Smart NIR II photo-thermal and colorimetric sensing techniques

Abstract

The invention relates to a method for quantitatively determining the concentration of active myrosinase in a sample. The method comprises the steps of (a) adding a portion of a sample comprising active myrosinase to a first analyte assay formulation for a first period of time to produce an analyte sample, (b) adding a portion of a sample comprising inactivated myrosinase to a second analyte assay formulation for a second period of time to produce an analyte reference sample, and (c) determining the concentration of active myrosinase in the sample. In an embodiment, the first analyte assay formulation includes a near-infrared absorbing and photo-thermal responsive compound. The invention also relates to a formulation for use in a method for the quantitative determination of the concentration of active myrosinase in a sample, as well as to a kit suitable for providing the above formulation.

Description

Technical Field

[0001] The present disclosure relates to methods of sensing myrosinase, and more particularly, to methods of quantifying the concentration of active myrosinase in a sample. Background Art

[0002] The listing or discussion of a previously published document in this specification should not be considered as an admission that the document is part of the prior art or is common general knowledge.

[0003] Myrosinase (Myr) is a member of the glycoside hydrolase family produced in commonly consumed cruciferous vegetables (such as broccoli, cauliflower, watercress, Brussels sprouts and cabbage), which can catalyze the hydrolysis of inactive glucosinolates (GL) into various compounds. Among the hydrolysates, isothiocyanates (ITCs) have powerful chemopreventive effects, including anti-inflammatory, antioxidant and anti-tumor effects. Studies have shown that increasing the intake of cruciferous vegetables can reduce the incidence of various diseases, which reveals the chemopreventive effect of the GLs-Myr-ITC system from the perspective of dietary intake. However, Myr is significantly inactivated by the storage, transportation and cooking processes of vegetables. Due to the advantages of dietary therapy and the vulnerability of the enzyme, it is very important to detect and profiling Myr to better utilize chemoprevention through daily consumption.

[0004] It has been reported that Myr is analyzed by measuring substrate (GL) consumption or the generation of specific products such as glucose (GO). Typical techniques mainly include spectrophotometry (UV), high performance liquid chromatography (HPLC), gas chromatography-mass spectrometry (GC-MS) and pH-salt determination. However, these methods also have some limitations in practical applications due to their complex and time-consuming protein purification steps. There are also some improved methods for detecting Myr, such as immunological methods and on-gel detection, to avoid tedious purification steps. Nevertheless, some disadvantages still exist, such as immunological methods may not be able to identify denatured Myr with no nutritional value, and on-gel detection requires a large amount of gel preparation, which is time-consuming and labor-intensive and is not suitable for on-site Myr testing. Therefore, although Myr from cruciferous vegetables has potential nutritional activation effects, the monitoring and profiling of Myr in food testing and nutritional evaluation is limited due to the lack of suitable analytical methods. There is currently no simple, rapid, effective and intuitive method for the analysis of black mustard enzyme profiles.

[0005] Therefore, there is a need to develop a sensing technology with intelligent display and comparison and use it for on-site screening in the food industry. Summary of the invention

[0006] Surprisingly it has been found that a simple method can be used to determine the level of myrosinase in a sample - whether the sample is from a vegetable or from a dietary supplement or other source. Aspects and embodiments of the invention are provided in the following numbered clauses.

[0007] 1. A method for quantitatively determining the concentration of active myrosinase in a sample, the method comprising the following steps:

[0008] (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:

[0009] providing glucose as a substrate for the active myrosinase enzyme as a reaction product;

[0010] Glucose oxidase;

[0011] Near-infrared absorbing and photothermal responsive compounds capable of forming charge transfer complexes or nanoparticle aggregates; and

[0012] Peroxidase or nanoparticles with peroxidase-like activity;

[0013] (b) adding a portion of the sample comprising inactivated myrosinase to a second analyte assay formulation for a second period of time to generate an analyte reference sample, the second analyte assay formulation being identical to the first analyte assay formulation and the second period of time being identical to the first period of time;

[0014] (c) determining the concentration of said active myrosinase in said sample by measuring one or more of the following:

[0015] (i) determining the red, green and blue color values ​​of the analyte sample and the red, green and blue color values ​​of the analyte reference sample, calculating the red / blue ratio of each of the analyte sample and the analyte reference sample, calculating the difference between the red / blue ratios to provide a sample red / blue ratio, and comparing the obtained sample red / blue ratio to a predetermined calibration curve of active myrosinase concentration based on the red / blue ratios obtained from active myrosinase of known concentrations;

[0016] (ii) obtaining an infrared image of the analyte sample and an infrared image of the analyte reference sample after irradiating the analyte sample and the analyte reference sample with laser for a third period of time, and calculating a temperature difference between a temperature obtained from the infrared image of the analyte sample and a temperature obtained from the infrared image of the analyte reference sample, and comparing the obtained difference value with a predetermined calibration curve of active myrosinase concentration based on temperatures obtained from infrared images of active myrosinase of known concentrations;

[0017] (iii) an absorbance spectrum of the analyte sample and an absorbance spectrum of the analyte reference sample, and calculating a difference between the absorbance spectrum of the analyte sample and the absorbance spectrum of the analyte reference sample, and comparing the obtained difference with a predetermined calibration curve of active myrosinase concentration based on the absorbance spectra of active myrosinase of known concentrations; and

[0018] (iv) obtaining temperature signals of the analyte sample and the analyte reference sample after irradiating the analyte sample and the analyte reference sample with laser for a third period of time, calculating a difference between the temperature signal of the analyte sample and the temperature signal of the analyte reference sample, and comparing the obtained difference value with a predetermined calibration curve of active myrosinase concentration based on temperature signals of active myrosinase with known concentrations.

[0019] 2. The method according to clause 1, wherein two or more of (i) to (iv) are used to determine the concentration of active myrosinase in the sample.

[0020] 3. The method according to clause 1 or clause 2, wherein the substrate for the active myrosinase providing glucose as a reaction product is a glucosinolate, optionally wherein the glucosinolate is selected from the group consisting of glucotropaeolin, gluconasturtiin, glucoraphanin and sinigrin.

[0021] 4. The method according to any one of the preceding claims, wherein the near-infrared absorption and photothermal response compound capable of forming a charge transfer complex or nanoparticle aggregate is selected from phenyl borate-modified gold nanoparticles, or one or more selected from the group consisting of perylene, F4TCNQ, tetracyanoquinodimethane (TCNQ) and 3,3',5,5'-tetramethylbenzidine, optionally wherein the near-infrared absorption and photothermal response compound capable of forming a charge transfer complex or nanoparticle aggregate is 3,3',5,5'-tetramethylbenzidine.

[0022] 5. The method according to any of the preceding clauses, wherein the nanoparticles having peroxidase-like activity are selected from silver, or more particularly, gold nanoparticles, optionally wherein the gold nanoparticles have one or more of the following properties:

[0023] (ai) absorption peak at about 527 nm;

[0024] (aii) an average diameter size of about 13 nm as determined by transmission electron microscopy;

[0025] (aiii) a zeta potential in aqueous solution of +20 to +30 mV, such as about +24.5 mV; and

[0026] (aiv) at about 1650cm -1 and about 3450cm -1 Infrared absorption peak at.

[0027] 6. A method according to any of the preceding clauses, wherein one or more of the following applies:

[0028] (bi) when the nanoparticles having peroxidase-like activity or the peroxidase are gold nanoparticles, they are present in the first analyte assay formulation and the second analyte assay formulation at a concentration of 0.02 to 0.8 nM, such as about 0.45 nM;

[0029] (bii) when the near infrared absorbing and photothermal responsive compound capable of forming a charge transfer complex or nanoparticle aggregate is 3,3',5,5'-tetramethylbenzidine, it is present in the first analyte assay formulation and the second analyte assay formulation at a concentration of 0.5 to 3 mM, such as about 1.5 mM;

[0030] (biii) the glucose oxidase is present in an amount of 5 U / mL to 25 U / mL;

[0031] (biv) the substrate for active myrosinase providing glucose as a reaction product is present in an amount of 0.05 mM to 0.75 mM.

[0032] 7. A method according to any of the preceding clauses, wherein the first analyte assay formulation and the second analyte assay formulation further comprise acetate buffer in an amount to provide a pH of 3.5 to 7.5, such as about 4.5.

[0033] 8. The method according to any of the preceding clauses, wherein the sample comprising active myrosinase and the sample comprising inactivated myrosinase are provided in a concentration of 1 to 20 mg / mL, such as about 10 mg / mL.

[0034] 9. The method according to any of the preceding clauses, wherein the sample comprising inactivated myrosinase is obtained by heating the sample comprising active myrosinase to a temperature suitable for denaturing the active myrosinase for a fourth period of time, optionally wherein one or both of the following apply:

[0035] The temperature is 80 to 120°C, such as about 100°C; and

[0036] The fourth period of time is 30 minutes to 2 hours, such as about 1 hour.

[0037] 10. A method according to any of the preceding clauses, wherein the sample comprising active myrosinase is incubated for a fifth period at a suitable temperature prior to use in the method, optionally wherein one or both of the following apply:

[0038] The temperature is 15 to 30°C, such as about 25°C; and

[0039] The fifth period of time is from 10 minutes to 1 hour, such as about 30 minutes.

[0040] 11. A method according to any of the preceding clauses, wherein when one or both of step (ii) and (iv) in clause 1 is used to determine the concentration of the active myrosinase in the sample, the laser is a near infrared laser, optionally wherein one or more of the following apply:

[0041] The laser provides a beam having a wavelength of 1000 to 1500 nm, such as about 1064 nm;

[0042] The laser has a power of 0.5 to 3 W / cm 2 , such as about 1 W / cm 2 power density; and

[0043] The third period of time is from 20 seconds to 5 minutes.

[0044] 12. A method according to any of the preceding clauses, wherein the first period of time and the second period of time are from 10 minutes to 1 hour, such as 30 minutes.

[0045] 13. A method according to any of the preceding clauses, wherein the absorption spectra of the analyte sample and the analyte reference sample are based on their absorbance at a specified wavelength or wavelength range in the near infrared range, optionally wherein the specified wavelength is a wavelength selected from 1,000 to 1,500 nm, such as 1064 nm.

[0046] 14. The method according to any of the preceding clauses, wherein the sample is obtained from a dietary supplement comprising myrosinase, wasabi (e.g. wasabi powder) or a cruciferous vegetable, optionally wherein the cruciferous vegetable is selected from broccoli, cauliflower, cabbage and Chinese cabbage.

[0047] 15. The method of any of the preceding clauses, wherein the first analyte assay preparation and the second analyte assay preparation comprise:

[0048] sinigrin;

[0049] Glucose oxidase;

[0050] 3,3',5,5'-Tetramethylbenzidine; and

[0051] Gold nanoparticles.

[0052] 16. The method according to any of the preceding clauses, wherein the method further comprises step (d), wherein step (d) is selected from one or more of the following:

[0053] (di) retaining or disposing of said batch based on the level of freshness of the batch from which said sample was drawn, said level of freshness of said batch being based on the concentration of said active myrosinase in said sample; and

[0054] (dii) marking the batch from which the sample was drawn with a specific level or amount of active myrosinase.

[0055] 17. A formulation for use in a method for quantitatively determining the concentration of active myrosinase in a sample, the formulation comprising:

[0056] providing glucose as a substrate for the active myrosinase enzyme as a reaction product;

[0057] Glucose oxidase;

[0058] Near-infrared absorbing and photothermal responsive compounds capable of forming charge transfer complexes or nanoparticle aggregates; and

[0059] Peroxidase or nanoparticles having peroxidase-like activity.

[0060] 18. A formulation according to clause 17, wherein the substrate for the active myrosinase providing glucose as a reaction product is a glucosinolate, optionally wherein the glucosinolate is selected from the group consisting of glucosinolate, natronigin, glucoraphanin and mesonaside.

[0061] 19. A formulation according to clause 17 or clause 18, wherein the near-infrared absorption and photothermal response compound capable of forming a charge transfer complex or nanoparticle aggregate is selected from phenyl borate-modified gold nanoparticles, or one or more selected from the group consisting of perylene, F4TCNQ, tetracyanoquinodimethane (TCNQ) and 3,3',5,5'-tetramethylbenzidine, optionally wherein the near-infrared absorption and photothermal response compound capable of forming a charge transfer complex or nanoparticle aggregate is 3,3',5,5'-tetramethylbenzidine.

[0062] 20. The formulation according to any one of claims 17 to 19, wherein the formulation comprises:

[0063] sinigrin;

[0064] Glucose oxidase;

[0065] 3,3',5,5'-Tetramethylbenzidine; and

[0066] Gold nanoparticles.

[0067] 21. A formulation according to any one of clauses 17 to 20, wherein the formulation further comprises an acetate buffer capable of generating an aqueous solution having a pH of 3.5 to 7.5, such as about 4.5.

[0068] 22. A kit of parts suitable for providing a formulation as described in any one of clauses 17 to 21, wherein the kit of parts comprises:

[0069] providing glucose as a substrate for an active myrosinase of the reaction product, optionally wherein the substrate for an active myrosinase of the reaction product providing glucose as a substrate is sinigrin;

[0070] Glucose oxidase;

[0071] Near-infrared absorbing and photothermal responsive compounds capable of forming charge transfer complexes or nanoparticle aggregates; and

[0072] Peroxidase or nanoparticles having peroxidase-like activity.

[0073] 23. The kit according to clause 22, wherein the kit comprises:

[0074] sinigrin;

[0075] Glucose oxidase;

[0076] 3,3',5,5'-Tetramethylbenzidine; and

[0077] Gold nanoparticles.

[0078] 24. A kit according to clause 22 or clause 23, wherein the kit further comprises an acetate buffer capable of generating an aqueous solution having a pH of 3.5 to 7.5, such as about 4.5. BRIEF DESCRIPTION OF THE DRAWINGS

[0079] Figure 1 Schematic diagram of fast and simple NIR-II photothermal response on-site Myr detection readout via smartphone.

[0080] Figure 2 The optimization of AuNP preparation time is depicted. (A) The system (TMB+H 2 O 2 + AuNP), and (B) absorbance at 1064 nm vs. preparation time of AuNP.

[0081] Figure 3 Depicted are (A) the size distribution of synthesized gold nanoparticles (AuNPs), the upper right inset is a transmission electron microscopy (TEM) image of AuNPs, and (B) the size distribution of AuNPs used for H 2 O 2 UV-vis-NIR spectra and colorimetric images of the detection, (C) Abs1064 nm at different pH values. The lower left inset is a TEM image of the charge transfer complex (CTC) at pH 4.5, and the upper right inset is a TEM image at pH 8.0, and (D) H 2 O 2 Detected temperature (ΔTemp) signal and infrared thermal image. The power density of 1064nm laser is 1W / cm 2 :(i) 3,3',5,5'-tetramethylbenzidine (TMB) only; (ii) TMB+AuNP; (iii) TMB+H 2 O 2 ; (iv) H 2 O 2 + AuNP; and (v) TMB + H 2 O 2 +AuNP;H 2 O 2 The final concentrations of , TMB and AuNPs were 0.25 mM, 1.5 mM and 0.45 nM, respectively.

[0082] Figure 4 The obtained absorption spectra of AuNPs are depicted.

[0083] Figure 5Depicted are (A) Fourier transform infrared (FTIR) spectra of the obtained AuNPs, and (B) zeta potential of the obtained AuNPs.

[0084] Figure 6 Depicted are (A) CTC formation and (B) TMB+H 2 O 2 +AuNP system absorption spectrum.

[0085] Figure 7 The optimization of AuNP concentration is described. (A) The system (TMB+H 2 O 2 + AuNP), and (B) absorbance at 1064 nm vs. c(AuNP). The upper left inset is a colorimetric image.

[0086] Figure 8 Optimization of TMB concentration is depicted. (A) UV-vis-NIR spectra, and (B) absorbance at 1064 nm at different TMB concentrations.

[0087] Fig. 9 Optimization of working temperature is depicted. (A) Effect of temperature on UV-vis-NIR spectra, and (B) Abs1064nm vs. temperature.

[0088] Fig.10 Depicted are (A) the concentrations of H 2 O 2 In the presence of TMB+H 2 O 2 + AuNP system, and (B) Abs1064 nm and c(H 2 O 2 ); the inset shows the relationship between H 2 O 2 Linear calibration plot of the assay.

[0089] Fig.11 Depicts TMB+H 2 O 2 (A) Absorption spectra and (B) Abs1064 nm vs. c(HRP) of the system. The inset is the linear relationship between Abs1064 nm and c(HRP).

[0090] Fig.12The optimization of the working conditions of TMB concentration, pH and temperature is depicted. (A) UV-Vis-NIR spectra and (B) absorbance at 1064nm at different TMB concentrations; (C) absorption spectra at different pH values ​​and (D) Abs1064nm at different pH values. The upper right inset is the system at pH 4.5 (TMB+H 2 O 2 + AuNPs), and the lower left inset is the TEM image at pH 8.0. (E) Effect of temperature on UV-Vis-NIR spectra and (F) Abs1064 nm vs. temperature.

[0091] Fig.13 Depicted are (A) UV-Vis-NIR spectra and colorimetric images, and (B) ΔTemp signals and infrared thermographs for Myr detection; (i) Sin+Myr+GOx+TMB, (ii) Sin+Myr TMB+AuNP, (iii) Sin+GOx+TMB+AuNP, (iv) Myr+GOx+TMB+AuNP, (v) Sin+Myr+GOx+AuNP, and (vi) Sin+Myr+GOx+TMB+AuNP. The final concentrations of Sin, Myr, GOx, TMB, and AuNP were 0.75 mM, 0.75 mg / mL, 0.5 mg / mL, 1.5 mM, and 0.45 nM, respectively. The power density of the 1064 nm laser was 1 W / cm 2 , (C) has Cu 2+ and ethylenediaminetetraacetic acid (EDTA) at 1064 nm; the inset shows the colorimetric image, and (D) the ΔTemp signal after being irradiated by a 1064 nm laser at 1 W / cm2 for 5 min; the inset is an infrared thermograph.

[0092] Fig.14 Depicted with Cu 2+ Absorption spectra of the Myr test system with EDTA.

[0093] Fig.15 Depicted are (A) UV-Vis-NIR spectra and colorimetric images and (B) temperature signals (ΔTemp) and infrared thermographs for GO detection (i) GO+GOx+TMB, (ii) GO+TMB+AuNP, (iii) GO+GOx+TMB+AuNP, and the concentrations of GO, GOx, TMB, and AuNP are 0.45 mM, 0.5 mg / mL, 1.5 mM, and 0.45 nM, respectively.

[0094] Fig.16Depicted are (A) the absorption spectra of the Sin+Myr+GOx+TMB+AuNP system with different Sin concentrations; the upper right inset is the corresponding colorimetric image, and (B) the relationship between Abs1064 nm and c(Sin). The final concentrations of Myr, GOx, TMB, and AuNPs were 0.5 mg / mL, 0.5 mg / mL, 1.5 mM, and 0.45 nM, respectively.

[0095] Fig.17 Depicted are (A) the absorption spectra of Myr at different concentrations; the corresponding colorimetric image is shown in the upper right, (B) the linear relationship between Abs1064 nm and Myr enzyme in the range of 0-172.5 mU / mL (i), and the relationship between temperature and the logarithm of Myr enzyme concentration (ii), (C) the absorbance of Myr and various interfering substances at 1064 nm; the inset shows the colorimetric image, and (D) the absorbance of Myr and various interfering substances at 1064 nm by 1064 nm laser at 1 W / cm 2 ΔTemp signal and infrared thermal image after 5 minutes of irradiation.

[0096] Fig.18 The graphs of Abs1064nm and c(Myr) are depicted. The linear equation of the fit is Abs 1064nm =4.39×10 -4 ×Myr(mU / mL)+0.0298,R 2 =0.99616, and the limit of detection (LOD) was estimated to be 2.96 mU / mL according to 3σ / s, where σ represents the standard deviation obtained from the blank sample and s is the slope of the calibration curve.

[0097] Fig.19 Depicted are (A) the concentrations of H 2 O 2 In the presence of TMB+H 2 O 2 + AuNP system, and (B) Abs1064 nm and c(H 2 O 2 ); the inset shows the relationship between H 2 O 2 Linear calibration plot of the assay.

[0098] Fig. 20 Depicted are (A) the absorption spectra of GO+GOx+TMB+AuNP system with increasing c(GO), and (B) the relationship between Abs1064 nm and c(GO); the inset shows the linear calibration plot for GO detection.

[0099] Fig.21The effects of amino acids (1 mM), small molecules (1 mM), and metal ions (1 mM) on Myr detection are depicted; c(Myr) is 0.75 mg / mL.

[0100] Fig. 22 The effects of amino acids (1 mM), small molecules (1 mM), and metal ions (1 mM) on Myr detection are depicted, with c(Myr) being 0.5 mg / mL.

[0101] Fig.23 Depicted are (A) the temperature response with increasing Myr concentration over 5 min, and (B) the temperature response at different times at a power of 1 W / cm 2 The corresponding infrared thermal images under 1064nm laser irradiation, and (C) thermal cycling stability of Sin+Myr+GOx+TMB+AuNP system.

[0102] Fig.24 Depicted are (A) a graph of B / R vs. c(Myr) in the range of 0-345 mU / mL, (B) presenting the linear relationship of B / R values ​​to Myr concentrations from 0 to 86.25 mU / mL, and (C) a linear calibration graph of B / R values ​​to the concentration of Myr in the range of 86.25-345 mU / mL.

[0103] Fig.25 (A) ΔAbs spectra and (B) temperature responses of various vegetables over a 5-min period are depicted. Carrot, lettuce, pakchoi, Chinese cabbage, cauliflower, green cabbage, and broccoli are represented by numbers i, ii, iii, iv, v, vi, and vii, respectively; the concentration of all vegetables is 10 mg / mL.

[0104] Fig.26 Depicted are (A) the ΔAbs of some vegetables at 1064 nm; the inset shows the colorimetric image, (B) the colorimetric images of several vegetables exposed to 1064 nm laser at 1 W / cm 2 Temperature after 5 min of irradiation; the inset is an infrared thermograph, (C) a diagram of the determination of Myr enzyme by the calculator, and (D) the B / R value and Myr concentration in the actual vegetable samples after processing by the calculator. Carrot, lettuce, Chinese cabbage, Chinese cabbage, cauliflower, green cabbage, and broccoli are represented by numbers i, ii, iii, iv, v, vi, and vii, respectively.

[0105] Fig. 27 Depicted are (A) absorption spectra and (B) Abs1064nm and temperature changes of 5 mg / mL broccoli and cabbage. Numbers i and ii represent broccoli and cabbage, respectively.

[0106] Fig.28Depicted are (A) the Δ absorbance of some types of vegetables at 1064 nm; the inset shows the colorimetric image, and (B) the absorption of several vegetables at 1064 nm laser at 1 W / cm 2 ΔTemp after 5 min of irradiation, the inset is the infrared thermal image. Carrot, lettuce, Chinese cabbage, Chinese cabbage, cauliflower, green cabbage, and broccoli are represented by numbers i, ii, iii, iv, v, vi, and vii, respectively.

[0107] Fig.29 A flow chart depicting image analysis and processing for the Calculator website.

[0108] Fig.30 Screenshots and diagrams depicting the developed "Calculator" interface. (A) Diagram of the "Calculator" user interface, (B) Screenshot of the initial "Calculator" interface, (C) Screenshot of "Calculator" image acquisition, and (D) Screenshot of "Calculator" data processing.

[0109] Fig.31 Depicted are (A) B / R vs. c(Myr) in the range of 0-345 mU / mL, (B) presenting the linear relationship between B / R values ​​and Myr concentrations from 0 to 86.25 mU / mL, (C) a linear calibration graph of B / R values ​​versus Myr concentrations in the range of 86.25-345 mU / mL, and (D) a graph of B / R values ​​vs. c(Myr) in actual vegetable samples after processing by the "calculator" website. The concentration of all vegetables was 10 mg / mL.

[0110] Fig.32 Depicted are (A) absorption spectra and (B) Abs1064 nm and temperature changes of wasabi powder (1 mg / mL) and dietary supplement (10 mg / mL). (C) Screenshot of Myr detection using a smartphone and the corresponding results in (D). Numbers i and ii represent dietary supplement (10 mg / mL) and wasabi powder (10 mg / mL), respectively.

[0111] Fig.33 A general EV extraction method is depicted.

[0112] Fig.34 The characteristics of the obtained EVs are depicted.

[0113] Fig.35 Depicted is the synthesis of engineered hybrid vesicles containing Myr.

[0114] Fig.36 Various Myr substrates for analytical and theranostic applications are depicted.

[0115] Fig.37The “homing effect” of EVs from different sources with respect to their affinity for lesions is depicted.

[0116] Fig.38 Different levels of imaging and theranostics evaluation are depicted. DETAILED DESCRIPTION

[0117] The present invention relates to a sensing technology that can accurately perform rapid on-site myrosinase analysis in real samples by using one or both of colorimetric and photothermal signals using a NIR-II absorption window, which in some cases can be accomplished using smart phone readout and smart data processing on a smart phone-based computing website. This colorimetric and temperature response-based sensing platform not only provides a new strategy for rapid qualitative and quantitative on-site myrosinase analysis (e.g., via smartphone readout), but also shows potential applications in the fields of food quality analysis and bacterial screening. This screening method does not necessarily require the use of expensive components, but can also use equipment involving near infrared II (NIR-II) lasers, cameras (IR and visible light), and computing systems to achieve rapid qualitative and quantitative on-site myrosinase analysis.

[0118] Figure 1 An example of the smart screening technology that has been developed is shown for profiling myrosinase from various substrates such as vegetables, food supplements, and bacteria. Briefly, myrosinase (Sin), myrosinase (Myr), and glucose oxidase (GOx) were first mixed for 30 min to generate H 2 O 2 , then 3,3',5,5'-tetramethylbenzidine (TMB) and gold nanoparticles (AuNP) were added and incubated at 45°C for 25min. After cooling to room temperature, the solution changed from colorless to blue due to the formation of a charge transfer complex (CTC), which has a wide absorption from the UV to NIR-II window. In addition, due to the photothermal effect of CTC after 1064nm laser irradiation, temperature signals and infrared thermographs can be obtained. Therefore, the combined detection of Myr by colorimetry and temperature can be achieved. In order to intelligently detect Myr in actual samples, a smartphone-based website named "Calculator" can be used to obtain the correlation between the blue / red (B / R) value of the colorimetric image and the Myr concentration. The Myr concentration in the actual sample can be read out via a smartphone and determined according to the formula.

[0119] Therefore, in a first aspect of the present invention, there is provided a method for quantitatively determining the concentration of active myrosinase in a sample, the method comprising the following steps:

[0120] (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:

[0121] providing glucose as a substrate for the active myrosinase enzyme as a reaction product;

[0122] Glucose oxidase;

[0123] Near-infrared absorbing and photothermal responsive compounds capable of forming charge transfer complexes or nanoparticle aggregates; and

[0124] Peroxidase or nanoparticles with peroxidase-like activity;

[0125] (b) adding a portion of the sample comprising inactivated myrosinase to a second analyte assay formulation for a second period of time to generate an analyte reference sample, the second analyte assay formulation being identical to the first analyte assay formulation and the second period of time being identical to the first period of time;

[0126] (c) determining the concentration of said active myrosinase in said sample by measuring one or more of the following:

[0127] (i) determining the red, green and blue color values ​​of the analyte sample and the red, green and blue color values ​​of the analyte reference sample, calculating the red / blue ratio of each of the analyte sample and the analyte reference sample, calculating the difference between the red / blue ratios to provide a sample red / blue ratio, and comparing the obtained sample red / blue ratio to a predetermined calibration curve of active myrosinase concentration based on the red / blue ratios obtained from active myrosinase of known concentrations;

[0128] (ii) obtaining an infrared image of the analyte sample and an infrared image of the analyte reference sample after irradiating the analyte sample and the analyte reference sample with laser for a third period of time, and calculating a temperature difference between a temperature obtained from the infrared image of the analyte sample and a temperature obtained from the infrared image of the analyte reference sample, and comparing the obtained difference value with a predetermined calibration curve of active myrosinase concentration based on temperatures obtained from infrared images of active myrosinase of known concentrations;

[0129] (iii) an absorbance spectrum of the analyte sample and an absorbance spectrum of the analyte reference sample, and calculating a difference between the absorbance spectrum of the analyte sample and the absorbance spectrum of the analyte reference sample, and comparing the obtained difference with a predetermined calibration curve of active myrosinase concentration based on the absorbance spectra of active myrosinase of known concentrations; and

[0130] (iv) obtaining temperature signals of the analyte sample and the analyte reference sample after irradiating the analyte sample and the analyte reference sample with laser for a third period of time, calculating a difference between the temperature signal of the analyte sample and the temperature signal of the analyte reference sample, and comparing the obtained difference value with a predetermined calibration curve of active myrosinase concentration based on temperature signals of active myrosinase with known concentrations.

[0131] In the embodiments herein, the word "comprising" may be interpreted as requiring the features mentioned, but not limiting the presence of other features. Alternatively, the word "comprising" may also refer to situations where it is intended that only the listed components / features exist (e.g., the word "comprising" may be replaced by the phrase "consisting of" or "consisting essentially of". It is explicitly contemplated that both broad and narrow interpretations may apply to all aspects and embodiments of the present invention. In other words, the word "comprising" and its synonyms may be preceded by the phrase "consisting of" or the phrase "consisting essentially of" or their synonyms, and vice versa.

[0132] The sample can be any suitable material containing or claiming to contain active myrosinase. Examples of such materials include, but are not limited to, dietary supplements comprising myrosinase, wasabi (e.g., wasabi powder), or cruciferous plants. Any suitable cruciferous plant can be tested using the methods disclosed herein. For example, the cruciferous plant can be, but is not limited to, broccoli, cauliflower, cabbage, and Chinese cabbage.

[0133] As mentioned herein, the method utilizes two samples derived from the same source material. One of the samples (i.e., the analyte sample) is used as is to maintain the activity of the myrosinase present in the sample. The other sample (i.e., the analyte reference sample) is obtained by inactivating the myrosinase by some form of denaturation. For example, a sample comprising an inactivated myrosinase can be obtained by heating a sample comprising an active myrosinase to a temperature suitable for denaturing the active myrosinase for a period of time (e.g., 30 minutes to 2 hours, e.g., about 1 hour). Suitable temperatures may include, but are not limited to, temperatures of 80 to 120° C., such as about 100° C.

[0134] As the selected samples may contain endogenous amounts of glucose, the analyte reference sample is used to determine the "background" level of glucose in the sample so that this does not affect the determination of the actual amount of myrosinase in the sample.

[0135] As mentioned herein, the method utilizes a first analyte determination formulation and a second analyte determination formulation. These formulations are substantially identical, the only difference being that one is added to a sample containing active myrosinase and the other is added to a sample having inactivated myrosinase. Both formulations include:

[0136] Providing glucose as a substrate for the active myrosinase enzyme as a reaction product

[0137] Glucose oxidase;

[0138] Near-infrared absorbing and photothermal responsive compounds capable of forming charge transfer complexes or nanoparticle aggregates; and

[0139] Peroxidase or nanoparticles having peroxidase-like activity.

[0140] Any substrate that can be provided to active myrosinase to provide glucose can be used herein. For example, the substrate can be a glucosinolate. Any suitable glucosinolate can be used herein. Examples of suitable glucosinolates include, but are not limited to, glucotropaeolin, gluconasturtiin, glucoraphanin, and mesona glycoside. In specific embodiments of the invention that may be mentioned herein, the glucosinolate can be mesona glycoside.

[0141] Near infrared absorption and photothermal response compounds capable of forming charge transfer complexes or nanoparticle aggregates can be any suitable material with these characteristics. Suitable nanoparticle aggregates can be gold nanoparticles modified by phenylboronic acid esters. Other suitable nanoparticles can be modified with thioketal, thioester or other boric acid esters. These materials are sensitive to active oxygen, and therefore will be cut into monomers by active oxygen (e.g. peroxide), causing the nanoparticle aggregates to lose structural integrity, and thereby providing photothermal response.

[0142] Suitable charge transfer complexes can be selected from, but not limited to, perylene, F4TCNQ, tetracyanoquinodimethane (TCNQ), 3,3',5,5'-tetramethylbenzidine (TMB) and combinations thereof (e.g., TMB-F4TCNQ and TMB-TCNQ). In a particular embodiment of the present invention, the near-infrared absorbing and photothermal responsive compound capable of forming a charge transfer complex or nanoparticle aggregate can be 3,3',5,5'-tetramethylbenzidine.

[0143] TCNQ has the following structure:

[0144]

[0145] F4TCNQ has the following structure:

[0146]

[0147] When peroxidase is present, any suitable peroxidase may be used. An example of a suitable peroxidase is horseradish peroxidase.

[0148] When nanoparticles with peroxidase-like activity are present, these nanoparticles may be silver, or more particularly, may be gold nanoparticles. Such silver and gold nanoparticles may be positively charged nanoparticles. In a particular embodiment of the invention, the gold nanoparticles may have one or more of the following properties:

[0149] (ai) absorption peak at about 527 nm;

[0150] (aii) an average diameter size of about 13 nm as determined by transmission electron microscopy;

[0151] (aiii) a zeta potential in aqueous solution of +20 to +30 mV, such as about +24.5 mV; and

[0152] (aiv) at about 1650cm -1 and about 3450cm -1 Silver nanoparticles with similar properties can also be used.

[0153] In embodiments of the invention where the nanoparticles or peroxidases having peroxidase-like activity are gold nanoparticles, they are present in the first analyte assay formulation and the second analyte assay formulation at a concentration of 0.02 to 0.8 nM, such as about 0.45 nM. Similar concentrations may be used for silver nanoparticles.

[0154] In embodiments of the invention where the near infrared absorbing and photothermally responsive compound capable of forming charge transfer complexes or nanoparticle aggregates is 3,3',5,5'-tetramethylbenzidine, it is present in the first analyte assay formulation and the second analyte assay formulation at a concentration of 0.5 to 3 mM, such as about 1.5 mM.

[0155] Glucose oxidase can be present in any suitable concentration in the first analyte assay formulation and the second analyte assay formulation. For example, glucose oxidase can be present in the first analyte assay formulation and the second analyte assay formulation in an amount of 5 U / mL to 25 U / mL.

[0156] The substrate for active myrosinase that provides glucose as a reaction product can be present in any suitable amount in the first analyte assay formulation and the second analyte assay formulation. For example, the substrate for active myrosinase that provides glucose as a reaction product can be present in an amount of 0.05 mM to 0.75 mM in the first analyte assay formulation and the second analyte assay formulation.

[0157] In certain embodiments of the invention, the first analyte assay formulation and the second analyte assay formulation may further include acetate buffer in an amount to provide a pH of 3.5 to 7.5, such as about 4.5. That is, the pH of the resulting solution should be within the pH range listed.

[0158] In certain embodiments of the invention, the sample comprising active myrosinase and the sample comprising inactive myrosinase may be provided at a concentration of 1 to 20 mg / mL, such as about 10 mg / mL.

[0159] In certain embodiments of the invention, a sample comprising active myrosinase may be incubated at a suitable temperature (e.g. 15 to 30° C., such as about 25° C.) for a period of time (e.g. 10 minutes to 1 hour, such as about 30 minutes) prior to use in the method. This may allow the myrosinase to be at a suitable level of activity prior to performing the determination of myrosinase levels.

[0160] The method disclosed herein is intended to allow the determination of the concentration of active myrosinase in a sample. This method relies on a cascade of reactions to provide the final result. This cascade can be summarized as:

[0161] (I) myrosinase (from the sample) is intended to hydrolyze the corresponding substrate (e.g., myrosinase) in the analyte sample to produce glucose;

[0162] (II) Glucose oxidase converts glucose into hydrogen peroxide;

[0163] (III) Peroxidase (e.g., horseradish peroxidase) or nanoparticles with peroxidase-like activity (e.g., positively charged gold nanoparticles or silver nanoparticles) utilize hydrogen peroxide to generate a colorimetric effect on NIR-II absorbing and photothermal responsive compounds (e.g., charge transfer complexes (such as 3,3',5,5'-tetramethylbenzidine (TMB), tetracyanoquinodimethane (TCNQ), F4TCNQ, perylene or combinations thereof, such as TMB-F4TCNQ and TMB-TCNQ) or nanoparticle aggregates (e.g., phenylboronic acid ester-modified gold nanoparticles).

[0164] The resulting color / thermal shift can then be interpreted as a means of determining the concentration of myrosinase. As previously mentioned, the sample may contain endogenous glucose. Thus, the analyte reference sample is used to provide a background level of glucose in the system, which can then be used to remove the effect of endogenous glucose (i.e., glucose not generated by myrosinase) from the analyte sample, thereby allowing the concentration of (active) myrosinase in the sample to be determined.

[0165] The determination step of the method may utilize one or more of the following:

[0166] (i) determining the red, green and blue color values ​​of the analyte sample and the red, green and blue color values ​​of the analyte reference sample, calculating the red / blue ratio of each of the analyte sample and the analyte reference sample, calculating the difference between the red / blue ratios to provide a sample red / blue ratio, and comparing the obtained sample red / blue ratio to a predetermined calibration curve of active myrosinase concentration based on the red / blue ratios obtained from active myrosinase of known concentrations;

[0167] (ii) obtaining an infrared image of the analyte sample and an infrared image of the analyte reference sample after irradiating the analyte sample and the analyte reference sample with laser for a third period of time, and calculating a temperature difference between a temperature obtained from the infrared image of the analyte sample and a temperature obtained from the infrared image of the analyte reference sample, and comparing the obtained difference value with a predetermined calibration curve of active myrosinase concentration based on temperatures obtained from infrared images of active myrosinase of known concentrations;

[0168] (iii) an absorbance spectrum of the analyte sample and an absorbance spectrum of the analyte reference sample, and calculating a difference between the absorbance spectrum of the analyte sample and the absorbance spectrum of the analyte reference sample, and comparing the obtained difference with a predetermined calibration curve of active myrosinase concentration based on the absorbance spectra of active myrosinase of known concentrations; and

[0169] (iv) obtaining temperature signals of the analyte sample and the analyte reference sample after irradiating the analyte sample and the analyte reference sample with laser for a third period of time, calculating a difference between the temperature signal of the analyte sample and the temperature signal of the analyte reference sample, and comparing the obtained difference value with a predetermined calibration curve of active myrosinase concentration based on temperature signals of active myrosinase with known concentrations.

[0170] For (i), the same red-green-blue (RGB) color value determination can be performed for a series of known concentrations of myrosinase to generate a red / blue ratio for each known concentration. These reference samples can then be used to establish relationships and calculation formulas to essentially provide a suitable calibration curve. Once the calibration curve is obtained, the analyte sample and the analyte reference sample are tested and the difference calculation is performed to provide the actual blue / red ratio. The formula used is:

[0171]

[0172] r 2 =0.9497

[0173] B refers to the blue value among the red, green, and blue (RGB) color values ​​of the obtained image. R refers to the red value among the red, green, and blue (RGB) color values ​​of the obtained image. (B / R) 2 Is the blue / red ratio squared. (B / R)3 is the cube of the blue / red ratio.

[0174] It is important to note that (i) relies solely on colorimetric sensing and therefore requires only a smartphone with a camera and access to an online calculator. This avoids the need for more expensive commercial equipment and allows for direct readout with intelligent assisted data processing (as explained in more detail in the Examples section below).

[0175] For (ii), a similar approach is used, but based on the temperature of the sample after laser excitation.

[0176] For (iii) and (iv), the difference can be obtained more directly from the absorption spectrum and the temperature signal, respectively.

[0177] The results of the measurement processes (i) to (iv) (eg, one or both of (i) and (ii)) can be compared with the true value based on the least squares method and verified with good repeatability.

[0178] It will be appreciated that two or more of the determination processes (i) to (iv) may be used to more accurately determine the concentration of active myrosinase in a sample. This may be achieved by any suitable means.

[0179] In an embodiment of the invention, wherein one or both of (ii) and (iv) of step (c) are used to determine the concentration of active myrosinase in a sample, the laser may be a near-infrared laser. In such embodiments, one or more of the following may apply:

[0180] The laser provides a beam having a wavelength of 1000 to 1500 nm, such as about 1064 nm;

[0181] The laser has a power of 0.5 to 3 W / cm 2 , such as about 1 W / cm 2 power density; and

[0182] The third period is from 20 seconds to 5 minutes.

[0183] In embodiments of the invention that may be mentioned herein, the first period of time and the second period of time are from 10 minutes to 1 hour, such as 30 minutes. It will be appreciated that any suitable time may be selected, but if a different time is selected, recalibration may be required, as active myrosinase will produce more or less glucose depending on whether the new time is longer or shorter than the previous time.

[0184] In embodiments of the invention, the absorption spectra of the analyte sample and the analyte reference sample may be based on their absorption at a specified wavelength or wavelength range in the near infrared range. For example, the specified wavelength may be a wavelength selected from 1,000 to 1,500 nm, such as 1064 nm.

[0185] In certain embodiments of the invention as may be mentioned herein, the first analyte preparation and the second analyte preparation may include:

[0186] sinigrin;

[0187] Glucose oxidase;

[0188] 3,3',5,5'-Tetramethylbenzidine; and

[0189] Gold nanoparticles.

[0190] It will be appreciated that the methods disclosed herein can be used to inspect and assess products such as dietary supplements and nutraceuticals that claim to have a certain level of myrosinase (eg, certain such products may claim to have 13 mg of myrosinase per serving).

[0191] Additionally, this method can be applied to:

[0192] Measuring myrosinase in dietary supplements, nutraceuticals, semi-processed foods and nutraceuticals;

[0193] · Evaluate dietary micronutrients of various vegetables for grading and dietary nutrient intake; and

[0194] Evaluate the freshness of a specific category (e.g., a batch of specific vegetables).

[0195] Therefore, in certain embodiments of the present invention, the method may further include step (d), which is selected from one or more of the following:

[0196] (di) retaining or disposing of said batch based on the level of freshness of the batch from which said sample was drawn, said level of freshness of said batch being based on the concentration of said active myrosinase in said sample; and

[0197] (dii) marking the batch from which the sample was drawn with a specific level or amount of active myrosinase.

[0198] In a second aspect of the present invention, there is provided a preparation for use in a method for quantitatively determining the concentration of active myrosinase in a sample, the preparation comprising:

[0199] providing glucose as a substrate for the active myrosinase enzyme as a reaction product;

[0200] Glucose oxidase;

[0201] Near-infrared absorbing and photothermal responsive compounds capable of forming charge transfer complexes or nanoparticle aggregates; and

[0202] Peroxidase or nanoparticles having peroxidase-like activity.

[0203] As the formulations (which correspond to the first analyte assay formulation and the second analyte assay formulation of the first aspect of the invention) have been described above, the various possible embodiments described above will not be repeated here for the sake of brevity.

[0204] Specific formulations according to this aspect may include:

[0205] sinigrin;

[0206] Glucose oxidase;

[0207] 3,3',5,5'-Tetramethylbenzidine; and

[0208] Gold nanoparticles.

[0209] In yet another aspect of the present invention, there is provided a kit suitable for providing the formulations described herein, wherein the kit comprises:

[0210] providing glucose as a substrate for an active myrosinase of the reaction product, optionally wherein the substrate for an active myrosinase of the reaction product providing glucose as a substrate is sinigrin;

[0211] Glucose oxidase;

[0212] Near-infrared absorbing and photothermal responsive compounds capable of forming charge transfer complexes or nanoparticle aggregates; and

[0213] Peroxidase or nanoparticles with peroxidase-like activity. Since the preparation and components are described in detail above, the discussion is omitted here for the sake of brevity.

[0214] Advantages associated with the disclosed technology are summarized in the numbered clauses below.

[0215] 1. The sensing technology disclosed herein allows for a simpler, more cost-effective and less labor-intensive on-site dietary myrosinase zymogram testing procedure. Through intelligent processing and visual output, sample screening can be easily achieved with low technical input.

[0216] 2. As an example of a chromogenic substrate, TMB was selected in the following examples because it has excellent sensitivity, good stability and anti-interference. As mentioned above, it also has dual functions because it can perform colorimetry and photothermal conversion simultaneously, thereby realizing dual-channel screening.

[0217] 3. As mentioned herein, compared with corresponding natural enzymes (e.g., horseradish peroxidase, HRP), the mimetic enzyme based on nanomaterials can be used for mediating colorimetric determination with higher stability, lower cost, wider operating temperature and pH range. For example, compared with the operating pH range of 5.0 to 8.0 of natural enzymes, the operating pH range of the mimetic enzyme based on nanomaterials can be 3.5 to 9.0. In the embodiment using this mimetic enzyme, it can contribute to more beneficial on-site spectrum analysis.

[0218] 4. The absorbance of the developer (eg TMB) at 1064 nm (ie NIR-II) can significantly exclude interfering signals from impurities in plant samples, which greatly expands the application scope of the screening system.

[0219] 5. It avoids protein extraction and purification, which helps to reduce costs and greatly shorten sample preparation time.

[0220] 6. With the assistance of smartphone readout, the smartphone can intelligently process the colorimetric images to obtain their RGB values. According to the relationship function between the B / R value and the myrosinase concentration, on-site rapid detection of myrosinase can be achieved with good results.

[0221] 7. The temperature signal and infrared thermal image generated by the photothermal effect of the charge transfer complex can further improve the credibility of the detection and make the results more reliable.

[0222] 8. The visual probe supports smartphone scanning and intelligently processes the RGB ratio, resulting in fast and reliable output.

[0223] 9. This sensing technology has been successfully applied to a wide range of real samples, including food ingredients, dietary supplements, condiments, and bacteria.

[0224] Further aspects and embodiments of the invention will now be discussed with reference to the following non-limiting examples.

[0225] Example

[0226] Material

[0227] Gold(III) chloride trihydrate, HAuCl 4 ·3H 2 O), chitosan (low viscosity, <200 mPa s), myrosinase (Sin), myrosinase (Myr), horseradish peroxidase (HRP), glucose oxidase (GOx), and a commercial GO detection kit were purchased from Sigma-Aldrich. TMB, H 2 O2 (3%), acetic acid, sodium acetate, KH 2 PO 4 , KCl, Na 2 HPO 4 and NaCl were purchased from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan). BroccoMax was purchased from Jarrow Formulas (Singapore). Dualspices Japanese wasabi powder was purchased from iHerb (USA). Cruciferous vegetables (broccoli, cauliflower, cabbage, Chinese cabbage, pakchoi, etc.) and non-cruciferous vegetables (lettuce and carrots) were purchased from local supermarkets. All chemical reagents were of analytical reagent grade and used as is without further treatment. Milli-Q water was used to prepare all solutions.

[0228] Analytical techniques

[0229] Ultraviolet-visible-near-infrared (UV-vis-NIR) spectroscopy

[0230] The UV-vis-NIR absorption spectra were measured by a Perkin Elmer Lambda 950 spectrometer.

[0231] Transmission Electron Microscopy (TEM)

[0232] The microstructure of the prepared AuNPs was characterized by TEM on a FEI EM208S TEM (Philips) with an accelerating voltage of 100 kV.

[0233] Dynamic Light Scattering (DLS) Measurements and Zeta Potential

[0234] Dynamic light scattering (DLS) measurements and zeta potential were estimated on a Brookhaven 90Plus nanoparticle size analyzer.

[0235] Fourier Transform Infrared (FTIR) Spectroscopy

[0236] FTIR spectroscopy was performed on a Bruker Vertex 80v vacuum FTIR spectrometer.

[0237] Temperature changes and infrared (IR) thermal images

[0238] The temperature changes and infrared thermal images were obtained by using an IR thermal imager (FLIR).

[0239] Example 1. Fast and simple NIRII photothermal response on-site Myr detection via smartphone readout

[0240] We developed a novel sensor that combines the photothermal effect with a colorimetric signal for rapid and precise on-site Myr analysis in the NIR-II window via smartphone readout. Figure 1 ). Briefly, Sin, Myr, and GOx were first mixed for 30 min to generate H 2 O 2 , TMB and AuNP were then added and incubated at 45°C for 25min. After cooling to room temperature, the solution changed from colorless to blue due to the formation of a charge transfer complex (CTC), which has a wide absorption range from UV to NIR-II window. In addition, due to the photothermal effect of CTC after irradiation with 1064nm laser, a temperature signal and an infrared thermal image will be obtained. Thus, the joint detection of Myr by colorimetry and temperature is achieved. In addition, in order to intelligently detect Myr in actual samples, we developed a smartphone-based website called "Calculator" to obtain the correlation between the B / R value of the colorimetric image and the Myr concentration. Then, the Myr concentration in the actual sample will be determined according to the formula read out by the smartphone. Specific details are provided in the following embodiments.

[0241] Example 2. Preparation of AuNPs

[0242] AuNPs were prepared according to a previous procedure with minor modifications (Wu, L. et al., J. Phys. Chem. C 2007, 112, 319-323).

[0243] Briefly, in diluted acetic acid (V CH3COOH :V H2O =1:9) in 0.228 mL of 25.39 mM HAuCl 4 and 4 mL of 2.254 × 10 -3 g / mL chitosan was added to 21.772 mL of H 2 O flask. Then, the mixture was heated to 120 ° C under vigorous stirring, the color of the solution turned red after 5 min, and the mixture was refluxed at 120 ° C for another 25 min under vigorous stirring. The AuNP sample can be used after the solution is cooled to room temperature. In order to evaluate the effect of different preparation times on the catalytic activity of AuNPs, the mixture was allowed to continue to react for 0, 5, 35 and 55 min after the solution turned red.

[0244] Example 3. Effect of AuNP preparation time on catalytic activity

[0245] The effect of preparation time on peroxidase-like (POD-like) activity was examined to obtain AuNPs with maximum catalytic activity. Figure 2It can be seen that after 10 minutes of reaction, a low-intensity peak appears at 1064 nm, and the intensity increases with the increase of reaction time, and the intensity decreases after 30 minutes; the absorbance at 1064 nm represents the degree of oxidation from TMB to CTC and the POD-like ability of AuNP. Therefore, when the time exceeds 30 minutes, the activity of AuNP decreases due to aggregation over time at high temperature. Therefore, 30 minutes is selected as the optimal preparation time of AuNP.

[0246] Example 4. Characterization of AuNPs

[0247] After obtaining the AuNPs with the highest catalytic activity, the characterization of the AuNPs was performed.

[0248] First, the morphology and size distribution of the prepared AuNPs were characterized by TEM. Figure 3 Inset in A, the average diameter is approximately -13 nm, the zeta potential of the solution was measured to be +19.2 mV, and the positive charge on the particles facilitates their dispersion throughout the medium. Figure 4 It shows that AuNP has a strong absorption peak at 527nm, which is almost consistent with previous studies (Wang, Z. et al., Nano Res. 2019, 12, 49-55). According to the formula c = A reported by Ray et al. 450 / ε 450 (ε 450 =2.18×10 8 )(Darbha, GK et al., J. Am. Chem. Soc. 2008, 130, 8038-8043), the AuNP concentration was estimated to be 4.5 nM.

[0249] In addition, FTIR was used to obtain the absorption bands arising from surface functional groups. Figure 5 The AuNPs are depicted to have positive charges; at 1650 and 3450 cm -1 The bands at are attributed to the presence of chitosan, and they represent amino and hydroxyl groups, respectively, which are crucial for stability in aqueous solutions (Wu, L. et al., J. Phys. Chem. C 2007, 112, 319-323). These results indicate that the synthesized AuNPs have the potential to act as POD-like enzymes.

[0250] Example 5. AuNP-catalyzed TMB and H 2 O 2 System validation for NIR-II photothermal and colorimetric sensing

[0251] Commercial H 2 O 2, GO and Myr detection were used to confirm the feasibility of the proposed method.

[0252] Results and Discussion

[0253] First, various known concentrations of Myr were mixed with sinigrin (Sin) and GOx to produce a series of samples containing different amounts of H 2 O 2 After TMB and AuNPs were added to the solution, H 2 O 2 It will decompose into ·OH, and TMB will be oxidized and converted into CTC, which has blue color and broad absorption from UV to NIR-II region (Jiang, C. et al., RSC Adv. 2017, 7, 44463-44469).

[0254] To illustrate the AuNP-TMB-H 2 O 2 The NIR-II driven photothermal and colorimetric effects of the system were investigated, and the absorption spectra of the system with different components were collected, such as Figure 3 B, and only in the presence of AuNPs, H 2 O 2 In the case of MgCl and TMB, a broad absorption spectrum extending into the NIR-II region appears (line (v)) accompanied by a significant color change from colorless to blue (v in the inset). Figure 3 B and its inset, this method can successfully detect H 2 O 2 In addition, as a control, no obvious absorbance and color changes were observed in the cases without AuNPs, TMB, or other conditions (i) or (iv). 2 O 2 Oxidized TMB facilitates the assembly of long straight nanoribbon structured CTCs with a broad absorption spectrum from UV to NIR-II regions (Wang, Z. et al., Nano Res. 2019, 12, 49-55; and Wang, Z. et al., ACS Nano 2019, 13, 5816-5825), such as Figure 6 A and Figure 3 As shown in the lower left inset of C.

[0255] In addition, the formation of CTCs is sensitive to changes in pH, with the color changing from blue to green. Figure 3As shown in Figures C and 6B, as the pH increases, the absorbance at 1064 nm first increases and then decreases, with the most satisfactory results at pH 4.5, and at higher pH values ​​(e.g., pH = 8.0), the morphology changes to short nanofragments (upper right inset) due to the generation of yellow diamine, as confirmed by our previous studies (Wang, Z. et al., Nano Res. 2019, 12, 49-55; and Wang, Z. et al., ACS Nano 2019, 13, 5816-5825).

[0256] Then, due to the absorbance of CTC at 1064nm, the temperature signal and infrared thermograph were obtained, which improved the detection accuracy. 2 The sample was irradiated with a 1064 nm laser for 5 min and the temperature change was recorded using FLIR ( Figure 3 D); only when the system contains H 2 O 2 , AuNPs and TMB, the temperature increased significantly by 24.2°C, and the corresponding infrared thermograph is shown in the inset (v). On the other hand, in the other systems (i-iv) where the components were missing, the temperature did not increase significantly. The results show that this response only occurs in H 2 O 2 This occurs when both AuNPs and CTCs are present, indicating that the synthesized AuNPs have POD-like activity and that the system has the potential to target H 2 O 2 NIR-II absorption-driven photothermal and colorimetric sensing capabilities.

[0257] The POD-like activity of AuNPs was also studied by varying the concentration of AuNPs. Figure 7 A shows that when the molar concentration of AuNPs increases from 0.025 to 0.9 nM, the absorbance at 1064 nm increases, and Figure 7 The inset of B shows that the color of the solution deepens with the increase of AuNP molar concentration; in the following steps, we choose 0.45nM as the optimal concentration of AuNP. The slope of the line is 0.13134 ( Figure 7 Illustration of B).

[0258] In this system, TMB is used to generate photothermal CTCs, so optimizing the TMB concentration is crucial. Figure 8 As shown in A, the absorbance at 1064 nm increased in the concentration range of 0.5 to 1.5 mM and decreased at higher concentrations. Figure 8B shows that the yield of CTC increases with increasing substrate concentration and reaches a maximum at 1.5 mM TMB concentration. When TMB levels are excessive, slight fading is observed, which may be due to further oxidation to form light yellow oxTMB 2+ (Gao, L. et al., Nat. Nanotechnol. 2007, 2, 577-583). Therefore, in our system, 1.5 mM is the optimal TMB concentration.

[0259] Then, we also studied the effect of temperature on the activity of AuNPs. Fig. 9 AB shows that the absorbance at 1064nm increases with increasing temperature in the range of 20 to 45°C, and then decreases from 45 to 80°C; therefore, 45°C was selected for the following experiments. It is worth noting that even when heated to 80°C, AuNPs still retain peroxidase activity. This is better than the natural HRP enzyme, which loses activity once the temperature exceeds 50°C (Pardini, A. et al., Food Chem. 2021, 355, 129634). This may be due to the fact that the external polymer chitosan is more resistant to high temperatures than small molecules. After optimizing all conditions, they were first evaluated for detecting commercial H 2 O 2 ability, and Fig.10 The results in this paper show that the system can detect H 2 O 2 , with a LOD of 0.68 μM, which is comparable to previously published results (Jiang, T. et al., Chem. Commun. 2009, 15, 1972-1974).

[0260] In comparison, the LOD and slope of HRP were 0.47 nM and 0.04644 nM, respectively ( Fig.11 ). The slope represents the increment of absorption per nanomole of natural or simulated enzyme. The larger the slope value, the higher the POD-like activity.

[0261] Example 6. Optimization of conditions

[0262] Optimization of conditions

[0263] First, the concentration of TMB was optimized as follows. Different concentrations of TMB (0.5, 1, 1.5, 2, 2.5, and 3 mM) were mixed with AuNPs and H 2 O 2 Then, the AuNP-catalyzed TMB-H 2 O 2Secondly, the pH of acetate buffer also affects the formation of CTCs and the activity of AuNPs. 2 O 2 The AuNPs were added to a series of buffers with different pH values ​​(3.5, 4, 4.5, 5.5, 7.4, 8.0), and the absorption spectra of the different samples were collected after cooling to room temperature. Finally, in order to study the effect of incubation temperature, TMB, H 2 O 2 and AuNPs were added to acetate buffer, and then the mixture was incubated at 20 °C, 35 °C, 45 °C, 60 °C, and 80 °C to obtain the absorption spectra.

[0264] Results and Discussion

[0265] In the nanozyme-mediated photothermal biosensing platform, TMB is used to generate photothermal CTCs, so it is crucial to optimize the TMB concentration to obtain the best detection performance. In general, given a constant AuNP concentration (0.45 nM) and reaction time (25 min), a series of TMB concentrations ranging from 0.5 to 3 mM were evaluated. Fig.12 As shown in AB, the absorbance at 1064nm (representing the characteristic peak of CTC) increases in the concentration range of 0.5 to 1.5mM and decreases at higher concentrations. The results show that given a fixed amount of catalyst, the yield of CTC increases with increasing substrate concentration and reaches a maximum at 1.5mM TMB concentration. When TMB levels are excessive, slight fading is observed, which may be due to further oxidation to form light yellow oxTMB 2+ (Stefan, L. et al., Nucleic Acids Res. 2012, 40, 8759-8772).

[0266] The effects of pH and temperature were evaluated to determine the optimal experimental conditions. To examine the effect of pH, acetate buffers were first prepared with pH varying from 3.5 to 8.0. Fig.12 The results in CD indicate that the POD-like activity of AuNPs is pH-dependent, and the most satisfactory results are obtained at pH 4.5. In addition, according to previous literature, at pH 4.5 and pH 8.0, the blue product CTC gradually decomposes with increasing pH, and the morphology changes from long straight nanoribbons ( Fig.12 D, upper right inset) into short nanofragments ( Fig.12D) (Josephy, PD et al., J. Biol. Chem. 1982, 257, 3669-3675; and Awano, H. et al., Bull. Chem. Soc. Jpn. 1990, 63, 2101-2103), which is due to the generation of yellow imide. At the same time, the solution color turned green, which is consistent with our previous results. Then, we also studied the effect of temperature (20-80 ° C) on the activity of AuNPs, Fig.12 EF shows that the absorbance at 1064nm increases with the increase of temperature in the range of 20 to 45°C, and then decreases from 45 to 80°C. AuNPs exhibit the highest POD-like activity at 45°C, so 45°C was selected for the following experiments. It is worth noting that AuNPs retain peroxidase activity even when heated to 80°C. This is better than the natural HRP enzyme, which loses activity once the temperature exceeds 50°C (Gao, L. et al., Nat. Nanotechnol. 2007, 2, 577-583). This may be due to the fact that the external polymer chitosan is more resistant to high temperatures than small molecules.

[0267] Example 7. Photothermal and colorimetric bioassays for Myr measurements

[0268] Photothermal and colorimetric assays for Myr enzyme detection

[0269] To GO, H 2 O 2 Typical spectral photothermal and colorimetric analysis of Myr and Myr was performed as follows. First, 2 O) in 300 μL of 5 mM TMB, in ddHO 2 100 μL of 4.5 nM AuNPs in ddHO 2 100 μL of different concentrations of HO 2 O 2 It was added to 500 μL of acetate buffer (pH=4.5), and the mixture was then incubated in a 45° C. water bath for 25 min, and then cooled to room temperature for absorption spectrum measurement and photothermal analysis.

[0270] For GO detection, a mixture of 90 μL GO and GOx (5 mg / mL, 10 μL) was reacted for 30 min. Then, acetate buffer containing TMB and AuNPs was added and incubated at 45 °C for 25 min. Finally, GO was analyzed using a UV-Vis-NIR spectrometer and a forward-looking infrared handheld thermal imager.

[0271] For Myr detection, Sin (2.5 mM, 30 μL) and GOx (5 mg / mL, 10 μL, according to previous literature (Jiang, C. et al., RSC Adv. 2017, 7, 44463-44469)) were first mixed with different concentrations of Myr and reacted at 37 °C for 30 min to generate H 2 O 2 Then, acetate buffer containing TMB and AuNPs was added to the above solution, incubated at 45°C for 25 min, and cooled to room temperature. Afterwards, the solution was used for the analysis of Myr enzyme activity.

[0272] Results and Discussion

[0273] Next, we investigated the feasibility of this approach for Myr testing, followed by optimization of conditions, and the results were Fig.13 A. There is no significant change in the absorption spectrum and image of sample (iv, containing incomplete components), while sample vi (including all components) shows obvious absorbance at 1064nm, accompanied by a distinct color change from colorless to blue. Consistent with the above results, H 2 O 2 It only appears in the presence of Myr and GOx, and then interacts with TMB under the catalysis of AuNPs to produce blue CTCs with NIR-II absorption; it has excellent photothermal conversion performance for temperature signal acquisition. After treating all samples with 1064nm laser, a sharp temperature improvement of nearly 22.5℃ and a significant infrared thermograph were obtained in sample vi due to the excellent photothermal conversion performance of CTCs ( Fig.13 B). In contrast, slight temperature fluctuations and negligible images were observed in the other samples (iv). These results suggest that the photothermal and colorimetric signals are derived solely from the formation of CTCs. In addition, the feasibility of analyzing Myr was further investigated, and Fig.13 The results of C and 14 show that when Cu 2+ After being added to the system containing Sin, Myr, GOx, TMB and AuNP, the absorbance at 1064nm decreased significantly. However, after adding EDTA to the system, the intensity recovered significantly because EDTA can react with Cu 2+ Combines and weakens Cu 2+ The corresponding temperature signal and infrared thermal image are as follows: Fig.13D. Similarly, the temperature increase significantly decreased from 22.1°C to 3.9°C and then increased to 19°C, and the infrared thermograph also changed significantly. All these results once again confirmed the feasibility of NIR-II absorption-driven photothermal and colorimetric sensing techniques and showed their potential for analyzing Myr in real samples by utilizing the NIR-II absorption of CTCs.

[0274] in addition, Fig.15 The successful detection of GO in the experiment also confirmed the feasibility of this method.

[0275] Example 8. Quantitative analysis of NIR-II absorption-driven photothermal and colorimetric sensing of Myr For the anti-interference experiments discussed below, Sin (2.5 mM, 30 μL) and GOx (5 mg / mL, 10 μL) were first mixed with different interfering substances and reacted at 37° C. for 30 min. Then, acetate buffer containing TMB and AuNPs was added to the above solution, incubated at 45° C. for 25 min, and cooled to room temperature, and then, the solution was used for photothermal and colorimetric analysis.

[0276] After optimizing all the conditions in the above examples, we applied this method to test Myr; however, previous studies have shown that high Gls concentrations have a significant inhibitory effect on Myr activity (Pardini, A. et al., Food Chem. 2021, 355, 129634). Before testing different Myr concentrations, it is necessary to determine the maximum Sin concentration that can be applied. Fig.16 It is shown that when the concentration of Sin exceeds 0.75 mM, the working efficiency decreases and the blue color becomes lighter, because the excess Sin binds to the active regulatory site of Myr, thereby inhibiting the activity of Myr to hydrolyze Sin. As a result, 0.75 mM was selected as the maximum Sin concentration for detecting different concentrations of commercial Myr and constructing a standard curve. Fig.17 As shown in AB and 18, when Sin is present at a concentration of 0.75 mM, Myr can be detected in the range of 0-345 mU / mL, and its linear detection range in the absorption spectrum is 0 to 172.5 mU / mL, and its LOD is 2.96 mU / mL. In contrast, our method shows an advantage in sensitivity, and its LOD is lower than the LOD of previously reported work (Finiguerra, MGet al., J. Agric. Food Chem. 2001, 49, 840-845; and Gonda, S. et al., Molecules 2018, 23, 2204). In addition, Fig.17Line (ii) in B also shows a linear relationship between the logarithm of temperature and Myr concentration, and its trend is similar to that described above (Fu, G. et al., Anal. Chem. 2018, 90, 5930-5937). These results further confirm that this method can be applied to Myr analysis in actual samples through both temperature and absorbance changes.

[0277] Fig.19 The results in this paper show that this method can detect H from 1μM to 2.5mM 2 O 2 , and its linear detection range is 1μM to 25μM. GO ( Fig. 20 A), whose LOD is 1.67 μM ( Fig. 20 B). Next, we applied this method to test Myr, however, previous literature showed that high Gls concentrations had a significant inhibitory effect on Myr activity (Román, J. et al., Food Chem. 2018, 254, 87-94; and Pardini, A. et al., Food Chem. 2021, 355, 129634).

[0278] More importantly, this simple and rapid method gets rid of the complicated sample preparation and measurement process, thus providing great potential for on-site detection. We can also qualitatively distinguish samples with different concentrations of Myr by observing the color with the naked eye, which is also conducive to on-site testing.

[0279] In addition, specificity is also an important indicator for evaluating the practicality of analytical techniques, so various interfering factors including amino acids, small molecules and metal ions are also measured by NIRII-driven photothermal and colorimetric sensing platforms. According to previous reports, high concentrations of vitamin C (Vc) and NaCl, amino acids and metal ions can inhibit the activity of Myr (Jwanny, EW et al., Phytochemistry 1995, 39, 1301-1303; and Ohtsuru, M. et al., Agric. Biol. Chem. 1979, 43, 2249-2255), while EDTA can alleviate the inhibitory effect of metal ions.

[0280] like Fig.17 C, we can see that Cu 2+ The inhibitory effect on Myr activity was the strongest, followed by other metal ions. 2+ When EDTA and Cu coexist in the system, 2+The binding of γ-glucose ions to Myr activity was less inhibited, thus weakening the inhibitory effect of metal ions. Vc, GSH, and Cys also inhibited Myr activity, which is consistent with previous reports. Fig.17 As shown in C, the absorbance of Myr at 1064 nm is significantly higher than that of other interfering molecules, and a significant color change is only observed for Myr. Its absorption spectrum is shown in Fig.21 In addition, Fig.17 D shows the corresponding temperature signal and infrared thermograph, indicating that after 1064 nm laser irradiation, the temperature increase of Myr is more obvious than that of other interfering factors. These results indicate that this method shows good specificity for Myr testing in complex matrix environments and has potential for on-site Myr testing. Their UV-vis-NIR absorption spectra are shown in Figure 2. Fig. 22 shown.

[0281] Example 9. Temperature response to Myr, thermal cycling stability and specificity of the Myr assay

[0282] Next, the thermal cycling stability and the temperature response to different Myr concentrations were discussed as described above; the reaction of TMB with H 2 O 2 The reaction produces blue CTCs with a broad absorption peak in the NIR-II region. Using this property, the samples were irradiated with a 1064 nm laser to obtain the temperature response of different Myr concentrations, such as Fig.23 As shown in A, we quantitatively analyzed Myr by temperature, for example, corresponding to 345 mU / mL, the temperature of Myr was about 49.8°C. In addition, the corresponding infrared thermograph ( Fig.23 B) to improve the accuracy of the method.

[0283] We also evaluated the thermal cycling stability used to detect Myr; Fig.23 As shown in Figure C, the method still has a good photothermal response even after 5 cycles of laser irradiation. This shows that the method proposed in the present disclosure can be used for Myr testing with desired thermal stability and can be used for Myr field testing.

[0284] Example 10. Intelligent on-site determination of Myr in real samples via smartphone

[0285] We planned to examine the feasibility of accurate analysis in our precise and dynamic system. The test results of commercial Myr can be used as a reference to make a preliminary qualitative judgment on the content of Myr in vegetables. To this end, we applied the method to test Myr in real samples, and we selected some cruciferous and non-cruciferous vegetables, as well as the dietary supplements BroccoMax and Dualspices Japanese wasabi powder as test samples.

[0286] Detection of Myr in real samples

[0287] Myr in dietary supplements and wasabi was measured by the following recommended method: ddH 2 5 mg / mL of broccoli or cabbage vegetables in ddHO or 2 The remaining actual sample of 10 mg / mL in O was heated at 100 °C for 1 h to inactivate Myr; other samples with the same concentration were incubated at room temperature (25 °C) for 30 min, and 90 μL of the sample was mixed with acetate buffer containing GOx (5 mg / mL, 10 μL), TMB (5 mM, 300 μL) and synthesized AuNPs (100 μL) and incubated for another 30 min. In order to obtain temperature signals and infrared thermographs, after we collected the absorption spectra, we used 1 W / cm 2 The samples were irradiated with 1064 nm laser.

[0288] For Myr analysis in Cruciferae, we first froze the plant material with liquid nitrogen for later Myr analysis (Keck, A Set al., J. Agric. Food Chem. 2003, 51, 3320-3327). Then, depending on the sample, 5 mg of powdered broccoli or cabbage or 10 mg of powdered vegetables were added to 1 mL of phosphate buffered saline (PBS) buffer and heated at 100 ° C for 1 h to inactivate endogenous Myr. Then, 60 μL of the sample was added to acetate buffer containing exogenous sinigrin (2.5 mM, 30 μL), GOx (5 mg / mL, 10 μL), TMB (5 mM, 300 μL) and AuNP (100 μL) for 30 min, and we collected their absorbance at 1064 nm, which can be used as the background signal of various vegetables (A b Then, the samples of the same concentration were incubated at room temperature for 30 min, and acetate buffer containing sinigrin (2.5 mM, 30 μL), GOx (5 mg / mL, 10 μL), TMB (5 mM, 300 μL), and synthesized AuNPs (100 μL) was mixed with 60 μL of the sample. The absorbance at 1064 nm was obtained and used as the detection signal (A d ), and Δ A It is used to evaluate the amount of endogenous Myr in vegetables. In addition, FLIR is used to obtain temperature signals and infrared thermal images to improve detection accuracy.

[0289] Background signal (A b ) was obtained from the sample treated at 100°C, and the detection signal (A d ) were collected from samples treated at room temperature;d Subtract A from b To estimate the amount of Myr, the result is an increased signal (ΔA). In addition, FLIR is used to obtain temperature signals and infrared thermographs. Considering the relationship between spectrum, colorimetric image, temperature signal and infrared thermograph, we associate the obtained image values ​​with Myr concentrations via a smart phone-based intelligent processing system "calculator" (see https: / / calculator-39f49.firebaseapp.com / # / ). Typically, by using a "calculator" with a formula, the obtained experimental image will be decomposed into digital pixels represented by red, green and blue colors (RGB) (Stefan, L. et al., Nucleic Acids Res. 2012, 40, 8759-8772). The ratio of different color intensity values, for example, the ratio of blue to red (B / R), will be used to verify the amount of Myr in real samples.

[0290] Results and Discussion

[0291] Considering the relationship between the spectrum, colorimetric image, temperature signal and infrared thermograph, we analyzed the relationship between the B / R value of the colorimetric image and the Myr concentration via a smartphone-based website called Calculator (https: / / calculator-39f49.firebaseapp.com / # / ), and then used the “Calculator” with formulas to quickly realize intelligent on-site analysis of Myr. The B / R value was used as a parameter for Myr quantification, and Fig.24 A shows that the ratio of B to R increases with the increase of Myr concentration. Fig.24 In B, the B / R value is 0-86.25mU / mL (R 2 =0.98883) in the range of Myr concentration, with a LOD of 2.48 mU / mL, and a B / R value exceeding 2.57692 (Myr concentration greater than 86.25 mU / mL). The B / R value is proportional to the Myr concentration, and its R 2 =0.99794( Fig.24 C). Therefore, the proposed method has considerable potential for the determination of Myr in a wide range of 0-345 mU / mL in two continuous linear ranges. After uploading and decomposing the colorimetric images through the "calculator", the Myr content in vegetables was obtained by reading out via a smartphone.

[0292] To determine the total Myr in vegetables, all samples were snap-frozen in liquid nitrogen, and to avoid interference from the samples themselves, we inactivated the samples at 100°C for 1 h to ensure the inactivation of Myr. Then, the Myr content in samples treated at room temperature and high temperature was determined using the method proposed in this disclosure.

[0293] like Fig.25 As shown in A, the absorption spectrum of the sample at room temperature (A d ) minus the absorption spectrum of the sample at high temperature (A b ) to obtain an absorption spectrum, called the Δ absorbance (ΔAbs) spectrum. Fig.26 A shows the ΔAbs of various vegetables including broccoli, cauliflower, green cabbage, Chinese cabbage, Chinese cabbage, lettuce and carrot at 1064nm. Among them, the ΔAbs1064nm value of broccoli is the largest, followed by green cabbage, and the value of carrot is the smallest. Then, their temperature changes ( Fig.25 B) and infrared thermography ( Fig.26 B) was also obtained by FLIR. Based on the established calibration curve, the amount of Myr per mg of actual sample is listed in Tables 1 and 2, respectively. Fig.26 and 27 Abs1064 nm and temperature were obtained.

[0294] Table 1. The amount of Myr per mg of actual sample calculated by the calibration curve of Abs1064 nm. The concentration of broccoli and cabbage was 5 mg / mL, and the concentration of other vegetables was 10 mg / mL. The results are based on Fig.18 The linear relationship between Abs1064 nm and Myr concentration is shown.

[0295]

[0296] Table 2. The amount of Myr per mg of actual sample calculated by the calibration curve of temperature. The concentration of broccoli and cabbage was 5 mg / mL, and the concentration of other vegetables was 10 mg / mL. The results were calculated based on the linear relationship between temperature and the logarithm of the Myr concentration, and the fitting equation is

[0297] Temp(℃)=2.96×lnMyr(mU / mL)+31.42(R 2 =0.98948).

[0298]

[0299] Fig.28 A shows the Δ absorbance at 1064 nm of various vegetables including broccoli, cauliflower, green cabbage, Chinese cabbage, Chinese cabbage, lettuce and carrot. Among them, the Abs1064 nm value of broccoli is the largest, followed by green cabbage, and the value of carrot is the smallest. Their infrared thermal images ( Fig.28B) is also obtained by FLIR. The test results of commercial Myr can be used as a reference to make a preliminary qualitative evaluation of the Myr concentration in vegetables.

[0300] For accurate intelligent on-site Myr analysis, a processing system "Calculator" involving conversion formulas was introduced to intelligently test Myr on-site. The image analysis and processing flow chart of the "Calculator" is Fig.29 , while the Calculator screenshot is shown in Fig.30 The B / R value was used as a parameter from the graphical visualization for quantification of real samples. Fig.29 As shown, the flowchart 2900 includes the following steps: obtaining a reference image 2910, identifying colors and decomposing the colors into RGB 2920, establishing a relationship between B / R value and Myr concentration and fitting a formula 2930, uploading an image of an actual sample and obtaining RGB 2940, obtaining the Myr concentration in the actual sample according to the formula 2950, ​​and saving the result 2960. After uploading and decomposing the colorimetric image using the "calculator", the Myr content in the vegetable is obtained by reading it out via a smartphone ( Fig.26 C) B / R values ​​and corresponding Myr concentrations Fig.26 D. The highest Myr content was found in broccoli, measured at 18.50 ± 1.58 mU / mg, while the Myr content in lettuce and carrot was below zero, indicating that these non-cruciferous vegetables do not contain Myr. Similarly, the Myr concentration calculated from the temperature corresponding to the infrared thermographs followed a consistent trend, as shown in Table 2, which further confirms the ability to synergistically screen by NIR-II activated photothermal and colorimetric signals.

[0301] The B / R value was used as a parameter for Myr quantification, and Fig.31 A shows that the ratio of B to R increases with the increase of Myr concentration. Fig.31 In B, the B / R value is 0 to 86.25 mU / mL (R 2 =0.98883) in the range of Myr concentration, with an LOD of 2.48 mU / mL. However, when the B / R value exceeded 2.57692 (Myr concentration was greater than 86.25 mU / mL), the B / R value was proportional to the Myr concentration, with an R 2 =0.99794( Fig.31 C). Therefore, the proposed method has considerable potential for the determination of Myr in a wide range of 0 to 345 mU / mL in two continuous linear ranges. After uploading and decomposing the colorimetric images using the "calculator", the Myr content in vegetables was obtained via smartphone readout. The B / R values ​​and the corresponding Myr concentrations were Fig.31D. The highest Myr content was found in broccoli, measured at 76.27 mU / mL, while the Myr content in lettuce and carrot was below zero, indicating that these non-cruciferous vegetables do not contain Myr.

[0302] In addition, if Fig.32 As shown in AB, the absorption spectra and temperature changes of dietary supplements and wasabi powder were also obtained. The results in Table 3 reflect that the amount of Myr in wasabi powder is much higher than that in dietary supplement BroccoMax. In addition, the Myr content was determined with the help of the "Calculator" and according to Fig.32 The CD results showed that the Myr content in 1 mg of BroccoMax was about 8.88 ± 0.26 mU, while the Myr concentration in 1 mg of wasabi powder was about 19.84 ± 0.27 mU, which once again confirmed the fact that the amount of Myr in wasabi powder was higher than that in BroccoMax. All these results show that the smartphone-based NIR-II absorption-driven photothermal and colorimetric sensing platform shows satisfactory performance in intelligent, convenient and non-interference detection of Myr in real samples.

[0303] Table 3. The amount of Myr per mg of real sample calculated from the calibration curve of Abs1064 nm and temperature. The concentrations of wasabi powder and dietary supplement were 1 mg / mL and 10 mg / mL, respectively.

[0304]

[0305] To achieve rapid on-site Myr analysis, we developed a smartphone-based intelligent processing system, named "Calculator", based on the relationship between B / R values ​​and Myr concentrations obtained from colorimetric or thermal images, and successfully applied it to accurately quantify Myr in real samples including various vegetables, dietary supplements, and commercial wasabi powder. The temperature-responsive and colorimetric sensing platform not only enables rapid qualitative and quantitative Myr on-site analysis via smartphone readout, but also demonstrates potential applications in the fields of food quality and nutritional analysis.

[0306] It can be understood that the method disclosed herein has some advantages as listed below:

[0307] (i) No protein extraction and purification is required, which significantly reduces costs and sample preparation time;

[0308] (ii) using absorbance at 1064 nm can exclude interference caused by impurities in plant samples, making it more suitable for the analysis of Myr in whole plant samples;

[0309] (iii) The temperature signal and infrared thermograph generated by the photothermal effect of CTC can further improve the credibility of detection and make the results more reliable; and

[0310] (iv) With the help of smartphone readout, the colorimetric image can be processed through a website named “Calculator” on the smartphone to obtain its RGB value related to the Myr concentration, and the relationship function between B / R value and Myr concentration is used to achieve rapid on-site Myr detection with good results. The colorimetric and temperature-responsive sensing platform not only enables rapid qualitative and quantitative Myr on-site analysis via smartphone readout, but also shows potential applications in the field of food quality analysis.

[0311] In summary, the present invention discloses a novel AuNP-catalyzed TMB and H 2 O 2 A novel method for rapid and non-interference detection of Myr with reaction and smartphone readout. Usually, H is generated under the catalysis of Myr and GOx. 2 O 2 , then in the above containing H 2 O 2 After adding TMB and AuNPs to the solution, CTCs with blue and NIR-II absorption are formed. Myr can be detected due to the colorimetric and photothermal effects of CTCs. For precise smart on-site Myr analysis, the relationship between the B / R value of the colorimetric image and the Myr concentration was analyzed using a website called "Calculator" on a smartphone. The proposed method is able to determine Myr in a wide range of 0-345 mU / mL in two continuous linear ranges. The amounts of Myr in 10 mg / mL broccoli, green cabbage, cauliflower, Chinese cabbage, pakchoi, dietary supplements, and wasabi powder were approximately 76.27, 30.07, 19.77, 2.65, 0.5, 18.66, and 88.97 mU / mL, respectively. Therefore, it is believed that the colorimetric and temperature-based sensing platform will provide a new technology for smart and precise on-site detection of Myr and open up new horizons for food quality evaluation.

[0312] Therefore, the present disclosure provides a new method for rapid and non-interference detection of Myr based on NIR-II absorption driven photothermal and colorimetric cooperative sensing system. This method has the following advantages:

[0313] (i) Using NIR-II absorbance at 1064 nm can largely exclude interference caused by impurities and chlorophyll in plant samples, making it more suitable for the analysis of Myr in real plant samples;

[0314] (ii) The temperature signal and near-infrared thermal image generated by the photothermal effect of CTC can further improve the credibility of the detection and make the results more reliable; and

[0315] (iii) With the help of smartphone readout, the smart processing system “calculator” can process the colorimetric image to obtain its RGB values ​​which are related to the Myr concentration and thus enable simple and rapid on-site Myr detection of complex and dynamic analytes with good results.

[0316] We believe that this NIR-II absorption-driven photothermal and colorimetric sensing platform will provide a new technology for intelligent and precise on-site detection of Myr and open up new horizons for food quality assessment and nutritional composition analysis, which will benefit related research areas in the future. In conclusion, the method disclosed in this paper provides a new method for on-site Myr analysis in actual food with the advantages of extraction-free, precise, cheap and intelligent. In addition, we believe it can be applied in the field of food quality evaluation.

[0317] Example 11. Engineering of extracellular vesicles of enteric bacteria by orthogonal labeling for diet-guided gastrointestinal theranostic therapy (theranostic)

[0318] Major gastrointestinal diseases, including inflammation and cancer, contribute to rising morbidity and mortality worldwide. There is a great need for gastrointestinal screening and local enrichment and targeted therapeutics. For example, we can take advantage of well-known enzymatic processes and extracellular vesicles (EVs) derived from the human gut microbiota. In addition, the accumulation of dietary-available gastrointestinal diagnostic and therapeutic integration methods can be used for screening and therapy.

[0319] EVs are heterogeneous, phospholipid bilayer-encapsulated bioparticles that regulate cellular communication. EVs derived from the gut microbiota that retain the functionality of the parent bacteria play an important role in inter- and intraspecies biological behavior. In this example, we isolated and purified EVs from the gut commensal Bacteroides thetaiotaomicron carrying bioactive enzymes, enhanced them through engineering methods, and created substrate-based probes with tissue affinity, bioorthogonality, fluorescence or drug functionality. Using engineered gut bacterial EVs and enzymatically activated glucosinolate probes, we can achieve localized regional accumulation of therapeutic agents for therapies in a specific gastrointestinal environment, including spatiotemporal monitoring of the intestine by NIR-II imaging and sustained therapeutic effects.

[0320] Bacterial culture medium or casein-free milk whey was sonicated for one hour and then centrifuged at 10,000 × g for 30 min at 4 ° C. The supernatant was filtered through a polyethersulfone (PES) membrane (0.22 μm pore size) (sartorius) to remove debris and cells. The filtered supernatant was centrifuged again at 10,000 × g for 30 min at 4 ° C to remove cell debris. The resulting supernatant was transferred to a 70mL polycarbonate bottle (Beckman coulter) using a pipette and centrifuged at 100,000 × g for 90 min at 4 ° C in an Optima XPN ultracentrifuge (Beckman coulter). The supernatant was completely removed with a pipette. The obtained crude EV was suspended in PBS.

[0321] A series of 10% to 90% sucrose solutions were prepared. The crude EVs were resuspended in 90% sucrose and added to a bottle of a 13.2-mL ultra-clear tube (Beckman coulter). Subsequently, a sucrose gradient solution from 80% to 10% was overlaid on top of the crude EV suspension. The gradient was ultracentrifuged at 144,000g for 17h at 4°C in a SW41 Ti rotor. After ultracentrifugation, the fractions were carefully collected from top to bottom and washed in PBS for 90min via ultracentrifugation at 100,000g in a 70mL polycarbonate bottle using a Rotor Type 45Ti rotor. After ultracentrifugation, the supernatant was poured out and the EVs were resuspended in PBS for use.

[0322] Protein concentration was quantified by bicinchoninic acid (BCA) assay (Thermo Fisher Scientific). A standard curve was drawn using serial dilutions of bovine serum albumin (BSA). Samples were measured in a minimum of three independent replicates and quantified using the standard curve. Total phosphate content was measured using the malachite green phosphate assay (Sigma-Aldrich).

[0323] EVs were negatively stained and observed using a transmission electron microscope (TEM). The obtained EVs were first mixed with an equal volume of 2% PFA. The mixture was deposited on a paraffin film to form droplets. In a sterile and dry environment, the Formvar-carbon-coated EM grid floated on the droplets for several hours. Subsequently, the Formvar-carbon-coated TEM grid was washed dropwise with PBS and DI water on a paraffin film and then transferred to a 1% glutaraldehyde droplet for 5 minutes. Finally, the Formvar-carbon-coated EM grid was washed in DI water to remove excess glutaraldehyde. The grid was air-dried and then analyzed using a JEM-1400 flash electron microscope and TEMJEOL 2010HR.

[0324] Results and Discussion

[0325] A general EV extraction method was developed ( Fig.33 ). Fig.34 Characterization results of EVs obtained from various sources are depicted.

[0326] Fig.35 Engineered enhancements to EVs are shown. Hybridization with artificial vesicles provides improved stability and biocompatibility. Genetic engineering of parental cells is performed to improve enzyme expression and activity.

[0327] Example 12. Development of a substrate-based multifunctional integrated diagnostic and therapeutic agent

[0328] To fully exploit the myrosinase-glucosinolate-based chemistry, a molecular library with various theranostic integrated substrates was established via simple and reliable click chemistry.

[0329] First, artificial aglycones containing azide functional groups were prepared according to a previous article (CP Glindemann et al., ChemBioChem 2019, 20, 2341). In short, azide-chlorooxime was synthesized according to the aldoxime pathway and then coupled with mercaptoglucose to produce thiohydroximate. After sulfation and deacetylation, substrate-based azide precursors were obtained. Next, various probes and therapeutic agents, including fluorescein, 5-fluorouracil, and cyanine probes with NIR-I / II fluorescence, were subsequently linked to aglycones to form artificial thioglucosides.

[0330] The prepared glucosinolates are in situ conjugated to biological sites after being hydrolyzed by myrosinase to achieve bioconjugation for myrosinase screening and targeted therapy.

[0331] Results and Discussion

[0332] A molecular library of Myr substrates for integrated diagnosis and treatment was developed for novel Myr activity screening technology and targeted therapy through bioconjugation ( Fig.36 ).

[0333] Example 13. Verification of the "homing effect" of lesion affinity

[0334] We plan to validate the homing effect of EVs as carriers of bioactive agents for targeted delivery and activation of prepared theranostic substrates.

[0335] The exosome suspension was diluted with PBS and a 2 mg / mL stock solution of lipid and membrane marker DiO DMSO was added to a final concentration of 20 μg / mL. The exosomes were stained for 1 h at room temperature, then washed with PBS and collected by centrifugation at 120000g for 70 min for 2 rounds. The stained exosome pellet was resuspended in 200 μL for cell culture.

[0336] MCF-7 cells, MDA-MB-231 cells, HeLa cells, HepG2 cells and A549 cells were cultured at 2×10 5 Cells / well were seeded in 35-mm diameter plastic-bottom μ-dishes (ibidi GmbH, Germany) in complete culture medium and incubated at 5% CO 2 and 37°C for 24 h. The cells were then incubated with 50 μL of the extracted exosome PBS suspension for 3 h. Subsequently, excess compounds were removed and the cells were washed 3 times with PBS and then incubated with Hoechst 33258 (2 μg / mL) for 15 min. Excess Hoechst 33258 was removed and the cells were washed again 3 times with PBS and immediately examined under a Zeiss LSM 800 confocal microscope.

[0337] Results and Discussion

[0338] Fig.37 The results obtained depict the targeting ability of EVs to the parental site. The "homing effect" of EVs was verified, and the results indicate that EVs are promising vesicles that carry bioactive compounds including myrosinase to achieve in situ activation of theranostic substrates.

[0339] Example 14. Evaluation of different levels of integrated imaging and diagnosis and treatment

[0340] The novel integrated diagnostic and therapeutic model following the myrosinase enzymatic process was further evaluated at different biological levels including bacteria, mammalian cells, and zebrafish models.

[0341] Bacteroides thetaiotaomicron (ATCC 29148) and Escherichia coli (E. coli) (ATCC 25922) were incubated in BHIS medium and LB medium at 37°C until mid-exponential phase. GL-Cy5 was then added to the medium to a final concentration of 100 μM. After 3 h of incubation, the bacteria were washed twice with PBS and then suspended in PBS to a final OD of 1. 600 =0.25. The bacterial solution was dropped on a glass slide and observed by CLSM. Real-time images were obtained by continuous CLSM shooting for 10 min. As described above, 640 nm excitation was used for GL-Cy5 labeling imaging of Bacteroides theta (B. theta) and Escherichia coli.

[0342] MCF-7 cells were cultured at 2×10 5 Cells / well were seeded in 35-mm diameter plastic-bottom μ culture dishes (ibidi GmbH, Germany) in complete culture medium and incubated at 5% CO 2 and 37°C for 24 h. Then, the cells were incubated with 50 μL of the extracted exosome PBS suspension for 3 h. Subsequently, the excess compound was removed and the cells were washed 3 times with PBS and then incubated with different probes for 15 min, including GL-Cy5 (10 μM), fluorescein (10 μM) and membrane markers (2 μg / mL). The excess probe was removed and the cells were washed again 3 times with PBS and immediately examined under a confocal microscope.

[0343] GFP oncogene-transfected zebrafish embryos were injected with the extracted exosome suspension at 12 hpf and incubated for 8 h at 28 °C. Afterwards, the embryos were incubated with GL-Cy5 for 16 h at 28 °C, and the yolk extension phenotype was observed using the bright field channel under a Zeiss LSM 800 confocal microscope.

[0344] Results and Discussion

[0345] Fig.38 The results of enzyme-activated ITC labeling at different biomass levels are depicted. The results suggest that this myrosinase-activated bioconjugation could be used for enzyme screening and targeted therapy.

[0346] Therefore, we disclose herein a novel diagnostic and therapeutic integration model inspired by the well-known enzymatic processes of the human gut microbiome to achieve localized regional accumulation for monitoring and treatment. In addition, the present disclosure provides innovative insights into potential strategies for non-invasive detection and targeted therapy of gastrointestinal lesions using extracellular vesicles derived from the gut microbiome.

Claims

1. A method for quantitatively determining the concentration of active myrosinase in a sample, the method comprising: The following steps are involved: (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: providing glucose as a substrate for the active myrosinase enzyme as a reaction product; Glucose oxidase; Near-infrared absorbing and photothermal responsive compounds capable of forming charge transfer complexes or nanoparticle aggregates; and Peroxidase or nanoparticles with 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 an analyte reference sample, the second analyte assay formulation being identical to the first analyte assay formulation and the second period of time being identical to the first period of time; (c) determining the concentration of said active myrosinase in said sample by measuring one or more of the following: (i) determining the red, green and blue color values ​​of the analyte sample and the red, green and blue color values ​​of the analyte reference sample, calculating the red / blue ratio of each of the analyte sample and the analyte reference sample, calculating the difference between the red / blue ratios to provide a sample red / blue ratio, and comparing the obtained sample red / blue ratio to a predetermined calibration curve of active myrosinase concentration based on the red / blue ratios obtained from active myrosinase of known concentrations; (ii) obtaining an infrared image of the analyte sample and an infrared image of the analyte reference sample after irradiating the analyte sample and the analyte reference sample with laser for a third period of time, and calculating a temperature difference between a temperature obtained from the infrared image of the analyte sample and a temperature obtained from the infrared image of the analyte reference sample, and comparing the obtained difference value with a predetermined calibration curve of active myrosinase concentration based on temperatures obtained from infrared images of active myrosinase of known concentrations; (iii) an absorbance spectrum of the analyte sample and an absorbance spectrum of the analyte reference sample, and calculating a difference between the absorbance spectrum of the analyte sample and the absorbance spectrum of the analyte reference sample, and comparing the obtained difference with a predetermined calibration curve of active myrosinase concentration based on the absorbance spectra of active myrosinase of known concentrations; and (iv) obtaining temperature signals of the analyte sample and the analyte reference sample after irradiating the analyte sample and the analyte reference sample with laser for a third period of time, calculating a difference between the temperature signal of the analyte sample and the temperature signal of the analyte reference sample, and comparing the obtained difference value with a predetermined calibration curve of active myrosinase concentration based on temperature signals of active myrosinase with known concentrations.

2. The method according to claim 1, in, Using two or more of (i) to (iv) to determine the concentration of active myrosinase in the sample.

3. The method according to claim 1 or claim 2, in, The substrate for the active myrosinase providing glucose as a reaction product is a glucosinolate, optionally wherein the glucosinolate is selected from the group consisting of glucosinolate, natroniin, glucoraphanin and mesonain.

4. The method according to any one of the preceding claims, in, The near-infrared absorption and photothermal response compound capable of forming a charge transfer complex or nanoparticle aggregate is selected from gold nanoparticles modified with phenyl borate, or one or more selected from the group consisting of perylene, F4TCNQ, tetracyanoquinodimethane (TCNQ) and 3,3',5,5'-tetramethylbenzidine, optionally wherein the near-infrared absorption and photothermal response compound capable of forming a charge transfer complex or nanoparticle aggregate is 3,3',5,5'-tetramethylbenzidine.

5. The method according to any one of the preceding claims, in, The nanoparticles having peroxidase-like activity are selected from silver, or more particularly, gold nanoparticles, optionally wherein the gold nanoparticles have one or more of the following properties: (ai) absorption peak at about 527 nm; (aii) an average diameter size of about 13 nm as determined by transmission electron microscopy; (aiii) a zeta potential in aqueous solution of +20 to +30 mV, such as about +24.5 mV; and (aiv) at about 1650cm -1 and about 3450cm -1 Infrared absorption peak at.

6. The method according to any one of the preceding claims, in, One or more of the following applies: (bi) when the nanoparticles having peroxidase-like activity or the peroxidase are gold nanoparticles, they are present in the first analyte assay formulation and the second analyte assay formulation at a concentration of 0.02 to 0.8 nM, such as about 0.45 nM; (bii) when the near infrared absorbing and photothermal responsive compound capable of forming a charge transfer complex or nanoparticle aggregate is 3,3',5,5'-tetramethylbenzidine, it is present in the first analyte assay formulation and the second analyte assay formulation at a concentration of 0.5 to 3 mM, such as about 1.5 mM; (biii) the glucose oxidase is present in an amount of 5 U / mL to 25 U / mL; (biv) the substrate for active myrosinase providing glucose as a reaction product is present in an amount of 0.05 mM to 0.75 mM.

7. The method according to any one of the preceding claims, in, The first analyte assay formulation and the second analyte assay formulation further include acetate buffer in an amount to provide a pH of 3.5 to 7.5, such as about 4.

5.

8. The method according to any one of the preceding claims, in, The sample comprising active myrosinase and the sample comprising inactive myrosinase are provided at a concentration of 1 to 20 mg / mL, such as about 10 mg / mL.

9. The method according to any one of the preceding claims, in, The sample comprising inactivated myrosinase is obtained by heating the sample comprising active myrosinase to a temperature suitable for denaturing the active myrosinase for a fourth period of time, optionally wherein one or both of the following apply: The temperature is 80 to 120°C, such as about 100°C; and The fourth period of time is 30 minutes to 2 hours, such as about 1 hour.

10. The method according to any one of the preceding claims, in, Prior to use in the method, the sample comprising active myrosinase is incubated at a suitable temperature for a fifth period of time, optionally wherein one or both of the following apply: The temperature is 15 to 30°C, such as about 25°C; and The fifth period of time is from 10 minutes to 1 hour, such as about 30 minutes.

11. The method according to any one of the preceding claims, in, When one or both of (ii) and (iv) of step (c) of claim 1 are used to determine the concentration of the active myrosinase in the sample, the laser is a near-infrared laser, optionally, wherein one or more of the following apply: The laser provides a beam having a wavelength of 1000 to 1500 nm, such as about 1064 nm; The laser has a power of 0.5 to 3 W / cm 2 , such as about 1 W / cm 2 power density; and The third period of time is from 20 seconds to 5 minutes.

12. The method according to any one of the preceding claims, in, The first period of time and the second period of time are 10 minutes to 1 hour, such as 30 minutes.

13. The method according to any one of the preceding claims, in, The absorption spectra of the analyte sample and the analyte reference sample are based on their absorbance at a specified wavelength or wavelength range in the near infrared range, optionally wherein the specified wavelength is a wavelength selected from 1,000 to 1,500 nm, such as 1064 nm.

14. The method according to any one of the preceding claims, in, The sample is obtained from a dietary supplement comprising myrosinase, wasabi (eg wasabi powder) or a cruciferous vegetable, optionally wherein the cruciferous vegetable is selected from broccoli, cauliflower, cabbage and Chinese cabbage.

15. The method according to any one of the preceding claims, in, The first analyte assay formulation and the second analyte assay formulation include: sinigrin; Glucose oxidase; 3,3',5,5'-Tetramethylbenzidine; and Gold nanoparticles.

16. The method according to any one of the preceding claims, in, The method further comprises step (d), wherein step (d) is selected from one or more of the following: (di) retaining or disposing of said batch based on the level of freshness of the batch from which said sample was drawn, said level of freshness of said batch being based on the concentration of said active myrosinase in said sample; and (dii) marking the batch from which the sample was drawn with a specific level or amount of active myrosinase.

17. A preparation for use in a method for quantitatively determining the concentration of active myrosinase in a sample, the preparation include: providing glucose as a substrate for the active myrosinase enzyme as a reaction product; Glucose oxidase; Near-infrared absorbing and photothermal responsive compounds capable of forming charge transfer complexes or nanoparticle aggregates; and Peroxidase or nanoparticles having peroxidase-like activity.

18. The preparation according to claim 17, in, The substrate for the active myrosinase providing glucose as a reaction product is a glucosinolate, optionally wherein the glucosinolate is selected from the group consisting of glucosinolate, natroniin, glucoraphanin and mesonain.

19. A formulation according to claim 17 or claim 18, in, The near-infrared absorption and photothermal response compound capable of forming a charge transfer complex or nanoparticle aggregate is selected from gold nanoparticles modified with phenyl borate, or one or more selected from the group consisting of perylene, F4TCNQ, tetracyanoquinodimethane (TCNQ) and 3,3',5,5'-tetramethylbenzidine, optionally wherein the near-infrared absorption and photothermal response compound capable of forming a charge transfer complex or nanoparticle aggregate is 3,3',5,5'-tetramethylbenzidine.

20. The formulation according to any one of claims 17 to 19, in, The preparation comprises: sinigrin; Glucose oxidase; 3,3',5,5'-Tetramethylbenzidine; and Gold nanoparticles.

21. The formulation according to any one of claims 17 to 20, in, The formulation also includes an acetate buffer capable of generating an aqueous solution having a pH of 3.5 to 7.5, such as about 4.

5.

22. A kit of parts suitable for providing a formulation as claimed in any one of claims 17 to 21, in, The kit comprises: providing glucose as a substrate for an active myrosinase of the reaction product, optionally wherein the substrate for an active myrosinase of the reaction product providing glucose as a substrate is sinigrin; Glucose oxidase; Near-infrared absorbing and photothermal responsive compounds capable of forming charge transfer complexes or nanoparticle aggregates; and Peroxidase or nanoparticles having peroxidase-like activity.

23. The kit according to claim 22, in, The kit comprises: sinigrin; Glucose oxidase; 3,3',5,5'-Tetramethylbenzidine; and Gold nanoparticles.

24. A kit according to claim 22 or claim 23, in, The kit also includes an acetate buffer capable of generating an aqueous solution having a pH of 3.5 to 7.5, such as about 4.5.

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