US-20260126382-A1 - INTELLIGENT NIR II PHOTOTHERMAL AND COLORIMETRIC SENSING TECHNOLOGY

US20260126382A1US 20260126382 A1US20260126382 A1US 20260126382A1US-20260126382-A1

Abstract

The current invention relates to a method of quantitatively determining the concentration of active myrosinase in a sample, the method comprising the steps of (a) adding a portion of the sample comprising active myrosinase to a first analyte assay formulation for a first period of time to generate an analyte sample, (b) adding a portion of the sample comprising inactivated myrosinase to a second analyte assay formulation for a second period of time to generate a analyte reference sample, and (c) determining the concentration of the active myrosinase in the sample. In an embodiment, the first anayte assasy formulation comprising a near infra-red absorbing and photothermally-responsive compound. The invention also relates to a formulation for use in a method of quantitatively determining the concentration of active myrosinase in a sample, and a kit of parts suitable to provide a formulation as aforementioned.

Inventors

Bengang XING
Ling QIAO
Wenchao LANG

Assignees

NANYANG TECHNOLOGICAL UNIVERSITY

Dates

Publication Date: 20260507
Application Date: 20231027
Priority Date: 20221027

Claims (20)

1 . A method of quantitatively determining the concentration of active myrosinase in a sample, the method comprising the steps of: (a) adding a portion of the sample comprising active myrosinase to a first analyte assay formulation for a first period of time to generate an analyte sample, the first analyte assay formulation comprising: a substrate for active myrosinase that provides glucose as a reaction product; a glucose oxidase; either a near infra-red absorbing and photothermally-responsive compound capable of forming a charge transfer complex or nanoparticle aggregates; and either a peroxidase or nanoparticles having peroxidase-like activity; (b) adding a portion of the sample comprising inactivated myrosinase to a second analyte assay formulation for a second period of time to generate a analyte reference sample, the second analyte assay formulation being the same as the first analyte assay formulation and the second period of time being the same as the first period of time; (c) determining the concentration of the active myrosinase in the sample by measuring one or more of: (i) Red Green Blue colour values in the analyte sample and Red Green Blue colour values in the analyte reference sample, calculating the Red/Blue ratio for each of the analyte sample and the analyte reference sample and calculating the difference between the Red/Blue ratios to provide a sample Red/Blue ratio and comparing the sample Red/Blue ratio obtained to a pre-determined calibration curve of active myrosinase concentration based on Red/Blue ratios obtained from known concentrations of active myrosinase; (ii) an infra-red image of the analyte sample and an infra-red image of the analyte reference sample obtained following irradiation of the analyte sample and the analyte reference same by a laser for a third period of time and calculating the temperature difference between a temperature obtained from the infra-red image of the analyte sample and a temperature obtained from the infra-red image of the analyte reference sample and comparing the difference value obtained to a pre-determined calibration curve of active myrosinase concentration based on temperatures obtained from infra-red images of known concentrations of active myrosinase; (iii) an absorbance spectra of the analyte sample and an absorbance spectra of the analyte reference sample and calculating the difference between the absorbance spectra of the analyte sample and the absorbance spectra of the analyte reference sample and comparing the difference value obtained to a pre-determined calibration curve of active myrosinase concentration based on absorbance spectra of known concentrations of active myrosinase; and (iv) a temperature signal obtained following irradiation of the analyte sample and the analyte reference sample by a laser for a third period of time and calculating the difference between the temperature signal of the analyte sample and the temperature signal of the analyte reference sample and comparing the difference value obtained to a pre-determined calibration curve of active myrosinase concentration based on temperature signals of known concentrations of active myrosinase.
2 . The method according to claim 1 , wherein two or more of (i) to (iv) are used to determine the concentration of active myrosinase in the sample.
3 . The method according to claim 1 , wherein the substrate for active myrosinase that provides glucose as a reaction product is a glucosinolate.
4 . The method according to claim 1 , wherein the near infra-red absorbing and photothermally-responsive compound capable of forming a charge transfer complex or nanoparticle aggregates is selected from phenylboronate decorated gold nanoparticles, or one or more of the group selected from perylene, F4TCNQ, tetracyanoquinodimethane (TCNQ), and 3,3′,5,5′-tetramethylbenzidine.
5 . The method according to claim 1 , wherein the nanoparticles having peroxidase-like activity are selected from silver or, more particularly, gold nanoparticles.
6 . The method according to claim 1 , wherein one or more of the following apply: (bi) when the nanoparticles having peroxidase-like activity or the peroxidase are gold nanoparticles, they are present in the first and second analyte assay formulations at a concentration of from 0.02 to 0.8 nM; (bii) when the near infra-red absorbing and photothermally-responsive compound capable of forming a charge transfer complex or nanoparticle aggregates is 3,3′,5,5′-tetramethylbenzidine, then it is present in the first and second analyte assay formulations at a concentration of from 0.5 to 3 mM; (biii) the glucose oxidase is present in an amount of from 5 U/mL to 25 U/mL; (biv) the substrate for active myrosinase that provides glucose as a reaction product is present in an amount of from 0.05 mM to 0.75 mM.
7 . The method according to claim 1 , wherein the first and second analyte assay formulations further comprise an acetate buffer in an amount that provides a pH of from 3.5 to 7.5.
8 . The method according to claim 1 , wherein the sample comprising active myrosinase and the sample comprising inactivated myrosinase is provided at a concentration of from 1 to 20 mg/mL.
9 . The method according to claim 1 , wherein the sample comprising inactivated myrosinase is obtained by heating a sample comprising active myrosinase to a temperature suitable to denature active myrosinase for a fourth period of time.
10 . The method according to claim 1 , wherein the sample comprising active myrosinase is subjected to incubation for a fifth period of time at a suitable temperature before use in the method.
11 . The method according to any claim 1 , wherein when one or both of (ii) and (iv) of step (c) in claim 1 are used to determine the concentration of the active myrosinase in the sample, then the laser is a near infra-red light laser.
12 . The method according to claim 1 , wherein the first and second periods of time are from 10 minutes to 1 hour.
13 . The method according to claim 1 , wherein the absorbance spectra of the analyte sample and the analyte reference sample are based on their absorbance at a specified wavelength or a wavelength range in the near infra-red range.
14 . The method according to claim 1 , wherein the sample is obtained from a dietary supplement comprising myrosinase, wasabi (e.g. wasabi powder), or a cruciferous plant.
15 . The method according to claim 1 , wherein the first and second analyte assay formulations comprise: sinigrin; a glucose oxidase; 3,3′,5,5′-tetramethylbenzidine; and gold nanoparticles.
16 . The method according to a claim 1 , wherein the method further comprises a step (d), which is selected from one or more of the following: (di) retaining or disposing of a batch from which the sample was drawn based on a level of freshness of the batch based on the concentration of the active myrosinase in the sample; and (dii) labelling a batch from which the sample was drawn with a specific level or amount of active myrosinase.
17 . A formulation for use in a method of quantitatively determining the concentration of active myrosinase in a sample, the formulation comprising: a substrate for active myrosinase that provides glucose as a reaction product; a glucose oxidase; either a near infra-red absorbing and photothermally-responsive compound capable of forming a charge transfer complex or nanoparticle aggregates; and either a peroxidase or nanoparticles having peroxidase-like activity.
18 . The formulation according to claim 17 , wherein the substrate for active myrosinase that provides glucose as a reaction product is a glucosinolate.
19 . The formulation according to claim 17 , wherein the near infra-red absorbing and photothermally-responsive compound capable of forming a charge transfer complex or nanoparticle aggregates is selected from phenylboronate decorated gold nanoparticles, or one or more of the group selected from perylene, F4TCNQ, tetracyanoquinodimethane (TCNQ), and 3,3′,5,5′-tetramethylbenzidine.
20 .- 21 . (canceled)

Description

FIELD OF INVENTION The present disclosure relates to methods of sensing of myrosinase, and more particularly relates to methods of quantitatively determining the concentration of active myrosinase in a sample. BACKGROUND The listing or discussion of a prior-published document in this specification should not necessarily be taken as an acknowledgement that the document is part of the state of the art or is common general knowledge. Myrosinase (Myr), a member of the glycoside hydrolase family produced in commonly consumed cruciferous vegetables such as broccoli, cauliflower, watercress, Brussels sprouts, and cabbage, can catalyze the hydrolysis of inactive glucosinolates (GLs) into various compounds. Among the hydrolyzed product, isothiocyanates (ITCs) possess powerful chemopreventive effects, including anti-inflammatory, antioxidant, and antitumor effects. Studies have shown that increased ingestion of cruciferous vegetables can reduce the incidence of various diseases, which reveal the chemopreventive effects of GLs-Myr-ITCs system from dietary intake. However, Myr is significantly inactivated through vegetable storage, transporting and cooking process. Owing to merits of diet therapy and vulnerability of enzyme, the detection and profiling of Myr to better utilize chemoprevention through daily consumption are significantly important. The analysis of Myr by measuring the substrate (GLs) consumption or specific product generation such as glucose (GO) have been reported. The classical techniques mainly include spectrophotometric assay (UV), high-performance liquid chromatography (HPLC), gas chromatography-mass spectrometry (GC-MS), and pH-salt assay. However, these methods also have some limitations in practical applications owing to their complex, time-consuming protein purification steps. There are also some revised methods for detecting Myr, such as immunological methods and on-gel detection, to avoid tedious purification steps. Nonetheless, some drawbacks persist, such as immunological methods that may not be capable of recognizing denatured Myr with no nutritive value, and on-gel detection which requires extensive gel preparations, which is in turn time-consuming, labor-intensive, and not suitable for onsite Myr testing. As such, despite the promising nutrition-activating potential of Myr derived from cruciferous vegetables, the monitoring and profiling of Myr in food inspection and nutrition evaluation is restricted due to the lack of suitable analysis methods. A simple, fast, effective, visualized myrosinase profiling method is still unavailable. Therefore, there exists a need to develop a sensing technology with intelligent display and comparison for onsite screening in food industry. SUMMARY OF INVENTION It has been surprisingly found that a simple method can be used to determine the level of myrosinase in a sample-whether from a vegetable or from a dietary supplement or other source. Aspects and embodiments of the invention are provided in the following numbered clauses. 1. A method of quantitatively determining the concentration of active myrosinase in a sample, the method comprising the steps of:(a) adding a portion of the sample comprising active myrosinase to a first analyte assay formulation for a first period of time to generate an analyte sample, the first analyte assay formulation comprising: a substrate for active myrosinase that provides glucose as a reaction product;a glucose oxidase;either a near infra-red absorbing and photothermally-responsive compound capable of forming a charge transfer complex or nanoparticle aggregates; andeither a peroxidase or nanoparticles having peroxidase-like activity; (b) adding a portion of the sample comprising inactivated myrosinase to a second analyte assay formulation for a second period of time to generate a analyte reference sample, the second analyte assay formulation being the same as the first analyte assay formulation and the second period of time being the same as the first period of time;(c) determining the concentration of the active myrosinase in the sample by measuring one or more of: (i) Red Green Blue colour values in the analyte sample and Red Green Blue colour values in the analyte reference sample, calculating the Red/Blue ratio for each of the analyte sample and the analyte reference sample and calculating the difference between the Red/Blue ratios to provide a sample Red/Blue ratio and comparing the sample Red/Blue ratio obtained to a pre-determined calibration curve of active myrosinase concentration based on Red/Blue ratios obtained from known concentrations of active myrosinase;(ii) an infra-red image of the analyte sample and an infra-red image of the analyte reference sample obtained following irradiation of the analyte sample and the analyte reference same by a laser for a third period of time and calculating the temperature difference between a temperature obtained from the infra-red image of the analyte