent.

In an embodiment, the kit is for diagnosing and or monitoring a disease that causes an increase in the concentration of L-asparagine amino acid in blood, such as leukemia disease.

The following figures provide preferred embodiments for illustrating the disclosure and should not be seen as limiting the scope of invention.

The invention relates to a fabric for detecting a L-asparagine amino acid in a sample as defined in claim <NUM>, wherein the fabric comprises a plurality of silica fibers; an ionic liquid linked to the plurality of silica fibers by a spacer arm, wherein the ionic liquid comprises a quaternary ammonium cation and chloride as counterion; and a L-asparaginase enzyme immobilized in the plurality of silica fibers through the ionic liquid; wherein the presence of L-asparagine in the sample is detectable by a change of color of the fabric after addition of a revealing solution. The use of said fabric as a sensor for detecting L-asparagine amino acid in an aqueous solution, a portable sensor for detecting L-asparagine amino acid in an aqueous solution comprising said fabric, the method for detecting L-asparagine amino acid using said sensor, and a kit comprising said fabric or sensor are also disclosed. Namely, the use of the fabric of the present disclosure for monitoring/diagnosing a disease that causes an increase in the concentration of L-asparagine amino acid in blood, in particular leukemia.

The present disclosure also relates to a fabric for immobilization of L-asparaginase enzyme, the fabric comprising a plurality of silica fibers; an ionic liquid supported in the silica fibers, wherein the ionic liquid comprises a quaternary ammonium cation and chloride as counterion; and wherein the L-asparaginase enzyme is immobilized on the plurality of silica fibers through the ionic liquid. The use of said fabric as a sensor for detecting L-asparagine amino acid in an aqueous solution, a portable sensor for detecting L-asparagine amino acid in an aqueous solution comprising said fabric, the method for detecting L-asparagine amino acid using said sensor, and a kit comprising said fabric or sensor are also disclosed.

The present disclosure relates to a colorimetric sensor for the visual detection of L-asparagine concentration ranges in aqueous solutions. The disclosed biosensor is based on a simple method that, by changing the color of the material specifically modified to attach the constituent responsible for the quantification of L-asparagine, allows the visual detection of high concentration ranges of L-asparagine in aqueous solutions. In an embodiment, the disclosed sensor can be used in the detection of L-asparagine in the pharmaceutical sector, industrial cooked starched based-foods and in chemical laboratories.

The aim of the present disclosure is to determine by color change the concentration levels of L-asparagine using a portable enzymatic biosensor made with silica fabric functionalized with ionic liquids to attach the L-asparaginase.

In an embodiment, the disclosed biosensor can be used in the pharmaceutical sector, chemical and food industries which need to determine the concentration levels of L-asparagine in aqueous solutions.

In an embodiment, silica fabric was functionalized with quaternary ammonium cations in addition to chloride as the counterion, resulting in a silica fabric functionalized with different functional groups. In particular, the silica fabric was functionalized with a quaternary ammonium cation selected from N,N-dimethylbutylammonium ([N<NUM>]+) and triethylammonium ([N<NUM>]+), in addition to chloride as the counterion, resulting in a silica fabric functionalized with N,N-dimethylbutylammonium chloride ([Si][N<NUM>]Cl) or triethylammonium chloride ([Si][N<NUM>]Cl), respectively. The abbreviations and chemical structures of the modified silica fabric are displayed in Table <NUM>.

In an embodiment, the silica fabric functionalization was performed through two main steps:.

Surprisingly, the functionalization with different ionic liquids did not significantly change the color of the functionalized fabric. In an embodiment, the color of the fabric after the functionalization with N,N-dimethylbutylammonium chloride ([Si][N<NUM>]Cl) is white-grey (L*:<NUM>, a*:<NUM>, and b*:<NUM>) upon addition of a aqueous solution without L-asparagine and white-grey (L*:<NUM>, a*:<NUM>, and b*:<NUM>) upon the addition of a commercial blood human serum sample without L-asparagine. In another embodiment, the color of the fabric after the functionalization with triethylammonium chloride ([Si][N<NUM>]Cl) is white-grey (L*:<NUM>, a*:<NUM>, and b*:<NUM>) upon addition of a aqueous solution without L-asparagine and white-grey (L*: <NUM>, a*:<NUM>, and b*:<NUM>) upon the addition of a commercial blood human serum sample without L-asparagine.

In an embodiment, all functionalized silica fabrics were characterized by elemental analysis, attenuated total reflectance-Fourier-transform infrared spectroscopy (ATR-FTIR), and scanning electron microscopy (SEM).

In an embodiment, the elemental analysis, i.e., the weight percentages (%w/w) of carbon (%C), hydrogen (%H), and nitrogen (%N) of [Si][C<NUM>]Cl and all functionalized silica fabrics, were determined through elemental analysis using the equipment TruSpec <NUM>-<NUM>-<NUM>, <NUM> of sample, combustion furnace temperature of <NUM>, and burner temperature of <NUM>. The detection method for carbon and hydrogen was infrared absorption, while for nitrogen was thermal conductivity. The elemental analysis of the modified silica fabric is displayed in Table <NUM>.

In an embodiment, the mass of IL per mass of material (mg. g-<NUM>) was obtained using the following formula (M - molar mass (g. mol-<NUM>); %N - weight percentage of nitrogen): <MAT>.

In an embodiment, spectra from attenuated total reflectance - Fourier-transform infrared (ATR-FTIR) spectroscopy was obtained using Perkin Elmer FT-IR System Spectrum BX equipment (Waltham, MA, USA) and a solid sample of all functionalized silica fabrics at <NUM> and in a range between <NUM>-<NUM>-<NUM>. Each sample was scanned <NUM> times with a resolution of <NUM>-<NUM>.

In an embodiment, scanning electron microscopy (SEM) was performed using a Hitachi SU-<NUM> microscope at the accelerating voltage of <NUM> kV. A carbon thin film deposition was used in order to increase the samples' conductivity. Fiber diameter of silica fabric and all functionalized silica fabrics were defined using the line tool in ImageJ (National Institute of Mental Health, Bethesda, Maryland, USA). The average fiber diameter of the modified silica fabric is displayed in Table <NUM>.

In an embodiment, the colorimetric biosensors were developed through two main steps: (i) L-Asparaginase physical adsorption on functionalized silica fabrics and (ii) revealing solution addition. In the first step, the enzyme L-asparaginase was immobilized on functionalized silica fabrics at room temperature through the addition of <NUM>µL of L-asparaginase solution (<NUM> mL-<NUM>) into <NUM> x <NUM> of functionalized silica fabrics: [Si][N<NUM>]Cl and [Si][N<NUM>]Cl. Subsequently, <NUM>µL of aqueous samples (solution of different L-asparagine concentration levels, <NUM>-<NUM> M, <NUM>-<NUM> M, <NUM>-<NUM> M, <NUM>-<NUM> M and <NUM>-<NUM> M, or commercial blood human serum with the same L-asparagine concentration levels) was added to the functionalized silica fabrics. Then, <NUM>µL of revealing solution, Nessler's reagent, is added. After <NUM> seconds, the resulting color of the functionalized silica fabrics, was dependent on the L-asparagine concentration levels of each sample.

In an embodiment, the resulting colors of functionalized silica fabrics were analyzed by a Portable Spectrophotometer CM-2300d (Konica Minolta Europe). Color changes were quantified using the color organization system, i.e., CIELAB (or CIE L* a* b*) color space, which expresses the color's lightness, red/green and yellow/blue intensity, as L*, a*, and b* values, respectively. These chromaticity coordinates were converted into RGB (Red, Green, Blue) color system using the conversion spreadsheets (available from http://colormine. org/convert/rgb-to-lab (accessed on <NUM> February <NUM>)).

The potential applications of the present biosensor for the quantification ranges of L-asparagine are in the pharmaceutical sector, food and chemical industries.

In an embodiment, in the pharmaceutical sector the biosensor can be used to determine concentration levels of L-asparagine in blood serum samples. The determined concentration of L-asparagine levels can be used to monitor the treatment response in disease therapy with L-asparaginase. L-asparagine concentration in leukemia blood serum samples is higher than detected in healthy blood serum samples. More specifically, the concentration of L-asparagine in leukemia blood serum samples varies from <NUM>-<NUM> to <NUM>-<NUM> M, while in healthy blood serum samples varies from <NUM>-<NUM> to <NUM>-<NUM> M [<NUM>-<NUM>]. In an embodiment, in food industries, starch-rich foods cooked at temperatures over <NUM> led to the formation of a carcinogenic compound, acrylamide, through the Maillard reaction between L-asparagine and carbonyl compounds. Thus, the present biosensor can be used to detect and quantify the L-asparagine levels in these foods, from a liquid food-derived sample, to avoid the formation of high levels of acrylamide (for example acrylamide carcinogenic levels).

In an embodiment, in chemical industries, the present biosensor can be applied in any chemical process that needs the detection of L-asparagine levels.

The disclosure should not be seen in any way restricted to the embodiments described and a person with ordinary skill in the art will foresee many possibilities to modifications thereof. The above-described embodiments are combinable.

Claim 1:
A fabric for detecting a L-asparagine amino acid in a sample, wherein the fabric comprises:
a plurality of silica fibers;
an ionic liquid linked to the plurality of silica fibers by a spacer arm, wherein the ionic liquid comprises a quaternary ammonium cation and a chloride as counterion; and
a L-asparaginase enzyme immobilized in the plurality of silica fibers through the ionic liquid;
wherein the spacer arm comprises a compound with formula CxHyClO<NUM>Si, selected from a list comprising (<NUM>-Chloropropyl)trimethoxysilane (C<NUM>H<NUM>ClO<NUM>Si), (<NUM>-Chloropropyl)triethoxysilane (C<NUM>H<NUM>ClO<NUM>Si), (Chloromethyl)trimethoxysilane (C<NUM>H<NUM>ClO<NUM>Si), (Chloromethyl)triethoxysilane (C<NUM>H<NUM>ClO<NUM>Si), (<NUM>-Chloroethyl)trimethoxysilane (C<NUM>H<NUM>ClO<NUM>Si), (<NUM>-Chloroethyl)triethoxysilane (C<NUM>H<NUM>ClO<NUM>Si), (<NUM>-Chlorobutyl)trimethoxysilane (C<NUM>H<NUM>ClO<NUM>Si), (<NUM>-Chlorobutyl)triethoxysilane (C<NUM>H<NUM>ClO<NUM>Si), or combinations thereof; and
wherein the fabric is configured to change color in the presence of L-asparagine in the sample after the addition of a revealing solution.