Alpha-keto acid light-excited free radical enhanced generation system for melting DNP and application of alpha-keto acid light-excited free radical enhanced generation system

Abstract

The invention belongs to the technical field of nuclear magnetic resonance and electron paramagnetic resonance, and particularly relates to an alpha-keto acid light-excited free radical enhanced generation system for dissolving DNP and application of the alpha-keto acid light-excited free radical enhanced generation system. The invention provides an application of hydrogen peroxide and / or titanium dioxide as a free radical enhancer in a molten dynamic nuclear polarization alpha-keto acid photoexcitation free radical enhanced generation system. According to the alpha-keto acid light excitation free radical enhancement generation system provided by the invention, the free radical enhancer can effectively induce alpha-keto acid molecules to generate free radical reaction under the ultraviolet / visible light illumination condition, so that the yield of transient free radicals is remarkably improved. The alpha-keto acid photoexcitation free radical enhanced generation system provided by the invention can provide a high-concentration free radical source meeting the technical requirements of DNP, promote the efficiency and rate improvement of an electron-nuclear polarization transfer process, and provide a key technical support for the application of a molten DNP technology in the fields of magnetic resonance imaging and the like.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of nuclear magnetic resonance and electron paramagnetic resonance, and particularly relates to a light-induced free radical enhancement generation system for dissolution DNP and application thereof. BACKGROUND

[0002] Dissolution dynamic nuclear polarization (d-DNP) technology is one of the core technologies for solving the problem of low sensitivity of nuclear magnetic resonance (NMR) and magnetic resonance imaging (MRI). The technology realizes high polarization of the observed nucleus through the double resonance of electrons and nuclei at low temperature, and can significantly improve the detection signal strength after dissolution in a high-temperature and high-pressure buffer solution, and has irreplaceable application value in the fields of biomedical metabolic tracing and molecular structure analysis.

[0003] Free radicals are the core medium for polarization transfer in d-DNP technology, and their performance directly determines the polarization efficiency. Traditional stable free radicals (such as TEMPO) will accelerate the polarization state relaxation of the probe after being dissolved, and cannot be directly introduced into the living body due to the problem of biological safety, so it is necessary to remove them after polarization by chemical reagents or physical filtration. This time-consuming and cumbersome step increases the complexity of operation and has a negative impact on the polarization efficiency of the probe. Therefore, the photosensitive free radical technology proposed by Arnaud Comment has become a research hotspot. The technology generates transient free radicals by ultraviolet light irradiation of photosensitive small molecules at low temperature (<77K), and remains stable at low temperature to realize polarization transfer. After being dissolved at high temperature, the free radicals can be self-quenched, realizing in-situ and on-demand generation of free radicals, which perfectly matches the technical requirements of d-DNP.

[0004] In the prior art, α-keto acids with α-dicarbonyl structure (such as pyruvic acid, α-ketoglutaric acid, oxalacetic acid, and oxalic acid) have been reported as photosensitive free radical precursors, Figure 13 The mechanism of pyruvic acid for ultraviolet light-induced generation of transient free radicals. The above-mentioned endogenous α-keto acid molecules can simultaneously act as MRI probes and photoexcited free radical precursors, can generate corresponding α-keto acid free radicals and directly realize polarization enhancement of the probe, and will not introduce other exogenous free radicals that affect the biological metabolic process. Literature and patents report that the conventional solvent system is a mixed solvent with a glycerol-water volume ratio of 6:4 (GW64). However, it is found in actual application that the system has the following key defects: α-keto acid can only produce a low concentration of transient free radicals (oxalic acid molecules cannot even produce transient free radicals) after being excited by ultraviolet light for a short time under the above-mentioned solvent ratio, which is far lower than the free radical concentration threshold required for efficient polarization transfer in d-DNP technology, resulting in low polarization efficiency and slow transfer speed, which cannot meet the actual detection requirements.

[0005] To solve the problem of insufficient photosensitive free radical yield, the prior art has tried to introduce an auxiliary agent to regulate, for example, the addition of an iron complex in an oxalic acid system can improve the free radical yield, but such an auxiliary agent has the limitations of poor biocompatibility and accelerating the relaxation of the signal of the probe molecule. In view of the biological safety and convenient source of alpha-keto acid molecules, it is urgent to develop an efficient and simple free radical enhancement scheme for a glycerol-water 6:4 solvent system to break through the bottleneck in the application of d-DNP. SUMMARY

[0006] The purpose of the present application is to provide an alpha-keto acid photoexcited free radical enhancement generation system for melting DNP and its application. The alpha-keto acid photoexcited free radical enhancement generation system for melting dynamic nuclear polarization constructed by the present application can specifically enhance the free radical generation efficiency of alpha-keto acid, provide a high-concentration free radical source that meets the requirements of DNP technology, promote the efficiency and rate of electron-nuclear polarization transfer, realize more efficient and faster electron-nuclear polarization transfer, and provide key technical support for the application of melting DNP technology in the field of magnetic resonance imaging.

[0007] To achieve the above purpose, the present application provides the following technical solutions: The present application provides the application of hydrogen peroxide and / or titanium dioxide as a free radical enhancer in an alpha-keto acid photoexcited free radical enhancement generation system for melting dynamic nuclear polarization.

[0008] The present application provides an alpha-keto acid photoexcited free radical enhancement generation system for melting dynamic nuclear polarization, which comprises a glassy reagent, an alpha-keto acid photosensitive precursor and a free radical enhancer. The glassy reagent is an alcohol-water system, the deuterium substitution rate of alcohol and water in the alcohol-water system is 0-100%, and the free radical enhancer comprises hydrogen peroxide and / or titanium dioxide, and the deuterium substitution rate of hydrogen peroxide is 0-100%.

[0009] Preferably, the glassy reagent comprises a glycerol-water system or an ethanol-water system; the volume ratio of glycerol to water in the glycerol-water system is 1:9-9:1; and the volume ratio of ethanol to water in the ethanol-water system is 1:9-9:1.

[0010] Preferably, the volume ratio of glycerol to water in the glycerol-water system is 6:4; and the volume ratio of glycerol to water in the ethanol-water system is 6:4.

[0011] Preferably, the alpha-keto acid photosensitive precursor comprises one or more of alpha-diketone, alpha-keto acid and alpha-keto acid derivative.

[0012] Preferably, the alpha-diketone comprises butanedione; the alpha-keto acid comprises one or more of pyruvic acid, alpha-ketoglutaric acid, oxaloacetic acid, oxalic acid and phenylglyoxylic acid; and the alpha-keto acid derivative comprises diethyl oxalate.

[0013] Preferably, when the radical enhancer is hydrogen peroxide, the hydrogen peroxide is used in the form of an aqueous hydrogen peroxide solution or a hydrogen peroxide generation system, the hydrogen peroxide generation system comprising a water and metal peroxide reaction system or a water photo and / or acoustic catalysis reaction system, the metal peroxide comprising sodium peroxide and / or calcium peroxide, and the water photo and / or acoustic catalysis reaction system comprising a water system in which hydrogen peroxide is generated by photo-illumination of a catalyst and / or by cavitation of ultrasound. The molar concentration of hydrogen peroxide in the alpha-keto acid photo-excited radical enhancement generation system is 1 µM to 100 mM.

[0014] Preferably, the particle size of the titanium dioxide is 10 to 500 nm; and when the radical enhancer is titanium dioxide, the mass concentration of titanium dioxide in the alpha-keto acid photo-excited radical enhancement generation system is 0.789 µg / L to 0.789 g / L.

[0015] The present application provides a photo-excited radical generation method for dynamic nuclear polarization melting, comprising the following steps: The alpha-keto acid photo-excited radical enhancement generation system described in the above technical solution is frozen to obtain microbeads. The microbeads are photo-excited to generate transient radicals.

[0016] Preferably, the diameter of the microbeads is 0.5 to 5 mm; the light source used for photo-excitation is ultraviolet light and / or visible light, the wavelength range of the light source is 200 to 800 nm, the light source is a continuous wave or a pulsed mode wave, and the light source is a coherent or incoherent light mode; when the wavelength range of the light source used for photo-excitation is 200 to 600 nm, the photo-excitation conditions comprise: the light illumination power is 2 to 40 W / cm 2 , and the light illumination time is 1 to 600 s; when the wavelength of the light source used for photo-excitation is 254 nm, the photo-excitation conditions comprise: the light illumination power is 0.6 to 25 mW / cm 2 , and the light illumination time is 5 to 60 min; and the photo-excitation is performed in a low-temperature environment, and the temperature of the low-temperature environment is < 120 K.

[0017] This invention provides the application of hydrogen peroxide and / or titanium dioxide as free radical enhancers in a molten dynamically nuclear polarized α-keto acid photoexcited free radical enhancement system. The invention provides a molten dynamically nuclear polarized α-keto acid photoexcited free radical enhancement system, comprising a glassy reagent, an α-keto acid photosensitizing precursor, and a free radical enhancer; the glassy reagent is an alcohol-water system, wherein the deuteration rates of the alcohol and water in the alcohol-water system are 0-100%, and the free radical enhancer comprises hydrogen peroxide and / or titanium dioxide, wherein the deuteration rate of the hydrogen peroxide is 0-100%. This invention addresses the shortcomings of existing technologies, such as low free radical yield, long illumination time, and uneven distribution of generated free radicals affecting polarization efficiency when photoexciting α-keto acid photosensitizing precursors (i.e., α-dicarbonyl molecules) in a glycerol-water volume ratio of 6:4 solvent system. This invention constructs a novel α-keto acid photoexcited free radical enhancement system by using hydrogen peroxide or titanium dioxide as a free radical enhancer. The α-keto acid photoexcited radical enhancement system provided by this invention, under ultraviolet / visible light irradiation, effectively induces free radical reactions in α-keto acid molecules, significantly improving the yield of transient free radicals. EPR detection confirms that its free radical concentration is several times or even orders of magnitude higher than that of the system without the enhancer. This α-keto acid photoexcited radical enhancement system provides a high-concentration free radical source that meets the requirements of DNP technology, promoting the efficiency and rate of electron-nuclear polarization transfer processes, and providing key technical support for the application of fused DNP technology in fields such as magnetic resonance imaging. Furthermore, the free radical enhancers used in this invention include hydrogen peroxide and / or titanium dioxide. Hydrogen peroxide and titanium dioxide can be easily removed without interfering with the probe molecule signal, thus not affecting polarization relaxation.

[0018] Compared with the prior art, the present invention has the following beneficial effects: (1) Significantly improved free radical yield: Hydrogen peroxide or titanium dioxide can enhance the photoexcitation free radical generation efficiency of α-keto acid photosensitive precursors through different mechanisms. Hydrogen peroxide can decompose under ultraviolet light to generate hydroxyl radicals (·OH), which can act as initiators to activate the homolytic cleavage reaction of α-keto acid photosensitive precursors; titanium dioxide, as a photocatalyst, can accelerate the electron transfer process of α-keto acid photosensitive precursors and promote the generation of free radicals after excitation by generating photogenerated electron-hole pairs. Both enhancers can increase the free radical concentration of the system by several times or even orders of magnitude, far exceeding the concentration threshold for efficient polarization transfer of DNP, suggesting that their electron-nucleus polarization transfer time is shorter and their polarization efficiency is higher; (2) Excellent compatibility of the enhancer: the free radical enhancer provided by the application has the characteristics of easy removal and high biological safety. Hydrogen peroxide can be naturally decomposed into water and oxygen during the heating and melting process, without the need for additional removal steps; titanium dioxide can be completely separated by simple centrifugation (speed 5000-8000 rpm, time 1-5 min) or by filtering through a filter membrane. After removal, there is no enhancer residue in the system, which neither affects the polarization relaxation process of the probe molecule nor meets the safety requirements of in-vivo magnetic resonance imaging and other application scenarios; (3) Simple process easy to promote: the glassy reagent (i.e. solvent), the alpha-keto acid photosensitive precursor and the free radical enhancer in the alpha-keto acid photo-excited free radical enhancement generation system provided by the application are all commercial products, which are convenient to source and low in cost; the operation process does not require special equipment, only needs to add an enhancer addition step in the existing photoexcitation process, and can be directly adapted to the existing melting DNP equipment, with good application and promotion prospects. BRIEF DESCRIPTION OF DRAWINGS

[0019] In order to more clearly illustrate the technical solutions in the embodiments of the application or the prior art, the following will briefly introduce the drawings needed to be used in the embodiments or the prior art description. Obviously, the drawings described below only relate to some embodiments of the present disclosure, rather than limit the application.

[0020] Figure 1 is an EPR spectrum of free radicals generated by a free radical generation system with hydrogen peroxide as an enhancer under ultraviolet light, Figure 1 , wherein a is an EPR spectrum of a pyruvic acid light irradiation system and a light irradiation system after adding hydrogen peroxide, Figure 1 , wherein b is a secondary integral spectrum of the EPR spectrum; Figure 2 is a quantitative relationship (standard curve) between the concentration of 4-hydroxy TEMPO free radicals and the secondary integral signal (DII) of the ESR spectrum; Figure 3 is the influence of different hydrogen peroxide concentrations on the yield of pyruvic acid free radicals; Figure 4 is the relationship between the irradiation time and the yield of free radicals under different hydrogen peroxide concentrations; Figure 5 is an EPR spectrum of free radicals generated by a free radical generation system with titanium dioxide as an enhancer under ultraviolet light, Figure 5 , wherein a is an EPR spectrum of a pyruvic acid light irradiation system and a light irradiation system after adding titanium dioxide, Figure 5 , wherein b is a secondary integral spectrum of the EPR spectrum; Figure 6 is the influence of different mass concentrations of titanium dioxide on the yield of pyruvic acid free radicals; Figure 7The relationship between the light irradiation time and the radical yield in the radical generation system with titanium dioxide as the enhancer; Figure 8 The influence of the deuterated reagent, Figure 8 a in the formula (1) is the EPR signal of the pyruvic acid system and the radical generated by the light excitation after adding deuterated / non-deuterated hydrogen peroxide, Figure 8 b in the formula (1) is the first integral of the EPR signal, Figure 8 c in the formula (1) is the radical concentration of the pyruvic acid system and after adding deuterated / non-deuterated hydrogen peroxide; Figure 9 The influence of hydrogen peroxide on the radical yield of the light excitation of other α-dicarbonyl structure molecules, Figure 9 a in the formula (2) is the EPR signal of the α-ketoglutaric acid system and the radical generated by the light excitation after adding hydrogen peroxide, Figure 9 b in the formula (2) is the EPR signal of the butanedione system and the radical generated by the light excitation after adding hydrogen peroxide; Figure 10 The influence of hydrogen peroxide on the radical yield of the light excitation of other α-dicarbonyl structure molecules with the extension of the light irradiation time, Figure 10 a in the formula (3) is the change trend of the radical concentration of the α-ketoglutaric acid system and the radical generated by the light excitation after adding hydrogen peroxide with the extension of the light irradiation time, Figure 10 b in the formula (3) is the change trend of the radical concentration of the butanedione system and the radical generated by the light excitation after adding hydrogen peroxide with the extension of the light irradiation time; Figure 11 The influence of titanium dioxide on the radical yield of the light excitation of other α-dicarbonyl structure molecules, Figure 11 a in the formula (4) is the EPR signal of the oxalic acid system and the radical generated by the light excitation after adding titanium dioxide, Figure 11 b in the formula (4) is the EPR signal of the diethyl oxalate system and the radical generated by the light excitation after adding titanium dioxide; Figure 12 The influence of titanium dioxide on the radical yield of the light excitation of oxalic acid molecules with the extension of the light irradiation time; Figure 13 The mechanism of the transient radical generated by the ultraviolet light irradiation of pyruvic acid. DETAILED DESCRIPTION

[0021] The application provides application of hydrogen peroxide and / or titanium dioxide as a radical enhancer in a melt dynamic nuclear polarization α-keto acid light excitation radical enhancement generation system.

[0022] The application provides an alpha-keto acid photo-excited free radical enhanced generation system for melt dynamic nuclear polarization, which comprises a glassy reagent, an alpha-keto acid photosensitive precursor and a free radical enhancer; the glassy reagent is an alcohol-water system, the deuterium substitution rate of alcohol in the alcohol-water system is 0-100 %, and the free radical enhancer comprises hydrogen peroxide and / or titanium dioxide, and the deuterium substitution rate of the hydrogen peroxide is 0-100 %.

[0023] In the application, all the preparation raw materials / components are commercially available products known to those skilled in the art without special instructions.

[0024] The alpha-keto acid photo-excited free radical enhanced generation system for melt dynamic nuclear polarization provided by the application comprises a glassy reagent. The glassy reagent is an alcohol-water system, and the deuterium substitution rate of alcohol in the alcohol-water system is 0-100 %. The deuterium substitution rate of water in the alcohol-water system is 0-100 %. In the examples, the water in the alcohol-water system can be deionized water. In the application, the glassy reagent preferably comprises a glycerol-water system or an ethanol-water system. The volume ratio of glycerol to water in the glycerol-water system is preferably 1:9-9:1, more preferably 3:7-8:2, further preferably 5:5-7:3, and in the examples, it can be 6:4. The volume ratio of ethanol to water in the ethanol-water system is preferably 1:9-9:1, more preferably 3:7-8:2, further preferably 5:5-7:3, and in the examples, it can be 6:4. The deuterium substitution rate of glycerol, ethanol and water in the glassy reagent of the application is 0-100 %.

[0025] The alpha-keto acid photo-excited free radical enhanced generation system for melt dynamic nuclear polarization provided by the application comprises an alpha-keto acid photosensitive precursor. The alpha-keto acid photosensitive precursor is an alpha-dicarbonyl structure molecule. The alpha-keto acid photosensitive precursor preferably comprises one or more of alpha-diketone, alpha-keto acid and alpha-keto acid derivative. The alpha-diketone preferably comprises butanedione. The alpha-keto acid preferably comprises one or more of pyruvic acid, alpha-ketoglutaric acid, oxalacetic acid, oxalic acid and phenylglyoxylic acid, and in the examples, it can be pyruvic acid. The alpha-keto acid derivative preferably comprises diethyl oxalate.

[0026] The application does not have special limitations on the molar concentration of the alpha-keto acid photosensitive precursor in the alpha-keto acid photo-excited free radical enhanced generation system, which is determined by the solubility of the alpha-keto acid photosensitive precursor. In the specific examples of the application, when the alpha-keto acid photosensitive precursor is specifically pyruvic acid, the molar concentration of pyruvic acid in the alpha-keto acid photo-excited free radical enhanced generation system can be 7.19 mol / L.

[0027] The alpha-keto acid photo-excited free radical enhanced production system for dynamic nuclear polarization melting provided by the present application comprises a free radical enhancer. The free radical enhancer comprises hydrogen peroxide and / or titanium dioxide. The deuterium enrichment of the hydrogen peroxide is 0-100%. The particle size of the titanium dioxide is preferably nanoscale. The titanium dioxide can be rutile titanium dioxide. The particle size of the titanium dioxide is preferably 10-500 nm, and in the embodiments, it can be 100-300 nm.

[0028] In the present application, when the free radical enhancer is hydrogen peroxide, the hydrogen peroxide is preferably used in the form of an aqueous hydrogen peroxide solution or a hydrogen peroxide generation system. The content of H2O2 in the aqueous hydrogen peroxide solution is preferably 1-10 wt%, and in the embodiments, it can be 3 wt%. The hydrogen peroxide generation system preferably comprises a water and metal peroxide reaction system or a water photo and / or acoustic catalytic reaction system. The metal peroxide preferably comprises sodium peroxide and / or calcium peroxide. The water photo and / or acoustic catalytic reaction system preferably generates hydrogen peroxide by light irradiation of a catalyst in the water system and / or generates hydrogen peroxide by cavitation of ultrasound. The present application does not have special limitations on the specific operation of the water and metal peroxide reaction system or the water photo and / or acoustic catalytic reaction system to generate hydrogen peroxide.

[0029] In the present application, the molar concentration of hydrogen peroxide in the alpha-keto acid photo-excited free radical enhanced production system is preferably 1 μM-100 mM, more preferably 10 μM-100 mM, further preferably 100 μM-1 mM, and in the embodiments, it can be 10 μM, 20 μM, 100 μM, 500 μM, 1 mM, 10 mM or 100 mM.

[0030] In the present application, when the free radical enhancer is titanium dioxide, the mass concentration of titanium dioxide in the alpha-keto acid photo-excited free radical enhanced production system is preferably 0.789 μg / L-0.789 g / L, and in the embodiments, it can be 78.9 μg / L, 7.89 mg / L or 0.789 g / L.

[0031] The application provides a preparation method of an alpha-keto acid photo-excited free radical enhancement generation system for melt dynamic nuclear polarization, and the method comprises the following steps: pre-mixing a glassy reagent and a free radical enhancer to obtain a mixed solvent containing the enhancer; and mixing the mixed solvent containing the enhancer and an alpha-keto acid photosensitive precursor to obtain the alpha-keto acid photo-excited free radical enhancement generation system (i.e. a system to be excited) for melt dynamic nuclear polarization. In the application, the pre-mixing is preferably performed at room temperature (i.e. without the need for temperature increase or decrease). The pre-mixing is preferably performed under vortex oscillation, and the pre-mixing time is preferably 1-5 min. In the application, the pre-mixing is performed under vortex oscillation, so that the free radical enhancer is uniformly dispersed in the glassy reagent.

[0032] The application provides a photo-excited free radical generation method for melt dynamic nuclear polarization, and the method comprises the following steps: The alpha-keto acid photo-excited free radical enhancement generation system is frozen to obtain microbeads. The microbeads are photo-excited to generate transient free radicals.

[0033] The alpha-keto acid photo-excited free radical enhancement generation system is frozen to obtain microbeads (i.e. frozen microbeads). In the application, the freezing temperature is preferably < 77 K. The freezing preferably comprises: dropping the alpha-keto acid photo-excited free radical enhancement generation system into liquid nitrogen to freeze into microbeads. In the application, the alpha-keto acid photo-excited free radical enhancement generation system is preferably taken out by a pipette and dropped into liquid nitrogen. In the application, the diameter of the microbeads is preferably 0.5-5 mm, and in the examples, the diameter can be 2-3 mm. The volume of each microbead can be 4-5 μL.

[0034] After obtaining the microbeads, the microbeads are photo-excited to generate transient free radicals. In the application, the photo-excitation is performed in a low-temperature environment, and the temperature of the low-temperature environment is preferably < 120 K, more preferably < 80 K, and in the examples, the temperature of the low-temperature environment can be < 77 K. In the application, the microbeads are preferably soaked in liquid nitrogen to form a low-temperature condition. In the examples of the application, the microbeads are preferably transferred to a finger-shaped quartz dewar filled with liquid nitrogen, and then the photo-excitation is performed.

[0035] In the application, the light source used for the photo-excitation is preferably ultraviolet light and / or visible light. In the application, the wavelength range of the light source is preferably 200-800 nm. The light source is preferably a continuous wave or a pulsed mode wave. The light source is preferably coherent or incoherent light mode. When the wavelength range of the light source used for the photo-excitation is 200-600 nm, the conditions of the photo-excitation preferably comprise: the light irradiation power is preferably 2-40 W / cm2 The illumination time is preferably 1~600 s, more preferably 5~500 s, and in this embodiment it can be 300 s. When the wavelength of the light source used for photoexcitation is preferably 254 nm, the photoexcitation conditions preferably include: the illumination power is preferably 0.6~25 mW / cm². 2 In the embodiment, it can be 25 mW / cm 2 The illumination time is preferably 5 to 60 min, more preferably 5 to 40 min, and even more preferably 5 to 35 min. In the embodiments, it can be 5 min, 10 min, 15 min, 20 min, 25 min, 30 min, or 35 min.

[0036] This invention uses a light source with a suitable wavelength range to irradiate microbeads for a suitable time to obtain the required concentration of photoinduced free radicals. Then, the microbeads are transferred to the polarizer of the fusion dynamic nuclear polarization system, where the photoinduced transient free radicals act as polarizing agents to polarize the low-γ nucleus of the target α-keto acid molecule.

[0037] To further illustrate the present invention, the technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0038] The technical and scientific terms used in the following embodiments have the same meanings as commonly understood by those skilled in the art. Unless otherwise specified, basic raw materials, reagents, and consumables are available from normal commercial sources. The room temperature described in this invention is 25–30°C. The electron paramagnetic resonance spectrometer used is a Bruker ELEXSYS E 580 or a Bruker A200. This invention provides a general and specific description of the materials and experimental methods used in the experiments. Although many organic synthesis materials and procedures used to achieve the objectives of this invention are well known in the art, they are still described herein as comprehensively as possible.

[0039] Example 1: Free radical generation system with hydrogen peroxide as an enhancer Step 1: Solvent preparation: Measure 6 mL of glycerol and 4 mL of deionized water, pour them into a 50 mL centrifuge tube, vortex for 5 min to obtain a homogeneous glycerol-water mixed solvent; Step 2: Addition of enhancer: Take a 3% hydrogen peroxide aqueous solution and slowly add it dropwise to the glycerol-water mixed solution prepared in Step 1. After the addition is complete, vortex at room temperature for 5 min to uniformly disperse the hydrogen peroxide and obtain a mixed system with a hydrogen peroxide concentration of 20 μM to 200 mM. Step 3: Precursor dissolution: Take pyruvate solution and add it to the above mixture at a volume ratio of 1:1. Vortex at room temperature for 5 min until fully mixed and homogeneous to form an excitation system with pyruvate concentration of 7.19 mol / L and hydrogen peroxide concentration of 10 μM~100 mM. Step 4: Low-temperature excitation: Using a pipette, pipette 5.00 μL of the system to be excited and drop it into a hemispherical low-temperature Dewar filled with liquid nitrogen, freezing it into microbeads with a diameter of 2-3 mm. Repeat this process until the number of microbeads produced meets the experimental requirements. Transfer a specified number (4-20 beads) of microbeads to a finger-shaped quartz Dewar filled with liquid nitrogen. Then, use a light source with a wavelength range of 280-480 nm and an illumination power of 2-40 W / cm². 2 The microbeads immersed in liquid nitrogen were irradiated for a specified time (5~500 s) using a light source (Dymax Bluewave 200), or a light source with a wavelength of 254 nm and an illumination power of 0.6~25 mW / cm² was used. 2 By irradiating microbeads immersed in liquid nitrogen with a light source (UVGo UVC-LED) for a specified time (5~40 min), the efficient generation of transient free radicals can be achieved.

[0040] Step 5: Free Radical Detection: A finger-shaped quartz Dewar containing the illuminated microbead sample was inserted into the resonant cavity of an electron paramagnetic resonance (EPR, Bruker A200) spectrometer. Fixed experimental parameters were tuned and set for X-band continuous wave EPR scanning. The EPR test parameters were as follows: temperature 77 K, microwave power 60.84 mW, modulation field amplitude 1 G, spectral width 3265~3465 G, single-point scan time 5.12 ms, number of scan points 512, and cumulative scans 5 times. Each sample group underwent the above experimental steps three times. Free radical detection revealed a significant increase in free radical concentration in the system with added hydrogen peroxide compared to the control group without added hydrogen peroxide (results are shown in Figure 1). Figure 1 As shown, Figure 1 In the figure, 'a' represents the EPR spectrum of the pyruvate-illuminated system and the system illuminated with added hydrogen peroxide. Figure 1 In this context, b represents the quadratic integral plot of the EPR spectral line.

[0041] Step 6: Standard Curve Plotting: The standard curve required for quantifying free radical concentration is plotted using known concentrations of stable free radicals (without photoexcitation) 4-hydroxy TEMPO (20.00, 40.00, 50.00, 80.00, 100.00 mM). Liquid nitrogen beads (four 5.00 μL frozen beads, total volume 20.00 μL) are dropped into the solution using the same method. EPR is then used for detection, baseline correction is performed, and the quadratic integral (DII) value is calculated. (e.g.) Figure 2 As shown, Figure 2This is a standard curve of 4-hydroxy TEMPO radical concentration versus ESR line double integral signal (DII).

[0042] Step 7: Quantitative Analysis of Free Radical Concentration: The concentration of free radicals generated by photoexcitation was calculated using TEMPO free radical concentration as a reference standard. Based on the above quantitative analysis of free radical concentration, the concentration of pyruvate free radicals in the free radical system with added hydrogen peroxide was 1 to 10 times higher than that in the control group without added hydrogen peroxide.

[0043] Example 2: Experiment on the relationship between hydrogen peroxide solutions of different concentrations and free radical production Experimental methods: Based on the basic system of Example 1, the solvent ratio (glycerol:water = 6:4, total volume 1 mL), pyruvate concentration (7.19 mol / L), and UV / Vis excitation conditions (wavelength range 280~480 nm, light power 2~40 W / cm²) were fixed. 2 The irradiation time was 300 s, and the low-temperature parameters (liquid nitrogen, <77K) were varied, with only the amount of hydrogen peroxide (3% aqueous solution) added. Seven concentration gradient groups were set up as follows: control group (0 μM), group 1 (10 μM), group 2 (100 μM), group 3 (500 μM), group 4 (1 mM), group 5 (10 mM), and group 6 (100 mM). At least three samples were prepared in parallel for each group. The excitation system was prepared according to the steps in Example 1 and excited by light irradiation. Using consistent EPR detection parameters (temperature 77 K, microwave power 60.84 mW, modulation field amplitude 1 G, scan width 200 G, total scan time 13.11 s) and quantitative methods (using 4-hydroxyTEMPO as a standard to plot a standard curve, and calculating the concentration by quadratic integration), the free radical concentrations of each group were measured, and the average values ​​were used for statistical analysis.

[0044] Experimental results: The effects of different hydrogen peroxide concentrations on pyruvate free radical production are shown in the following data. Figure 3 As shown, the relative yield is calculated with the free radical concentration of the control group as 1. The results show that the relationship between hydrogen peroxide concentration and free radical yield initially increases, then stabilizes, and then decreases: when the concentration increases from 10 μM to 100 μM, the free radical concentration increases from 26.66 mM to 48.01 mM, and the relative yield increases from 1.68 to 3.06; when the concentration is in the range of 100 μM to 1 mM, the free radical concentration remains at 3.05–3.12 mM, reaching a peak plateau; when the concentration increases to 10 mM, the free radical concentration decreases to 2.21 mM, presumably due to the consumption of free radicals by side reactions triggered by high-concentration hydrogen peroxide. In summary, the optimal concentration range for hydrogen peroxide addition is 100 μM to 1 mM.

[0045] Example 3: Experiment on the relationship between illumination time and free radical production Experimental Methods: Based on the basic system of Example 1, with fixed solvent ratio (glycerol:water = 6:4, total volume 1 mL), pyruvate concentration (7.19 mol / L), and low temperature parameters (liquid nitrogen, <77K), ultraviolet excitation conditions with relatively weak light power (wavelength 254 nm, power 25 mW / cm²) were selected. 2 The experiment was conducted to obtain light-time dependent information by irradiation time (5–35 min). Only two variables were set: the final concentration of hydrogen peroxide (3% aqueous solution) and the irradiation time. Five gradient groups of hydrogen peroxide concentration were set as follows: control group (0 μM), group 1 (10 μM), group 2 (100 μM), group 3 (1 mM), and group 4 (10 mM). Each concentration group corresponded to seven irradiation time gradients: 5 min, 10 min, 15 min, 20 min, 25 min, 30 min, and 35 min. At least three samples were prepared in parallel for each group. The excitation system was prepared according to the steps in Example 1 and excitation was completed by irradiation. Consistent EPR detection parameters (temperature 77 K, microwave power 60.84 mW, modulation field amplitude 1 G, scan width 200 G, total scan time 13.11 s) and quantitative methods (using 4-hydroxyTEMPO as a standard to plot a standard curve, and calculating the concentration by quadratic integration) were used to determine the free radical concentration in each group and plot the trend of free radical concentration with increasing irradiation time.

[0046] Experimental results: The relationship between light exposure time and free radical production under different hydrogen peroxide concentrations is as follows: Figure 4 As shown, the relative yield is calculated with the free radical concentration of the control group after 5 minutes of illumination as 1. The results indicate that the free radical system using hydrogen peroxide as an enhancer exhibits hydrogen peroxide concentration dependence and illumination time saturation characteristics. Under the same illumination time, the trend of free radical concentration with hydrogen peroxide concentration was consistent with that in Example 2. The free radical yield was relatively high in the range of 100 μM to 1 mM. The free radical concentrations of Group 2 (100 μM), Group 3 (1 mM), and Group 4 (10 mM) were significantly higher than those of other groups. Under the same concentration, the free radical concentration first increased rapidly with the extension of illumination time and then tended to stabilize. The low concentration groups (control group and Group 1) had a shorter saturation time (saturation began at 15-20 min, and there was no significant change in yield after the extension of illumination time). The high-efficiency concentration groups (Group 2, Group 3, and Group 4) continued to increase the free radical yield after 20-25 min and finally saturated at 30-35 min. The optimal combination was Group 2 (100 μM) with illumination for 35 min. At this time, the free radical concentration reached 62.66 mM, and the relative yield was 9.58 times, with a significant increase in free radical yield.

[0047] Example 4: Free radical generation system with titanium dioxide as a reinforcing agent Step 1: Solvent preparation: Measure 6 mL of glycerol and 4 mL of deionized water, pour them into a 50 mL centrifuge tube, vortex for 5 min to obtain a homogeneous glycerol-water mixed solvent; Step 2: Addition of reinforcing agent: Weigh the corresponding mass of nano-titanium dioxide (particle size 10~500 nm), add it to the glycerol-water mixed solvent prepared in Step 1 above, and vortex at room temperature for 5 min to uniformly disperse the titanium dioxide, so as to obtain a mixed system with a nano-titanium dioxide mass concentration of 1.578 μg / L~1.578 g / L. Step 3: Precursor dissolution: Take pyruvate solution and add it to the above mixture at a volume ratio of 1:1. Vortex at room temperature for 5 min until fully mixed and homogeneous to form an excitation system with pyruvate concentration of 7.19 mol / L and nano titanium dioxide mass concentration of 0.789 μg / L~0.789 g / L. Step 4: Low-temperature excitation: Using a pipette, pipette 5.00 μL of the system to be excited and drop it into a hemispherical low-temperature Dewar filled with liquid nitrogen, freezing it into microbeads with a diameter of 2-3 mm. Repeat this process until the number of microbeads produced meets the experimental requirements. Transfer a specified number (4-20 beads) of microbeads to a finger-shaped quartz Dewar filled with liquid nitrogen. Then, use a light source with a wavelength range of 280-480 nm and an illumination power of 2-40 W / cm². 2 The microbeads immersed in liquid nitrogen were irradiated for a specified time (5~500 s) using a light source (Dymax Bluewave 200), or a light source with a wavelength of 254 nm and an illumination power of 0.6~25 mW / cm² was used. 2 By irradiating microbeads immersed in liquid nitrogen with a light source (UVGo UVC-LED) for a specified time (5~40 min), the efficient generation of transient free radicals can be achieved.

[0048] Step 5: Free Radical Detection: A finger-shaped quartz Dewar containing the illuminated microbead sample was inserted into the resonant cavity of an electron paramagnetic resonance (EPR, Bruker A200) spectrometer. Fixed experimental parameters were tuned and set for X-band continuous wave EPR scanning. The EPR test parameters were as follows: temperature 77 K, microwave power 60.84 mW, modulation field amplitude 1 G, spectral width 3265~3465 G, single-point scan time 5.12 ms, number of scan points 512, and cumulative scan count 5. Each sample group underwent the above experimental steps three times. Free radical detection revealed that the free radical concentration in the titanium dioxide-added system was significantly increased compared to the control group without titanium dioxide (e.g., ...). Figure 5 As shown, Figure 5 In the figure, 'a' represents the EPR spectrum of the pyruvate-illuminated system and the system after adding titanium dioxide. Figure 5In the diagram, b represents the quadratic integral plot of the EPR spectral lines. Figure 5 The molar concentration of titanium dioxide in the sample is 100 μM, which corresponds to a mass concentration of 7.89 mg / L.

[0049] Step 6: Quantitative analysis of free radical concentration: The concentration of free radicals generated by photoexcitation was calculated using the aforementioned TEMPO free radical concentration standard curve as a reference.

[0050] Example 5: Experiment on the relationship between titanium dioxide of different mass concentrations and free radical production Experimental Methods: Based on the basic system of Example 4, the experimental parameters were fixed as follows: solvent ratio of glycerol-water = 6:4 (total volume 1 mL), pyruvate concentration 7.19 mol / L, UV / Vis excitation conditions of wavelength range 280~480 nm, and light power of 2~40 W / cm². 2 The irradiation time was 300 s, and the low-temperature environment was liquid nitrogen (<77K). Only the amount of nano-titanium dioxide (particle size 100~300nm, rutile) added was varied, and four mass concentration gradient groups were set up as follows: control group (0 μg / L), group 1 (78.9 μg / L), group 2 (7.89 mg / L), and group 3 (0.789 g / L). At least three samples were prepared in parallel for each group. The excitation system was prepared according to the steps in Example 4 and excited by light irradiation. During the addition of the reinforcing agent, vortexing for 5 min was required to ensure uniform dispersion of the nano-titanium dioxide. Using the same EPR detection parameters as in Example 4 (temperature 77 K, microwave power 60.84 mW, modulation field amplitude 1 G, scan width 200 G, total scan time 13.11 s) and quantitative method (using 4-hydroxyTEMPO as a standard to plot a standard curve, and calculating the concentration by quadratic integration), the free radical concentrations of each group were measured, and the average values ​​were used for statistical analysis.

[0051] Experimental results: The effect of different mass concentrations of titanium dioxide on the production of pyruvate free radicals is shown in the following data. Figure 6As shown, the relative yield is calculated with the free radical concentration of the control group as 1. The results show that the free radical concentrations in all experimental groups with added titanium dioxide were significantly higher than those in the control group, and there were no significant differences between different concentration groups. After adding a low concentration of titanium dioxide (78.9 μg / L), the free radical concentration increased from 15.58 mM to 50.66 mM, with a relative yield increase of 3.25 times. This suggests that the presence of titanium dioxide increases the number of photocatalytic active sites, promoting free radical generation. With higher concentrations of titanium dioxide (7.89 mg / L~0.789 g / L), the free radical concentration remained between 43.19 and 50.66 mM, with no significant fluctuations between groups. This suggests that the number of catalytic active sites in the titanium dioxide system at low concentrations is sufficient to match the reaction requirements. Based on the combined data from each group and considering the titanium dioxide removal steps required after d-DNP dissolution, a lower titanium dioxide concentration (78.9 μg / L) can be selected. This allows the system to achieve a high free radical concentration while avoiding negative impacts on probe molecule signal relaxation after dissolution.

[0052] Example 6: Experiment on the relationship between illumination time and free radical production Experimental Methods: Based on the basic system of Example 4, the experimental parameters were fixed as follows: solvent ratio of glycerol-water = 6:4 (total volume 1 mL), pyruvate concentration 7.19 mol / L, and ultraviolet excitation conditions with relatively weak light power (wavelength 254 nm, power 25 mW / cm²) were selected. 2 The irradiation time was 5–35 min, and the low-temperature environment was liquid nitrogen (<77 K). Only the irradiation time was set as a variable, and two control groups were set up: a control group (0 μg / L titanium dioxide) and an experimental group (78.9 μg / L titanium dioxide, a typical low-concentration value from Experiment 3). Each group corresponded to 7 irradiation time gradients: 5 min, 10 min, 15 min, 20 min, 25 min, 30 min, and 35 min. At least 3 samples were prepared in parallel for each group. The system to be excited was prepared according to the steps in Example 4 and excited by light irradiation. During the addition of the reinforcing agent, vortexing was required for 5 min to ensure uniform dispersion of the nano-titanium dioxide. Using the same EPR detection parameters as in Example 4 (temperature 77 K, microwave power 60.84 mW, modulation field amplitude 1 G, scan width 200 G, total scan time 13.11 s) and quantitative methods (using 4-hydroxyTEMPO as a standard to plot a standard curve, and calculating the concentration by quadratic integration), the free radical concentration of each group was measured, and the trend of free radical concentration with increasing irradiation time was plotted.

[0053] Experimental results: The relationship between illumination time and free radical production in 78.9 μg / L titanium dioxide and the control group is as follows: Figure 7As shown, the relative yield is calculated by taking the free radical concentration of the control group after 5 min of illumination as 1. The results showed that the free radical concentration of the experimental group (78.9 μg / L) was significantly higher than that of the control group at all time points, and exhibited a characteristic of "gradually increasing with the extension of illumination time": as the illumination time increased from 5 min to 25 min, the free radical concentration of the experimental group increased from 7.33 mM to 31.63 mM, and the relative yield increased from 1.36 times to 5.88 times. After 25 min, the upward trend slowed down, but no saturation was observed. The free radical concentration of the control group increased slowly with the extension of illumination time, and a plateau was observed after 25 min, with the saturated free radical concentration being only 15.61~18.32 mM, indicating that the promoting effect of single illumination on free radical generation is limited. In summary, when 78.9 μg / L titanium dioxide was combined with 35 min of low-power illumination, the free radical concentration reached 43.06 mM, and the free radical yield was increased by 8.00 times compared with the control group.

[0054] Example 7: Effect of deuterated reagent on free radical production in a free radical generation system enhanced by hydrogen peroxide Experimental methods: Based on the basic system of Example 1, the solvent ratio (glycerol:water = 6:4, total volume 1 mL), pyruvate concentration (7.19 mol / L), hydrogen peroxide addition (to maintain a final concentration of 100 μM), and UV / Vis excitation conditions (wavelength range 280–480 nm, light power 2–40 W / cm²) were fixed. 2 The experiment used a controlled irradiation time of 180 s and low-temperature parameters (liquid nitrogen, <77K). Deuterated reagents were used instead of the standard reagents. Three experimental groups were set up: control group (pyruvate + glycerol + water, PGW), group 1 (pyruvate + glycerol + water + hydrogen peroxide, PGW-H2O2), and group 2 (pyruvate + glycerol + water + deuterated hydrogen peroxide, PGW-D2O2). At least three samples were prepared in parallel for each group. The excitation system was prepared according to the steps in Example 1 and excited by light irradiation. Consistent EPR detection parameters (temperature 77 K, microwave power 30.03 mW, modulation field amplitude 1 G, scan width 200 G, total scan time 51.2 s) and quantitative methods (using 4-hydroxyTEMPO as a standard to plot a standard curve, and calculating the concentration through quadratic integration) were used to determine the free radical concentrations of each group, and the average values ​​were used for statistical analysis.

[0055] Experimental results: The effect of deuterated hydrogen peroxide on the production of pyruvate free radicals is shown in the following data. Figure 8 As shown, Figure 8 This is due to the influence of deuterated reagents. Figure 8 In the figure, 'a' represents the EPR signal of free radicals generated by photoexcitation of the pyruvate system and the addition of deuterated / undeuterated hydrogen peroxide. Figure 8 In this context, b represents the first integral of the EPR signal.Figure 8 In the figure, 'c' represents the pyruvate system and the free radical concentration after adding deuterated / undeuterated hydrogen peroxide. The relative yield is calculated with the control group's free radical concentration as 1. The results show that deuterated hydrogen peroxide yields pyruvate free radicals comparable to undeuterated hydrogen peroxide; furthermore, the baseline width of pyruvate free radicals excited by the deuterated hydrogen peroxide group is narrower, theoretically making it more suitable for d-DNP applications. In conclusion, the strategy of deuterating hydrogen peroxide is feasible and can reduce the free radical baseline width to some extent without affecting the free radical yield.

[0056] Example 8: Hydrogen peroxide enhances the free radical yield of other α-dicarbonyl molecular structures. Experimental methods: Based on the basic system of Example 1, the solvent ratio (ethanol:water = 6:4, total volume 1 mL), the amount of hydrogen peroxide added (to maintain a final concentration of 20 μM), and the UV / Vis excitation conditions (wavelength range of 280–480 nm, illumination power of 2–40 W / cm²) were fixed. 2 The experiment used a single irradiation time of 180 s and low temperature parameters (liquid nitrogen, <77K). Only molecules with different α-dicarbonyl structures were used as free radical precursors. The free radical precursor group was set up as the control group and the free radical precursor + hydrogen peroxide group was set up as the experimental group. The free radical precursors used were as follows: α-ketoglutarate (α-KG, 0.5 M) and butanedione (BTO, 2.3 M).

[0057] At least three samples were prepared in parallel for each group. The system to be excited was prepared according to the steps in Example 1 and excited by light irradiation. Using consistent EPR detection parameters (temperature 77 K, microwave power 30.03 mW, modulation field amplitude 1 G, scan width 200 G, total scan time 51.2 s) and quantitative methods (using 4-hydroxyTEMPO as a standard to plot a standard curve, and calculating the concentration by quadratic integration), the free radical concentrations of each group were measured and the average values ​​were taken for statistical analysis.

[0058] Experimental results: Data on the effect of hydrogen peroxide on the free radical yield of other α-dicarbonyl molecular structures are as follows: Figure 9 , Figure 10 As shown, Figure 9 The effect of hydrogen peroxide on the photoexcited radical yield of other α-dicarbonyl molecules. Figure 9 In this context, 'a' represents the α-ketoglutarate system and the EPR signal of the free radicals generated by photoexcitation after the addition of hydrogen peroxide. Figure 9 In the figure, b represents the butanedione system and the EPR signal of the free radicals generated by photoexcitation after the addition of hydrogen peroxide. Figure 10 The effect of hydrogen peroxide on the photoexcited radical yield of other α-dicarbonyl molecules with prolonged light exposure time. Figure 10In the figure, 'a' represents the α-ketoglutaric acid system and the trend of the concentration of free radicals generated by photoexcitation after the addition of hydrogen peroxide as a function of light exposure time. Figure 10 In the figure, b represents the trend of the concentration of free radicals generated by photoexcitation after the addition of hydrogen peroxide in the dimethyl ethyl ketone system as a function of light exposure time. The relative yield is calculated with the free radical concentration of the control group as 1. The results show that hydrogen peroxide can increase the free radical yield of other α-dicarbonyl molecules: for α-ketoglutaric acid, the addition of 20 μM hydrogen peroxide increased the relative yield of photoexcited free radicals by 1.29–1.55 times, and the free radical concentration after light exposure saturation increased from 15.08 mM to 24.04 mM; for dimethyl ethyl ketone, the addition of 20 μM hydrogen peroxide increased the relative yield of photoexcited free radicals by 1.53–1.69 times, ultimately reaching a free radical concentration of approximately 7.95 mM.

[0059] Example 9: Free radical generation system using titanium dioxide as a reinforcing agent; experiment on the enhancement of free radical production of other α-dicarbonyl molecular structures by titanium dioxide. Experimental Methods: Based on the basic system of Example 2, the experimental parameters were fixed as follows: solvent ratio of ethanol-water = 6:4 (total volume 1 mL); UV / Vis excitation conditions: wavelength range of 280-480 nm; light power of 2-40 W / cm². 2 The single irradiation time was 180 s, and the low-temperature environment was liquid nitrogen (<77K). Different α-dicarbonyl molecules were used as free radical precursors. The free radical precursor group was set as the control group and the free radical precursor + titanium dioxide group was set as the experimental group. The free radical precursors used were as follows: oxalic acid (Oxa, 1 M) and diethyl oxalate (DEO, 1 M).

[0060] At least three samples were prepared in parallel for each group. The excitation system was prepared according to the steps in Example 2 and excited by light irradiation. During the addition of the reinforcing agent, vortexing for 5 min was required to ensure uniform dispersion of the nano-titanium dioxide. Using the same EPR detection parameters (temperature 77 K, microwave power 30.03 mW, modulation field amplitude 1 G, scan width 200 G, total scan time 51.2 s) and quantitative method (using 4-hydroxyTEMPO as a standard to plot a standard curve, and calculating the concentration by quadratic integration), the free radical concentration of each group was measured and the average value was used for statistical analysis.

[0061] Experimental results: The data on the effect of titanium dioxide on the free radical yield of other α-dicarbonyl molecular structures are as follows: Figure 11 , Figure 12 As shown, Figure 11 The effect of titanium dioxide on the photoexcited free radical yield of other α-dicarbonyl molecules. Figure 11 In the figure, 'a' represents the oxalic acid system and the EPR signal of the free radicals generated by photoexcitation after the addition of titanium dioxide.Figure 11 In the figure, b represents the diethyl oxalate system and the EPR signal of the free radicals generated by photoexcitation after the addition of titanium dioxide. Figure 12 The effect of titanium dioxide on the photoexcited radical yield of oxalic acid molecules with prolonged illumination time was investigated. The relative yield was calculated with the radical concentration of the control group as 1. The results show that titanium dioxide can increase the radical yield of other α-dicarbonyl molecules: for oxalic acid molecules, direct illumination or the addition of hydrogen peroxide to the oxalic acid system did not produce an effective concentration of radicals, but the addition of titanium dioxide effectively generated photoexcited radicals, achieving a breakthrough from "no" to "some," with the radical concentration reaching 20.83 mM after illumination time saturation; for diethyl oxalate molecules, the addition of titanium dioxide also enabled the generation of diethyl oxalate photoexcited radicals.

[0062] In summary, the above embodiments of the present invention have verified the feasibility of using hydrogen peroxide and titanium dioxide as free radical enhancers to improve the yield of photoexcited pyruvate free radicals through a photoexcited pyruvate system. Furthermore, the embodiments of the present invention, using pyruvate as a model α-keto acid, demonstrate that the addition of hydrogen peroxide or titanium dioxide to the photoexcited free radical system can achieve a several-fold to order-of-magnitude increase in the concentration of photoexcited free radicals compared to the system without the enhancer (wherein pyruvate can achieve a several-fold increase; the change from no signal to free radical generation in oxalic acid can be considered an order-of-magnitude characteristic).

[0063] As shown in the above embodiments, this invention provides an α-keto acid photoexcited radical enhancement system for molten DNP. This invention constructs a novel photoexcited radical generation system by introducing hydrogen peroxide or titanium dioxide as a radical enhancer into the α-keto acid photoexcited radical enhancement system. Under ultraviolet / visible light irradiation, the radical enhancer effectively induces free radical reactions in α-keto acid molecules, significantly improving the yield of transient free radicals. EPR detection confirms that the free radical concentration is several times or even orders of magnitude higher than that of the system without the enhancer. The enhancement system of this invention can provide a high-concentration free radical source that meets the requirements of DNP technology, promoting the efficiency and rate of the electron-nuclear polarization transfer process, and providing key technical support for the application of molten DNP technology in fields such as magnetic resonance imaging.

[0064] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. Other embodiments can be obtained based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. Use of hydrogen peroxide and / or titanium dioxide as a radical enhancer in a melt dynamic nuclear polarization alpha-keto acid photoexcited radical enhanced generation system.

2. An alpha-keto acid photo-activated radical-enhanced production system for melt dynamic nuclear polarization, characterized in that, The glassy reagent is an alcohol-water system, the deuterium enrichment of alcohol and water in the alcohol-water system is 0-100%, and the radical enhancer comprises hydrogen peroxide and / or titanium dioxide, and the deuterium enrichment of hydrogen peroxide is 0-100%.

3. The alpha-keto acid photo-activated radical generating system according to claim 2, wherein The glassy reagent comprises a glycerol-water system or an ethanol-water system, the volume ratio of glycerol to water in the glycerol-water system is 1:9-9:1, and the volume ratio of ethanol to water in the ethanol-water system is 1:9-9:

1.

4. The alpha-keto acid photo-activated radical generating system according to claim 3, wherein The volume ratio of glycerol to water in the glycerol-water system is 6:4, and the volume ratio of glycerol to water in the ethanol-water system is 6:

4.

5. The alpha-keto acid photo-activated radical generating system according to claim 2, wherein The alpha-keto acid photosensitive precursor comprises one or more of alpha-diketone, alpha-keto acid and alpha-keto acid derivative.

6. The alpha-keto acid photo-activated radical generating system according to claim 5, wherein The alpha-diketone comprises butanedione, the alpha-keto acid comprises one or more of pyruvic acid, alpha-ketoglutaric acid, oxalacetic acid, oxalic acid and benzoyl formic acid, and the alpha-keto acid derivative comprises diethyl oxalate.

7. The alpha-keto acid photo-activated radical generating system according to claim 2, wherein When the radical enhancer is hydrogen peroxide, the hydrogen peroxide is used in the form of a hydrogen peroxide aqueous solution or a hydrogen peroxide generation system, the hydrogen peroxide generation system comprises a water and metal peroxide reaction system or a water photo and / or acoustic catalytic reaction system, the metal peroxide comprises sodium peroxide and / or calcium peroxide, and the water photo and / or acoustic catalytic reaction system comprises a water system in which hydrogen peroxide is generated by light irradiation of a catalyst and / or by cavitation of ultrasound; The molar concentration of hydrogen peroxide in the alpha-keto acid photoexcited radical enhanced generation system is 1 μM-100 mM.

8. The alpha-keto acid photo-activated radical generating system according to claim 2, wherein The particle size of titanium dioxide is 10-500 nm, and when the radical enhancer is titanium dioxide, the mass concentration of titanium dioxide in the alpha-keto acid photoexcited radical enhanced generation system is 0.789 μg / L-0.789 g / L.

9. A photo-excitation radical generation method for dynamic nuclear polarization melting, characterized by, The method comprises the following steps: The alpha-keto acid photoexcited radical enhanced generation system according to any one of claims 2-8 is frozen to obtain microbeads; The microbeads are photoexcited to generate transient radicals.

10. The method of claim 9, wherein the laser is a frequency- doubled Nd:YAG laser. The diameter of the microbeads is 0.5-5 mm; the light source used for the photoexcitation is ultraviolet light and / or visible light, the wavelength range of the light source is 200-800 nm, the light source is a continuous wave or a pulsed mode wave, and the light source is a coherent or incoherent light mode; when the wavelength range of the light source used for the photoexcitation is 200-600 nm, the photoexcitation conditions include: the light irradiation power is 2-40 W / cm 2 , the light irradiation time is 1-600 s; when the wavelength of the light source used for the photoexcitation is 254 nm, the photoexcitation conditions include: the light irradiation power is 0.6-25 mW / cm 2 , the light irradiation time is 5-60 min; the photoexcitation is carried out in a low-temperature environment, and the temperature of the low-temperature environment is <120 K.

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