CN117884166A - Hydrogel kit for rapid quantitative detection of hydrogen peroxide and sarcosine, and preparation method and application thereof - Google Patents
Hydrogel kit for rapid quantitative detection of hydrogen peroxide and sarcosine, and preparation method and application thereof Download PDFInfo
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- CN117884166A CN117884166A CN202410081508.2A CN202410081508A CN117884166A CN 117884166 A CN117884166 A CN 117884166A CN 202410081508 A CN202410081508 A CN 202410081508A CN 117884166 A CN117884166 A CN 117884166A
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- sarcosine
- hydrogen peroxide
- hydrogel
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- FSYKKLYZXJSNPZ-UHFFFAOYSA-N sarcosine Chemical compound C[NH2+]CC([O-])=O FSYKKLYZXJSNPZ-UHFFFAOYSA-N 0.000 title claims abstract description 162
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- 238000005303 weighing Methods 0.000 description 1
Abstract
The invention provides a hydrogel kit for rapid quantitative detection of hydrogen peroxide and sarcosine, and a preparation method and application thereof, and belongs to the technical field of chemical detection. Adding cobalt nitrogen chlorine doped nano enzyme, 3', 5' -tetramethyl benzidine, hydrogel and acetic acid-sodium acetate buffer solution into deionized water, heating, stirring, mixing uniformly, cooling and solidifying to obtain the hydrogel kit for rapid quantitative detection of hydrogen peroxide and sarcosine. The invention provides a new thought for detecting hydrogen peroxide in food samples and sarcosine in biological samples, and lays a foundation for clinically analyzing and detecting other disease biomarkers except for prostate cancer.
Description
Technical Field
The invention relates to the technical field of chemical detection, in particular to a hydrogel kit for rapid quantitative detection of hydrogen peroxide and sarcosine, and a preparation method and application thereof.
Background
Hydrogen peroxide is widely used in disinfectant bactericides in food industry enterprises due to its strong oxidizing property, and is mainly used for sterilizing packages of dairy products. The use of hydrogen peroxide as an additive to retard the growth of microorganisms in milk has emerged in some areas to extend its freshness and prevent spoilage. Because of their adverse effects, which can have a damaging effect on human health, the concentration of hydrogen peroxide in finished milk is clearly defined in most countries, even if such substances are kept in forbidden limits. Several methods for measuring hydrogen peroxide in different types of samples have been reported, such as high performance liquid chromatography, colorimetry, surface enhanced raman scattering, chemiluminescence, fluorescence, and electrochemistry. The method is unfavorable for the rapid and accurate detection of the actual sample due to expensive instruments and strong specialization and limited operation places.
Prostate cancer (PCa) is one of the common tumors threatening the health of men, with mortality inferior to lung cancer. The main detection means of prostate cancer is to measure serum prostate specific antigen, which is an important index for diagnosing prostate cancer production and metastasis. However, in recent years, it has been found that this index is not completely reliable, and it is not possible to accurately determine whether a tumor is malignant or benign. Sarcosine (Sarcosine) is a methylated derivative of glycine, whose primary physiological function is to promote the synthesis of Adenosine Triphosphate (ATP), which is rarely found in urine. It has been found that the urine sarcosine level of prostate cancer patients is specifically increased and can be used as a marker for diagnosing prostate cancer. At present, various methods for detecting sarcosine, such as a colorimetry, a fluorescence method, an electrochemical method, an enzyme-linked immunosorbent assay and a high performance liquid chromatography, are available, the traditional measuring method is relatively expensive in analytical instrument, long in analytical time and needs professional testers to operate in fixed test places, so that inconvenient influence is caused for rapid diagnosis and treatment of diseases, and observation of self indexes by patients is not facilitated.
For detection analysis of the biomarker and other objects to be detected by using a natural enzyme-linked immunosorbent assay, in order to overcome the defect that natural enzymes are easy to denature in non-mild environments, the nanomaterial with the similar nature of the natural enzymes has the advantages of easy preparation, controllable size, adjustable catalytic activity, relatively stable non-mild detection environments, customizable composition structure and good biocompatibility, so that the nanomaterial has wide application in the aspects of disease treatment, tissue engineering, biosensing, food safety and environmental protection.
Colorimetric sensing analysis is a method for analyzing and detecting a target object by analyzing the color depth of a colored substance solution based on the color change of the solution caused by the target object. Colorimetric sensors have wide application due to their simplicity of operation, low cost, ease of visualization, and the need for complex instrumentation, which has led to their widespread use in such areas as detection of drugs in biological samples, disease biomarkers, etc., food quality control, contaminant trace detection, etc. The combination of the nano material and the traditional colorimetric method makes up the short plate of the traditional colorimetric method, so that the short plate has lower detection limit and higher sensitivity.
In recent years, eutectic solvents have received attention as a new generation of green alternative solvents to organic solvents and ionic liquids. When the components are mixed in a certain proportion, strong hydrogen bond interaction is established, and uniform and stable liquid is formed. One of the most widely used ingredients for forming the eutectic solvent is choline chloride, which can be combined with a variety of hydrogen bond donors, including urea, renewable carboxylic acids (e.g., oxalic acid, citric acid, succinic acid, or amino acids), or renewable polyols such as glycerol. The eutectic solvent has the advantages of good chemical stability, low volatility, simple preparation, no toxicity, biodegradability, good biocompatibility, low price and the like.
Hydrogels are novel materials composed of crosslinked three-dimensional (3D) networks of hydrophilic polymers, which, unlike two-dimensional materials, can absorb large amounts of liquids or fluids, can readily encapsulate bioactive molecules (e.g., nucleotides, proteins, antibodies, and drugs) and retain their bioactivity. Thus small molecule substances can be incorporated into the crosslinked network to tailor its structure while maintaining its 3D structure. In recent years, the advantages of biocompatibility, transparency, visibility, stretchability, swelling, conductivity and the like have been widely applied to the directions of tissue repair and regeneration, biosensors, contaminant recovery, drug delivery and the like.
At present, the content of the sarcosine is generally measured by liquid-gas chromatography in parallel and mass spectrometry, and the method uses expensive instruments, has complex operation and is not convenient for large-scale popularization and application. Enzymes are highly efficient biocatalysts, and almost all reactions in living beings involve enzymes. Enzyme analysis has wide application in clinical diagnosis, biology, food, chemistry, environment and other fields. The enzyme catalytic reaction is paid attention to because of the characteristics of high efficiency, specificity, mild conditions and the like. However, natural enzymes have limited sources, difficult purification and high price, and the experimental conditions and the operation environment are demanding in order to maintain the activity of the natural enzymes, so that the application of the natural enzymes is greatly limited, and therefore, the development and application research of the mimic enzymes are attracting more and more attention.
Therefore, development of a novel stimulus-responsive hydrogel based on eutectic solvents for colorimetric detection of hydrogen peroxide and sarcosine is of great significance for food safety and detection of clinical diseases.
Disclosure of Invention
The invention aims to provide a hydrogel kit for rapid quantitative detection of hydrogen peroxide and sarcosine, a preparation method and application thereof, and the hydrogel kit can be changed in color by dripping the sarcosine and sarcosine oxidase or hydrogen peroxide, and the convenient visual detection can be realized by converting an Image acquired by a smart phone into color intensity through Image J software, so that the use of a traditional expensive detection instrument is avoided, and inaccurate color is converted into data through software, so that a certain precision is improved. The analysis strategy provides a new thought for detecting hydrogen peroxide in food samples and sarcosine in biological samples, and lays a foundation for clinically analyzing and detecting other disease biomarkers except for the prostate cancer.
The technical scheme of the invention is realized as follows:
the invention provides cobalt nitrogen chlorine doped nano enzyme which is prepared by the following method: and (3) mixing choline chloride, proline and cobalt salt, heating and stirring to form a uniform transparent solution, cooling to room temperature, adding deionized water, performing ultrasonic treatment to completely dissolve the solution, transferring the mixture into a high-pressure reaction kettle, performing heating reaction to completely, cooling to room temperature, centrifuging, filtering, and freeze-drying the obtained solution to obtain the cobalt nitrogen chlorine doped nano-enzyme.
As a further improvement of the invention, the molar ratio of the choline chloride, the proline and the cobalt salt is 0.5-1.5:0.5-1.5:0.5-1.5, wherein the temperature of heating and stirring is 45-55 ℃, the power of ultrasonic is 1000-1200W, the time of ultrasonic is 5-15min, the temperature of heating reaction is 170-190 ℃, the time is 5-7h, the rotating speed of centrifugation is 5000-7000r/min, the time is 10-20min, and the filter membrane for filtration is a 0.22 mu m microporous filter membrane; the cobalt salt is at least one selected from cobalt chloride, cobalt sulfate and cobalt nitrate.
The invention further protects a rapid quantitative detection system for hydrogen peroxide and sarcosine, which comprises the cobalt nitrogen chlorine doped nano enzyme, 3', 5' -tetramethyl benzidine, acetic acid-sodium acetate buffer solution and deionized water, wherein the mass ratio of the cobalt nitrogen chlorine doped nano enzyme to the 3,3', 5' -tetramethyl benzidine to the acetic acid-sodium acetate buffer solution to the deionized water is (2-4): 2-3:3-5:5-7, wherein the concentration of the acetic acid-sodium acetate buffer solution is 0.1-0.3mmol/L.
The invention further protects a use method of the rapid quantitative detection system for hydrogen peroxide and sarcosine, a sample to be detected is diluted and then added into the detection system, the mixture is uniformly mixed, the mixture is incubated for 5 to 15 minutes at a temperature of between 36 and 38 ℃, and the ultraviolet absorption spectrum of the solution at 652nm is recorded for detection; the regression equation of sarcosine is y=0.00093x+0.2096, the linear range is 1 mu mol/L-200 mu mol/L, and the detection limit is 0.80 mu mol/L; regression equation for hydrogen peroxide was y=0.00097x+0.0552, linear range was 1. Mu. Mol/L-1000. Mu. Mol/L, and detection limit was 0.31. Mu. Mol/L.
The invention further provides a hydrogel kit for rapid quantitative detection of hydrogen peroxide and sarcosine, and the preparation method comprises the following steps: adding the cobalt nitrogen chlorine doped nano enzyme, 3', 5' -tetramethyl benzidine, hydrogel and acetic acid-sodium acetate buffer solution into deionized water, heating, stirring, mixing uniformly, cooling and solidifying to obtain the hydrogel kit for rapid quantitative detection of hydrogen peroxide and sarcosine.
As a further improvement of the invention, the mass ratio of the cobalt nitrogen chlorine doped nano enzyme to the 3,3', 5' -tetramethyl benzidine to the hydrogel to the acetic acid-sodium acetate buffer solution to the deionized water is 2-4:2-3:50-70:3-5:5-7, wherein the concentration of the acetic acid-sodium acetate buffer solution is 0.1-0.3mmol/L.
As a further improvement of the invention, the hydrogel is at least one selected from peptone, agar powder and modified hydrogel; the preparation method of the modified hydrogel comprises the following steps:
t1 preparation of carboxyl/aldehyde chitosan: dissolving chitosan in acid liquor, adding alpha-ketoglutaric acid, heating and stirring for reaction, then adding sodium borohydride, continuing the reaction, adding ethanol for precipitation, centrifuging, dissolving the solid in the acid liquor, adding sodium periodate, stirring for reaction in a dark place, adding ethanol for precipitation, centrifuging, washing, and drying to obtain carboxyl/aldehyde chitosan;
T2. preparation of modified chitosan: dissolving the carboxyl/aldehyde chitosan prepared in the step T1, 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide and N-hydroxy thiosuccinimide in water, uniformly mixing, adding a catalyst, heating and stirring for reaction, adding ethanol for precipitation, centrifuging, washing and drying to prepare modified chitosan;
t3. tannic acid modified polyvinyl alcohol: dissolving polyvinyl alcohol in water, adding tannic acid and a catalyst, stirring at room temperature for reaction, dialyzing, and drying dialyzate to obtain tannic acid modified polyvinyl alcohol;
t4. preparation of modified hydrogel: uniformly mixing the modified chitosan prepared in the step T2, the tannic acid modified polyvinyl alcohol prepared in the step T3 and water, adding acetic acid to adjust the pH value to 3.5-4.5, obtaining gel, repeatedly freezing and thawing, drying, and crushing to obtain the dried modified hydrogel.
As a further improvement of the invention, the acid liquor in the step T1 is 0.5-1.5wt% of acetic acid or HCl solution, and the mass ratio of the chitosan, the alpha-ketoglutaric acid and the sodium borohydride is 1-2:2-4:0.5-0.9, wherein the temperature of the heating and stirring reaction is 35-40 ℃, the time is 20-22h, the time of the continuous reaction is 5-7h, and the mass ratio of the solid to the sodium periodate is 2-4:1-3, wherein the light-shielding stirring reaction time is 1-2h, the ethanol content in the system is 80-85wt% after adding ethanol, and the precipitation time is 3-5h; in the step T2, the dopamine hydrochloride, carboxyl/aldehyde chitosan, 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide, 3-5 parts by weight of N-hydroxysulfosuccinimide and a catalyst with the mass ratio of 10-12:15-20:2-3:3-5:0.5-1 are prepared from Tris-HCl solution with pH value of 8.5-9, wherein the temperature of the heating and stirring reaction is 40-45 ℃ and the time is 2-4h; in the step T3, the mass ratio of the polyvinyl alcohol to the tannic acid to the catalyst is 10-15:5-7:0.2-0.5, the catalyst is Tris-HCl solution with pH value of 8.5-9, the room temperature stirring reaction time is 5-7h, the aperture of a dialysis bag for dialysis is 1.5-2kDa, and the time is 3-5h; and (3) in the step (T4), the mass ratio of the modified chitosan to the tannic acid modified polyvinyl alcohol to the water is 15-20:12-17:100, the repeated freezing and thawing method is that the gel is frozen for 1-2 hours at the temperature of-20 to-25 ℃, the gel is restored to the room temperature for 3-5 hours, and the operation is repeated for 3-5 times.
The invention further provides application of the hydrogel kit for rapid quantitative detection of hydrogen peroxide and sarcosine in detection of the content of hydrogen peroxide and/or sarcosine.
The invention further protects the use method of the hydrogel kit for rapid quantitative detection of hydrogen peroxide and sarcosine, the sample to be detected is diluted and dripped into the kit, the reaction is carried out for 80-100min at room temperature, the color after the reaction is photographed, and the picture is imported into Image J software and is converted into data information, so that the content measurement result is obtained; the regression equation of sarcosine is y=0.0015x+0.2435, the linear range is 1 mu mol/L-500 mu mol/L, and the detection limit is 0.40 mu mol/L; regression equation of hydrogen peroxide is y=0.00099x+0.2912, linear range is 1. Mu. Mol/L-800. Mu. Mol/L, and detection limit is 0.61. Mu. Mol/L.
The invention further provides a modified hydrogel, and the preparation method comprises the following steps:
t1 preparation of carboxyl/aldehyde chitosan: dissolving 1-2 parts by weight of chitosan in 0.5-1.5wt% acetic acid or HCl solution, adding 2-4 parts by weight of alpha-ketoglutaric acid, heating to 35-40 ℃, stirring for reacting for 20-22h, then adding 0.5-0.9 part by weight of sodium borohydride, continuing to react for 5-7h, adding ethanol until the ethanol content in the system is 80-85wt%, precipitating for 3-5h, centrifuging, dissolving 2-4 parts by weight of solid in 100 parts by weight of 0.5-1.5wt% acetic acid or HCl solution, adding 1-3 parts by weight of sodium periodate, stirring for reacting for 1-2h in a dark place, adding ethanol until the ethanol content in the system is 80-85wt%, precipitating for 3-5h, centrifuging, washing, and drying to obtain carboxyl/aldehyde chitosan;
T2. preparation of modified chitosan: dissolving 15-20 parts by weight of carboxyl/aldehyde chitosan prepared in the step T1, 2-3 parts by weight of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide and 3-5 parts by weight of N-hydroxysulfosuccinimide in 500 parts by weight of water, adding 10-12 parts by weight of dopamine hydrochloride, stirring and mixing uniformly, adding 0.5-1 part by weight of catalyst, heating to 40-45 ℃, stirring and reacting for 2-4h, adding ethanol until the ethanol content in the system is 80-85wt%, precipitating for 3-5h, centrifuging, washing and drying to prepare modified chitosan;
the catalyst is Tris-HCl solution with pH=8.5-9;
t3. tannic acid modified polyvinyl alcohol: dissolving 10-15 parts by weight of polyvinyl alcohol in 200 parts by weight of water, adding 5-7 parts by weight of tannic acid and 0.2-0.5 part by weight of catalyst, stirring at room temperature for reacting for 5-7 hours, dialyzing for 3-5 hours by using a dialysis bag with the aperture of 1.5-2kDa, and drying the dialyzate to obtain tannic acid modified polyvinyl alcohol;
the catalyst is Tris-HCl solution with pH=8.5-9;
t4. preparation of modified hydrogel: uniformly mixing 15-20 parts by weight of the modified chitosan prepared in the step T2, 12-17 parts by weight of the tannic acid modified polyvinyl alcohol prepared in the step T3 and 100 parts by weight of water, adding acetic acid to adjust the pH value to 3.5-4.5 to obtain gel, freezing the gel at the temperature of-20 to-25 ℃ for 1-2 hours, recovering the temperature to room temperature for 3-5 hours, repeating the operation for 3-5 times, drying the product, and crushing to obtain the dried modified hydrogel.
The invention has the following beneficial effects:
the cobalt nitrogen chlorine doped nano enzyme prepared by the invention has peroxidase simulation activity, can promote hydrogen peroxide to generate active oxygen metabolites (ROS) with strong oxidability, further catalyze the oxidation of a substrate, and can effectively catalyze a colorless substrate to generate a colored compound or a fluorescent product. The sarcosine is oxidized to generate hydrogen peroxide under the action of sarcosine oxidase, and the generated hydrogen peroxide generates active oxygen metabolites (ROS) with strong oxidability under the action of cobalt nitrogen and chlorine doped nano enzyme, so that TMB is catalyzed to generate oxTMB, and an oxTMB ultraviolet absorption peak can appear at 652nm, so that TMB becomes blue, and a foundation is laid for qualitatively and quantitatively detecting hydrogen peroxide and sarcosine.
According to the invention, cobalt nitrogen chlorine doped nano enzyme, 3', 5' -tetramethyl benzidine (TMB) serving as a chromogenic substrate and acetic acid-sodium acetate buffer solution serving as a chromogenic environment are mixed into a hydrogel network to prepare a response kit. When hydrogen peroxide and/or sarcosine exist in a sample to be detected, under the catalysis of cobalt nitrogen chlorine doped nano enzyme, TMB changes color, so that hydrogel becomes blue visible to the naked eye, and corresponding color intensity after the sample to be detected is added is recorded through Image J software, so that the hydrogen peroxide and the sarcosine can be automatically and rapidly detected, and the effect of visual detection is achieved. The portable hydrogel kit prepared by the method can be successfully applied to hydrogen peroxide detection in human urine sarcosine and milk.
The invention prepares a modified hydrogel, respectively introduces hydrophobic groups and hydrophilic residues into a reticular structure of a water-soluble polymer, combines the hydrophilic residues with water molecules to connect water inside a network, swells the hydrophobic residues when meeting water to form a three-dimensional reticular polymer taking water as a dispersion medium, has the advantages of low toxicity, biocompatibility, good antibacterial property, long service life, large drug loading capacity, high strength, super toughness, economy and effectiveness, biodegradability, oxidation resistance, ultraviolet resistance and the like in structural and physical characteristics similar to natural extracellular matrixes, adopts physical crosslinking (repeated freeze thawing), and can avoid toxic residues based on physical interactions (hydrophobic, electrostatic and hydrogen bonding) among different polymer chains without using a crosslinking agent.
The prepared modified hydrogel is frozen at low temperature and thawed at room temperature, and the operation is repeated in such a way that the movement of polyvinyl alcohol and chitosan main chain molecular chains in the hydrogel is limited at low temperature to begin crystallization, the formed crystals form physical crosslinking points in the hydrogel, the molecular chains are thawed at room temperature, the crystals begin to grow, the number of freeze thawing cycles is increased, the number of crystal nuclei can be increased, the crystal structure is perfected, the mechanical property of the hydrogel can be obviously improved, and meanwhile, the physical crosslinking without chemical reagents can be realized.
The chitosan is subjected to carboxylation and hydroformylation modification due to poor solubility, so that the carboxyl and aldehyde groups on the upper chain of the molecular chain of the chitosan are greatly improved, and on the other hand, the carboxyl and aldehyde groups of the chitosan can react with amino groups in the polydopamine, so that the chitosan is modified by the polydopamine without a cross-linking agent, and the modified chitosan is prepared;
in addition, the tannic acid is used for modifying the polyvinyl alcohol to prepare the tannic acid modified polyvinyl alcohol, and after the tannic acid modified polyvinyl alcohol is mixed with the modified chitosan, a hydrogel network is constructed based on physical crosslinking of non-covalent interactions (hydrogen bonds and hydrophobic interactions), so that the tannic acid modified polyvinyl alcohol has the advantages of being high in mechanical strength, good in water retention, low in toxicity, biocompatible, good in antibacterial property, long in service life, large in drug loading capacity, high in strength, super-tough, economical, effective, biodegradable, oxidation-resistant, ultraviolet-resistant and the like.
The invention constructs a sensing platform for selectively colorimetric detecting hydrogen peroxide and sarcosine, and the prepared cobalt nitrogen chlorine doped nano-enzyme hydrogel kit adopts physically crosslinked hydrogel as a carrier, so that the kit can load abundant components, does not need to consume toxic crosslinking agents, and has the advantages of low toxicity, biocompatibility, good antibacterial property, long service life, large drug loading capacity, high strength, super toughness, biodegradability, antioxidation, ultraviolet resistance and the like. The kit can be changed in color by dripping sarcosine and sarcosine oxidase or hydrogen peroxide, the Image acquired by the smart phone can be converted into color intensity through Image J software, so that convenient visual detection can be realized, the use of a traditional expensive detection instrument is avoided, and inaccurate color is converted into data through software, so that a certain precision is improved. The analysis strategy provides a new thought for detecting hydrogen peroxide in food samples and sarcosine in biological samples, and lays a foundation for clinically analyzing and detecting other disease biomarkers except for the prostate cancer.
Drawings
In order to more clearly illustrate the embodiments of the invention or the solutions of the prior art, the drawings that are necessary for the description of the embodiments or the prior art will be briefly described, it being obvious that the drawings in the description below are only some embodiments of the invention and that other drawings can be obtained from them without inventive effort for a person skilled in the art.
In fig. 1, (a) is a TEM image of the cobalt nitrogen chlorine doped nano-enzyme and (b) is a fourier transform infrared spectrogram of the cobalt nitrogen chlorine doped nano-enzyme;
in fig. 2, (a) is a full XPS spectrum of cobalt nitrogen chlorine doped nanoenzyme; (b) a high resolution XPS spectrum of C1 s; (c) a high resolution XPS profile of N1 s; (d) high resolution XPS spectrum for O1 s; (e) high resolution XPS spectrum of Cl 2 p; (f) high resolution XPS spectrum of Co 2 p;
FIG. 3 is a graph showing the ultraviolet absorption spectrum of different reaction systems: (a) TMB is an ultraviolet absorption spectrum diagram of different substrate reaction systems, (b) OPD is an ultraviolet absorption spectrum diagram of different substrate reaction systems, and (c) APTS is an ultraviolet absorption spectrum diagram of different substrate reaction systems (hydrogen peroxide + substrate, cobalt nitrogen chlorine doped carbon dots + hydrogen peroxide + substrate);
FIG. 4 is a graph showing the comparison of enzymatic reactions of cobalt nitrogen chlorine doped nanoenzymes with different substrates: (a) a graph of concentration versus reaction rate for hydrogen peroxide as a substrate, (b) a double reciprocal plot fit result for hydrogen peroxide as a substrate, (c) a graph of concentration versus reaction rate for TMB as a substrate, and (d) a double reciprocal plot fit result for TMB as a substrate;
FIG. 5 is a graph of EPR results for three reactive oxygen species: (a) 1 O 2 - EPR results of (b). OH, (c). O 2 - EPR results of (2);
FIG. 6 is a graph showing the effect of different reaction conditions on hydrogen peroxide detection: (a) molar ratio of the metal eutectic solvent, (b) reaction time, (c) reaction temperature, (d) PH of the reaction system, (e) concentration of chromogenic substrate TMB, (f) concentration of hydrogen peroxide;
in FIG. 7, (a) is 1-1000. Mu. Mol.L -1 An ultraviolet absorption diagram of hydrogen peroxide, (b) a linear diagram;
FIG. 8 is a graph showing ultraviolet absorption spectra of different reaction systems using TMB as a substrate (TMB+cobalt nitrogen chlorine doped carbon dot, TMB+cobalt nitrogen chlorine doped carbon dot+sarcosine oxidase, TMB+cobalt nitrogen chlorine doped carbon dot+sarcosine+sarcosine oxidase);
FIG. 9 is a graph showing the effect of different reaction conditions on sarcosine detection: (a) concentration of sarcosine oxidase, (b) reaction time;
FIG. 10 is 1-200. Mu. Mol.L -1 An ultraviolet absorbance plot (a) linear plot of sarcosine;
in FIG. 11, (a) is 1 day color change of the storage kit at room temperature and 4 ℃ and (b) is 1-7 days color change of the storage kit at 4 ℃;
FIG. 12 is a graph showing the effect of different reaction times on the detection of hydrogen peroxide by a hydrogel kit;
FIG. 13 is 1-800. Mu. Mol.L -1 Linear diagram of hydrogen peroxide;
FIG. 14 is 1-500. Mu. Mol.L -1 A linear plot of sarcosine;
FIG. 15 is 200. Mu. Mol.L -1 Hydrogel selectivities for sarcosine, amino acids, metal ions, and saccharides.
Detailed Description
The following description of the technical solutions in the embodiments of the present invention will be clear and complete, and it is obvious that the described embodiments are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Chitosan, deacetylation degree 95%, shanghai microphone Biochemical technology Co., ltd.
Preparation example 1A modified hydrogel
The preparation method comprises the following steps:
t1 preparation of carboxyl/aldehyde chitosan: dissolving 1 part by weight of chitosan in 0.5wt% acetic acid solution, adding 2 parts by weight of alpha-ketoglutaric acid, heating to 35 ℃, stirring for reacting for 20 hours, then adding 0.5 part by weight of sodium borohydride, continuing to react for 5 hours, adding ethanol until the ethanol content in the system is 80wt%, precipitating for 3 hours, centrifuging, dissolving 2 parts by weight of solid in 100 parts by weight of 0.5wt% acetic acid solution, adding 1 part by weight of sodium periodate, stirring for reacting for 1 hour in a dark place, adding ethanol until the ethanol content in the system is 80wt%, precipitating for 3 hours, centrifuging, washing, and drying to obtain carboxyl/aldehyde chitosan;
T2. preparation of modified chitosan: dissolving 15 parts by weight of carboxyl/aldehyde chitosan prepared in the step T1, 2 parts by weight of 1- (3-dimethylaminopropyl) -3-Ethylcarbodiimide (EDC) and 3-5 parts by weight of N-hydroxysulfosuccinimide (NHS) in 500 parts by weight of water, adding 10 parts by weight of dopamine hydrochloride, uniformly stirring and mixing, adding 0.5 part by weight of catalyst, heating to 40 ℃, stirring and reacting for 2 hours, adding ethanol until the ethanol content in the system is 80wt%, precipitating for 3 hours, centrifuging, washing and drying to obtain modified chitosan;
the catalyst is Tris-HCl solution with pH=8.5;
t3. tannic acid modified polyvinyl alcohol: dissolving 10 parts by weight of polyvinyl alcohol in 200 parts by weight of water, adding 5 parts by weight of tannic acid and 0.2 part by weight of catalyst, stirring at room temperature for reaction for 5 hours, dialyzing for 3 hours by using a dialysis bag with the aperture of 1.5kDa, and drying the dialyzate to obtain tannic acid modified polyvinyl alcohol;
the catalyst is Tris-HCl solution with pH=8.5;
t4. preparation of modified hydrogel: uniformly mixing 15 parts by weight of the modified chitosan prepared in the step T2, 12 parts by weight of the tannic acid modified polyvinyl alcohol prepared in the step T3 and 100 parts by weight of water, adding acetic acid to adjust the pH value to 3.5 to obtain gel, freezing the gel at the temperature of-20 ℃ for 1h, recovering the gel to room temperature for 3h, repeating the operation for 3 times, drying the product, and crushing to obtain the dried modified hydrogel.
PREPARATION EXAMPLE 2A modified hydrogel
The preparation method comprises the following steps:
t1 preparation of carboxyl/aldehyde chitosan: dissolving 2 parts by weight of chitosan in 1.5wt% of HCl solution, adding 4 parts by weight of alpha-ketoglutaric acid, heating to 40 ℃, stirring for reacting for 22 hours, then adding 0.9 part by weight of sodium borohydride, continuing to react for 7 hours, adding ethanol until the ethanol content in the system is 85wt%, precipitating for 5 hours, centrifuging, dissolving 4 parts by weight of solid in 100 parts by weight of 1.5wt% of HCl solution, adding 3 parts by weight of sodium periodate, stirring for reacting for 2 hours in a dark place, adding ethanol until the ethanol content in the system is 85wt%, precipitating for 5 hours, centrifuging, washing, and drying to obtain carboxyl/aldehyde chitosan;
t2. preparation of modified chitosan: dissolving 20 parts by weight of carboxyl/aldehyde chitosan prepared in the step T1, 3 parts by weight of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide and 5 parts by weight of N-hydroxysulfosuccinimide in 500 parts by weight of water, adding 12 parts by weight of dopamine hydrochloride, stirring and mixing uniformly, adding 1 part by weight of catalyst, heating to 45 ℃, stirring and reacting for 4 hours, adding ethanol until the ethanol content in the system is 85wt%, precipitating for 5 hours, centrifuging, washing and drying to prepare modified chitosan;
The catalyst is Tris-HCl solution with pH=9;
t3. tannic acid modified polyvinyl alcohol: dissolving 15 parts by weight of polyvinyl alcohol in 200 parts by weight of water, adding 7 parts by weight of tannic acid and 0.5 part by weight of catalyst, stirring at room temperature for reaction for 7 hours, dialyzing for 5 hours by using a dialysis bag with the aperture of 2kDa, and drying dialyzate to obtain tannic acid modified polyvinyl alcohol;
the catalyst is Tris-HCl solution with pH=9;
t4. preparation of modified hydrogel: uniformly mixing 20 parts by weight of the modified chitosan prepared in the step T2, 17 parts by weight of the tannic acid modified polyvinyl alcohol prepared in the step T3 and 100 parts by weight of water, adding acetic acid to adjust the pH value to 4.5 to obtain gel, freezing the gel at the temperature of-25 ℃ for 2 hours, recovering the gel to room temperature for 5 hours, repeating the operation for 5 times, drying the product, and crushing to obtain the dried modified hydrogel.
PREPARATION EXAMPLE 3A modified hydrogel
The preparation method comprises the following steps:
t1 preparation of carboxyl/aldehyde chitosan: dissolving 1.5 parts by weight of chitosan in 100 parts by weight of acetic acid solution with the concentration of 1wt%, adding 3 parts by weight of alpha-ketoglutaric acid, heating to 37 ℃, stirring for reaction for 21 hours, then adding 0.8 part by weight of sodium borohydride, continuing to react for 6 hours, adding ethanol until the ethanol content in the system is 82wt%, precipitating for 4 hours, centrifuging, dissolving 3 parts by weight of solid in 100 parts by weight of acetic acid solution with the concentration of 1wt%, adding 2 parts by weight of sodium periodate, stirring for reaction for 1.5 hours in a dark place, adding ethanol until the ethanol content in the system is 82wt%, precipitating for 4 hours, centrifuging, washing, and drying to obtain carboxyl/aldehyde chitosan;
T2. preparation of modified chitosan: dissolving 17 parts by weight of carboxyl/aldehyde chitosan prepared in the step T1, 2.5 parts by weight of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide and 4 parts by weight of N-hydroxysulfosuccinimide in 500 parts by weight of water, adding 11 parts by weight of dopamine hydrochloride, stirring and mixing uniformly, adding 0.7 part by weight of catalyst, heating to 42 ℃, stirring and reacting for 3 hours, adding ethanol until the ethanol content in the system is 82wt%, precipitating for 4 hours, centrifuging, washing and drying to prepare modified chitosan;
the catalyst is Tris-HCl solution with pH=8.7;
t3. tannic acid modified polyvinyl alcohol: dissolving 12 parts by weight of polyvinyl alcohol in 200 parts by weight of water, adding 6 parts by weight of tannic acid and 0.35 part by weight of catalyst, stirring at room temperature for reaction for 6 hours, dialyzing for 4 hours by using a dialysis bag with the aperture of 2kDa, and drying dialyzate to obtain tannic acid modified polyvinyl alcohol;
the catalyst is Tris-HCl solution with pH=8.7;
t4. preparation of modified hydrogel: mixing 17 parts by weight of the modified chitosan prepared in the step T2, 15 parts by weight of the tannic acid modified polyvinyl alcohol prepared in the step T3 and 100 parts by weight of water uniformly, adding acetic acid to adjust the pH value to 4 to obtain gel, freezing the gel at the temperature of-22 ℃ for 1.5 hours, recovering the gel to room temperature for 4 hours, repeating the operation for 4 times, drying the product, and crushing to obtain the dried modified hydrogel.
Test example 1
The modified hydrogels prepared in preparation examples 1 to 3 of the present invention were subjected to performance test.
1. Swelling Property
The dried modified hydrogel was weighed and recorded as m 0 . Soaking in PBS buffer solution with pH=7.4, incubating at 37deg.C for 12 hr, taking out swollen hydrogel sample, removing excessive water on the surface of hydrogel sample with filter paper, and recording the mass as m 1 。
Swelling ratio (%) = (m) 1 -m 0 )/m 0 ×100％。
2. Degradation performance:
the dried modified hydrogel was weighed and recorded as m 0 The method comprises the steps of carrying out a first treatment on the surface of the The dried modified hydrogel was then placed in a 48-well plate, soaked in PBS buffer containing 50U/mL lysozyme at ph=7.4, and incubated at 37 ℃. Taking out the hydrogel sample after 14d, washing with water, freeze-drying, weighing, and marking as m 2 。
Degradation rate (%) = (m) 2 -m 0 )/m 0 ×100％。
3. In vitro cytotoxicity test:
the dried modified hydrogels were immersed in PBS buffer at ph=7.4, placed at 37 ℃ and incubated for 12h, and tested according to IS010993-5 standard test methods.
4. Mechanical property measurement:
the dried modified hydrogel is prepared into a sample according to a plastic film tensile property test method (GB 13022-91), and the tensile strength and the elongation at break of the sample are measured by an electronic universal tester, wherein the tensile speed is 25mm/min, and the gauge length is 80mm.
The results are shown in Table 1.
TABLE 1
As is clear from the above table, the modified hydrogels prepared in preparation examples 1 to 3 of the present invention have good comprehensive properties.
Example 1 preparation method of cobalt Nitrogen chlorine doped nanoenzyme
The method comprises the following steps:
0.1mol of choline chloride (ChCl), 0.1mol of proline, 0.1mol of CoCl 2 ·6H 2 O is placed in an conical flask, heated to 50 ℃, stirred to form a uniform transparent solution, cooled to room temperature to obtain a dark blue viscous liquid, and added into 200mL of deionized water, and subjected to ultrasonic treatment at 1100W for 10min to completely dissolve the dark blue viscous liquid. The mixture was then transferred to an autoclave, heated to 180℃in an oven, and reacted for 6h. After cooling, the solution was centrifuged at 6000r/min for 15min and filtered through a 0.22 μm microporous filter. Finally, the obtained solution is freeze-dried to obtain dark blue cobalt nitrogen chlorine doped nano enzyme powder, the specific characterization result is shown as figures 1-2, lattice fringes with the size of 0.22nm are observed, the doped material has obvious lattice structure (figure 1 a), absorption peaks (figure 1 b) corresponding to vibration such as C-H, N-H, C=C/C=N/C=O, C-N, C-O, co-O, C-Cl and the like appear in a Fourier infrared spectrogram, the material mainly consists of Co, N and Cl elements (figure 2 a), and peak fitting data of each element and groups appearing in infrared are shown as full-scan XPS spectrum The analytical results were consistent (FIGS. 2 b-f), which all demonstrated that cobalt nitrogen chlorine doped nanoenzymes were successfully synthesized.
The enzyme activity verification result of the prepared cobalt nitrogen chlorine doped nano enzyme is as follows:
to three typical chromogenic substrates, including 3,3', 5' -Tetramethylbenzidine (TMB), o-phenylenediamine (OPD) and 2,2' -bianno-bis (3-ethylbenzothiopyrroline-6 sulfonic Acid) (ABTS), hydrogen peroxide, nanoenzyme, and mixtures of both were added, respectively, and the results are shown in FIG. 3. When the nano-enzyme and the hydrogen peroxide exist simultaneously, three substances respectively show obvious absorption peaks at about 652 nm, 450 nm and 405nm, and corresponding blue, yellow and green color development effects are generated (figure 3), so that the synthesized cobalt nitrogen chlorine doped nano-enzyme has peroxidase-like activity.
The steady state dynamics verification result of the prepared cobalt nitrogen chlorine doped nano enzyme is as follows:
FIG. 4 shows that as the substrate concentration increases, the initial rate of reaction gradually increases and then stabilizes at high concentrations, demonstrating that the steady state kinetics of the cobalt nitrogen chlorine doped nanoenzyme conforms to the Michaelis-Menten kinetics curve. When hydrogen peroxide is used as a substrate, K m Value sum V max The values are 0.72 mmol.L respectively -1 And 2.12X10 -8 mol·L -1 ·s -1 (FIGS. 4 a-b), K when TMB is used as a substrate m Value sum V max The values were 0.27 mmol.L, respectively -1 And 2.28X10 -8 mol·L -1 ·s -1 (FIGS. 4 c-d) while comparing K to horseradish peroxidase m Smaller. The results prove that the cobalt nitrogen chlorine doped nano-enzyme has higher affinity to hydrogen peroxide and TMB.
The Electron Paramagnetic Resonance (EPR) research result of the prepared cobalt nitrogen chlorine doped nano enzyme is as follows:
as shown in the EPR results of FIG. 5, for singlet oxygen 1 O 2 - ) Hydroxyl radical (. OH) and superoxide radical (. O) 2 - ) Performing a test, wherein 1 O 2 - A 1:1:1 triplet signal peak is generated, OH produces a spectral signal with a relative intensity of 1:2:2:1, &O 2 - Quadruple signals of 1:1:1:1 appear, which indicate that cobalt nitrogen chlorine doped nano enzyme generates three active oxygen when catalyzing hydrogen peroxide, thereby generating the active effect of peroxidase.
Example 2 detection of Hydrogen peroxide
The method comprises the following steps:
(1) The hydrogen peroxide measurement process is operated as follows:
100 mu L of the mixture is concentrated to 1.5 mg.mL -1 100 mu L of the nano-enzyme solution with the concentration of 10 mmol.L -1 TMB of (1) at a concentration of 100. Mu.L of 1-1000. Mu. Mol.L -1 500 μl ph=4 concentration of 0.2mol·l -1 After vortexing in a centrifuge tube for 1min, incubating for 40min at 50 ℃, and recording the ultraviolet absorption spectrum of the solution at 652 nm. Triplicate samples were prepared for each sample.
(2) The conditions for hydrogen peroxide detection are optimized as follows:
the detection conditions are optimized from the two aspects of material synthesis and reaction conditions during testing, including the raw material molar ratio of the metal eutectic solvent during material synthesis, and important factors affecting the catalytic activity of the nano enzyme, such as pH, temperature, reaction time, hydrogen peroxide concentration and chromogenic substrate TMB concentration. When the raw material mole ratio of the metal eutectic solvent is 1:1: 1. the pH is 4.0, the reaction temperature is 50 ℃, the ultraviolet absorption value is maximum when the reaction time is 40min, and the analysis performance of the method is optimal. The optimization results are shown in fig. 6.
(3) Linear range of detection of hydrogen peroxide, limit of detection:
under the optimal detection conditions obtained in the step (2), detection is performed according to the operation of the step (1). The ultraviolet absorption intensity of the final mixed solution gradually increased with increasing hydrogen peroxide concentration to 1. Mu. Mol.L -1 -1000μmol·L -1 Within the range, the absorption value has good linear relation with the hydrogen peroxide concentration, and the regression equation is y=0.00097x+0.0552 (R 2 = 0.9907) (fig. 7). Simultaneously, 11 blank samples are prepared in parallel, the standard deviation is calculated, and hydrogen peroxide is obtained by substituting the linear slope obtained according to the formula 3 sigma/k The detection limit of (C) is 0.31. Mu. Mol.L -1 。
EXAMPLE 3 detection of sarcosine
The method comprises the following steps:
(1) The determination process of sarcosine is operated as follows:
50 mu L of the mixture is concentrated to 1-200 mu mol.L -1 50. Mu.L of sarcosine solution with a concentration of 1.0 mg.mL -1 After vortexing in a centrifuge tube for 1min, incubating at 37deg.C for 10min, and adding 100 μl of 1.5 mg/mL -1 100 mu L of the nano-enzyme solution with the concentration of 10 mmol.L -1 500 μL PH=4 concentration of 0.2 mol.L) -1 After vortexing for 1min, incubating for 40min at 50 ℃, and recording the ultraviolet absorption spectrum of the solution at 652 nm. Triplicate samples were prepared for each sample.
Feasibility verification of sarcosine detection, the results were as follows:
the results of adding nanoenzyme, nanoenzyme+sarcosine mixture, nanoenzyme+sarcosine oxidase mixture, nanoenzyme+sarcosine+sarcosine oxidase mixture to TMB as chromogenic substrate are shown in FIG. 8. When the nano enzyme, the sarcosine and the sarcosine oxidase exist simultaneously, obvious absorption peaks appear at about 652nm, and a blue product is generated (an illustration in fig. 8), which proves the reasonability of detecting the sarcosine by the method.
(2) The conditions for sarcosine detection are optimized as follows:
Factors affecting the enzymatic reaction process, including sarcosine oxidase concentration and reaction time, were examined as opposed to hydrogen peroxide determination. When the concentration of sarcosine oxidase is 1.0 mg.mL and the reaction time is 10min, the ultraviolet absorption value reaches the maximum, and the analysis performance of the method reaches the optimum. The optimization results are shown in fig. 9.
(3) Linear range of detection of sarcosine, limit of detection:
under the optimal detection conditions obtained in the above step (2) and the optimization of the hydrogen peroxide detection conditions, the detection is performed according to the operation of step (1). The ultraviolet absorption intensity of the final mixed solution gradually increases with the increase of the sarcosine concentration, and is 1 mu mol.L -1 -200μmol·L -1 Within the range, the absorption value and the sarcosine concentration have good linear relation, and the regression equation is y=0.00093x+0.2096 (R 2 =0.9945) (fig. 10). Simultaneously, 11 blank samples are prepared in parallel, the standard deviation is calculated, and the detection limit of the sarcosine obtained by substituting the linear slope obtained according to the formula 3 sigma/k is calculated to be 0.80 mu mol L -1 。
Example 4 detection of Hydrogen peroxide in milk
The method comprises the following steps:
mixing milk with trichloroacetic acid by vortex, centrifuging, collecting supernatant, filtering with 0.22 μm microporous membrane, diluting the obtained liquid 10 times, collecting 100 μl diluted milk sample, and adding 100 μl of 1.5 mg/mL -1 100 mu L of the nano-enzyme solution with the concentration of 10 mmol.L -1 500 μL PH=4 concentration of 0.2 mol.L) -1 After vortexing for 1min, incubating for 40min at 50 ℃, recording ultraviolet absorption spectrum of the solution at 652nm for detection, preparing triplicate samples for each sample in parallel, and taking average value. Milk is added with different concentrations (100, 400 and 700 mu mol.L) -1 ) The average recovery rate of hydrogen peroxide is between 95.44 and 108.79%, the RSD is less than 3.4%, and the data are shown in Table 2;
TABLE 2 detection of Hydrogen peroxide in actual samples based on colorimetric platforms
Example 5 detection of sarcosine in human urine
The method comprises the following steps:
centrifuging urine, collecting supernatant, filtering with 0.22 μm microporous membrane, collecting 50 μl of treated urine sample, adding 50 μl sarcosine oxidase, swirling for 1min, mixing, incubating at 37deg.C for 10min, and adding 100 μl of 1.5 mg/mL -1 100 mu L of the nano-enzyme solution with the concentration of 10 mmol.L -1 500 μL PH=4 concentration of 0.2 mol.L) -1 Is stirred for 1min, is evenly mixed and is incubated for 40min at 50 ℃, and the dissolution is recordedThe solution was tested by UV absorption at 652nm and triplicate samples were prepared for each sample and averaged. Urine is added with different concentrations (50, 100 and 200 mu mol.L) -1 ) The average recovery rate of sarcosine solution is 92.19-105.95%, RSD is less than 5.4%, and the data are shown in Table 3;
TABLE 3 detection of sarcosine in actual samples based on colorimetric platforms
Example 6 preparation of hydrogel kit for rapid quantitative detection of Hydrogen peroxide and sarcosine
The method comprises the following steps:
the concentration of 2mL is 1.5 mg.mL -1 1mL of the nano-enzyme solution with the concentration of 10 mmol.L -1 60mg of the modified hydrogel prepared in preparation example 3, 2mL of the modified hydrogel having a pH=4 concentration of 0.2 mol.L -1 Heating and uniformly mixing 6mL of deionized water, dripping 200 mu L of the mixture into a 5mL centrifugal tube cover, cooling and solidifying to obtain the cobalt nitrogen chlorine doped nano enzyme-based hydrogel kit, and storing at 4 ℃ for later use.
The stability of the hydrogel kit was evaluated. After storage at room temperature and 4 ℃ for 1 day, the hydrogel color change was recorded by photographing, and Image J software was introduced to convert the color intensity into data for analysis, and fig. 11a shows that the hydrogel color changed significantly after storage at room temperature for 1 day, but not after storage at 4 ℃ for a further investigation of the hydrogel color change from 1 to 7 days after storage at 4 ℃ as shown in fig. 11b. These findings indicate that the color of the hydrogel does not change by more than 20% after 7 days of cold storage at 4 ℃ and can be stably stored.
Example 7 detection of Hydrogen peroxide by hydrogel kit for rapid quantitative detection of Hydrogen peroxide and sarcosine
The method comprises the following steps:
(1) The hydrogen peroxide measurement process is operated as follows:
100 mu L of hydrogen peroxide with the concentration of 1-1000 mu mol.L-1 is dripped into the prepared hydrogel kit, the kit is placed for 90min at room temperature, a smart phone is used for photographing and recording colors, pictures are imported into Image J software and converted into data information, and 3 samples are prepared in parallel.
(2) The conditions for hydrogen peroxide detection are optimized as follows:
based on the prepared hydrogel kit, the hydrogel development time is optimized, and the graph result shows that the color intensity reaches the maximum when the reaction time is 90min, and the reaction is the most complete. The optimization results are shown in fig. 12.
(3) Linear range of detection of hydrogen peroxide, limit of detection:
under the optimal detection conditions obtained in the step (2), detection is performed according to the operation of the step (1). The final color intensity gradually increased with increasing hydrogen peroxide concentration at 1. Mu. Mol.L -1 -800μmol·L -1 In the range, the absorption value and the hydrogen peroxide concentration have good linear relation, and the regression equation is y=0.00099x+0.2912 (R 2 = 0.9906) (fig. 13). Simultaneously, 11 blank samples are prepared in parallel, the standard deviation is calculated, and the detection limit of hydrogen peroxide is calculated to be 0.61 mu mol L according to the formula 3 sigma/k and the linear slope obtained by substitution -1 。
Example 8 detection of sarcosine by hydrogel kit for rapid quantitative detection of Hydrogen peroxide and sarcosine
The method comprises the following steps:
(1) The determination process of sarcosine is operated as follows:
50 mu L of the mixture is concentrated to 1-500 mu mol.L -1 50. Mu.L of sarcosine solution with a concentration of 1.0 mg.mL -1 The sarcosine oxidase solution is stirred in a centrifuge tube for 1min, then incubated for 10min at 37 ℃, then dripped into the prepared hydrogel kit, placed for 90min at room temperature, photographed by a smart phone to record colors, and the pictures are imported into Image J software to be converted into data information. Triplicate samples were prepared for each sample.
(2) Linear range of detection of sarcosine, limit of detection:
under the optimal detection conditions obtained in the optimization of the hydrogen peroxide detection conditions, the detection is performed according to the operation of step (1). Most preferably, the first to fourthThe final color intensity gradually increased with increasing sarcosine concentration at 1. Mu. Mol.L -1 -500μmol·L -1 Within the range, the absorption value and the sarcosine concentration have good linear relation, and the regression equation is y=0.0015x+0.2435 (R 2 = 0.9978) (fig. 14). Simultaneously, 11 blank samples are prepared in parallel, the standard deviation is calculated, and the detection limit of the sarcosine obtained by substituting the linear slope obtained according to the formula 3 sigma/k is calculated to be 0.40 mu mol L -1 。
(3) Selectivity of detection of sarcosine:
200 mu mol/L -1 Amino acids, metal ions and saccharides were added to the reaction system instead of sarcosine, and the change in color of the hydrogel was observed. The results show that other interfering substances hardly change the color of the hydrogel (fig. 15), which shows a high selectivity for the detection of sarcosine.
Example 9 hydrogel kit for rapid quantitative detection of hydrogen peroxide and sarcosine the detection of hydrogen peroxide in milk is specifically as follows:
mixing milk and trichloroacetic acid vortex, centrifuging, collecting supernatant, filtering with 0.22 μm microporous filter membrane, diluting the obtained liquid 10 times, taking 100 μl diluted milk sample, dripping into prepared hydrogel kit, standing at room temperature for 90min, photographing with smart phone, recording color, transferring picture into Image J software, converting into data information, preparing three samples in parallel, and taking average value. Milk is added with different concentrations (100, 400 and 600 mu mol.L) -1 ) The average recovery rate of hydrogen peroxide is between 96.97-104.30%, RSD is less than 6.8%, and the data are shown in Table 4;
TABLE 4 Hydrogen peroxide detection in actual samples
Example 10 detection of sarcosine in human urine by hydrogel kit for rapid quantitative detection of Hydrogen peroxide and sarcosine
The method comprises the following steps:
centrifuging urine, collecting supernatant, filtering with a 0.22 mu m microporous filter membrane, dripping 50 mu L of treated urine sample into the prepared hydrogel kit, standing for 90min at room temperature, photographing with a smart phone, recording color, transferring the picture into Image J software, converting into data information, preparing three samples in parallel, and taking an average value. Urine was added at various concentrations (50, 200 and 400. Mu. Mol.L) -1 ) The average recovery rate is 102.50-107.37%, RSD is less than 8.6%, and the data are shown in Table 5;
TABLE 5 detection of sarcosine in actual samples
The foregoing description of the preferred embodiments of the invention is not intended to be limiting, but rather is intended to cover all modifications, equivalents, alternatives, and improvements that fall within the spirit and scope of the invention.
Claims (10)
1. The cobalt nitrogen chlorine doped nano enzyme is characterized by being prepared by the following method: and (3) mixing choline chloride, proline and cobalt salt, heating and stirring to form a uniform transparent solution, cooling to room temperature, adding deionized water, performing ultrasonic treatment to completely dissolve the solution, transferring the mixture into a high-pressure reaction kettle, performing heating reaction to completely, cooling to room temperature, centrifuging, filtering, and freeze-drying the obtained solution to obtain the cobalt nitrogen chlorine doped nano-enzyme.
2. The cobalt nitrogen chlorine doped nano-enzyme according to claim 1, wherein the molar ratio of choline chloride, proline and cobalt salt is 0.5-1.5:0.5-1.5:0.5-1.5, wherein the temperature of heating and stirring is 45-55 ℃, the power of ultrasonic is 1000-1200W, the time of ultrasonic is 5-15min, the temperature of heating reaction is 170-190 ℃, the time is 5-7h, the rotating speed of centrifugation is 5000-7000r/min, the time is 10-20min, and the filter membrane for filtration is a 0.22 mu m microporous filter membrane; the cobalt salt is at least one selected from cobalt chloride, cobalt sulfate and cobalt nitrate.
3. The rapid quantitative detection system for the hydrogen peroxide and the sarcosine is characterized by comprising the cobalt nitrogen chlorine doped nano enzyme, 3', 5' -tetramethyl benzidine, acetic acid-sodium acetate buffer solution and deionized water according to the following weight ratio of 2-4:2-3:3-5:5-7, wherein the concentration of the acetic acid-sodium acetate buffer solution is 0.1-0.3mmol/L.
4. A method for using the rapid quantitative detection system for hydrogen peroxide and sarcosine according to claim 3, wherein the sample to be detected is diluted and then added into the detection system, the mixture is uniformly mixed, incubated for 5-15min at 36-38 ℃, and the ultraviolet absorption spectrum of the solution at 652nm is recorded for detection; the regression equation of sarcosine is y=0.00093x+0.2096, the linear range is 1 mu mol/L-200 mu mol/L, and the detection limit is 0.80 mu mol/L; regression equation for hydrogen peroxide was y=0.00097x+0.0552, linear range was 1. Mu. Mol/L-1000. Mu. Mol/L, and detection limit was 0.31. Mu. Mol/L.
5. The hydrogel kit for rapid quantitative detection of hydrogen peroxide and sarcosine is characterized by comprising the following steps of: adding the cobalt nitrogen chlorine doped nano enzyme, 3', 5' -tetramethyl benzidine, hydrogel and acetic acid-sodium acetate buffer solution according to claim 1 or 2 into deionized water, heating, stirring, mixing uniformly, cooling and solidifying to obtain the hydrogel kit for rapid quantitative detection of hydrogen peroxide and sarcosine.
6. The hydrogel kit for rapid quantitative detection of hydrogen peroxide and sarcosine according to claim 5, wherein the mass ratio of cobalt nitrogen chlorine doped nano-enzyme to 3,3', 5' -tetramethylbenzidine to hydrogel to acetic acid-sodium acetate buffer to deionized water is 2-4:2-3:50-70:3-5:5-7, wherein the concentration of the acetic acid-sodium acetate buffer solution is 0.1-0.3mmol/L.
7. The kit for rapid quantitative detection of hydrogen peroxide and sarcosine according to claim 5, wherein the hydrogel is at least one selected from peptone, agar powder, and modified hydrogel; the preparation method of the modified hydrogel comprises the following steps:
t1 preparation of carboxyl/aldehyde chitosan: dissolving chitosan in acid liquor, adding alpha-ketoglutaric acid, heating and stirring for reaction, then adding sodium borohydride, continuing the reaction, adding ethanol for precipitation, centrifuging, dissolving the solid in the acid liquor, adding sodium periodate, stirring for reaction in a dark place, adding ethanol for precipitation, centrifuging, washing, and drying to obtain carboxyl/aldehyde chitosan;
T2. preparation of modified chitosan: dissolving the carboxyl/aldehyde chitosan prepared in the step T1, 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide and N-hydroxy thiosuccinimide in water, uniformly mixing, adding a catalyst, heating and stirring for reaction, adding ethanol for precipitation, centrifuging, washing and drying to prepare modified chitosan;
t3. tannic acid modified polyvinyl alcohol: dissolving polyvinyl alcohol in water, adding tannic acid and a catalyst, stirring at room temperature for reaction, dialyzing, and drying dialyzate to obtain tannic acid modified polyvinyl alcohol;
t4. preparation of modified hydrogel: uniformly mixing the modified chitosan prepared in the step T2, the tannic acid modified polyvinyl alcohol prepared in the step T3 and water, adding acetic acid to adjust the pH value to 3.5-4.5, obtaining gel, repeatedly freezing and thawing, drying, and crushing to obtain the dried modified hydrogel.
8. The preparation method according to claim 7, wherein the acid solution in the step T1 is 0.5-1.5wt% acetic acid or HCl solution, and the mass ratio of the chitosan, the alpha-ketoglutaric acid and the sodium borohydride is 1-2:2-4:0.5-0.9, wherein the temperature of the heating and stirring reaction is 35-40 ℃, the time is 20-22h, the time of the continuous reaction is 5-7h, and the mass ratio of the solid to the sodium periodate is 2-4:1-3, wherein the light-shielding stirring reaction time is 1-2h, the ethanol content in the system is 80-85wt% after adding ethanol, and the precipitation time is 3-5h; in the step T2, the dopamine hydrochloride, carboxyl/aldehyde chitosan, 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide, 3-5 parts by weight of N-hydroxysulfosuccinimide and a catalyst with the mass ratio of 10-12:15-20:2-3:3-5:0.5-1 are prepared from Tris-HCl solution with pH value of 8.5-9, wherein the temperature of the heating and stirring reaction is 40-45 ℃ and the time is 2-4h; in the step T3, the mass ratio of the polyvinyl alcohol to the tannic acid to the catalyst is 10-15:5-7:0.2-0.5, the catalyst is Tris-HCl solution with pH value of 8.5-9, the room temperature stirring reaction time is 5-7h, the aperture of a dialysis bag for dialysis is 1.5-2kDa, and the time is 3-5h; and (3) in the step (T4), the mass ratio of the modified chitosan to the tannic acid modified polyvinyl alcohol to the water is 15-20:12-17:100, the repeated freezing and thawing method is that the gel is frozen for 1-2 hours at the temperature of-20 to-25 ℃, the gel is restored to the room temperature for 3-5 hours, and the operation is repeated for 3-5 times.
9. Use of a hydrogel kit for rapid quantitative detection of hydrogen peroxide and sarcosine according to any one of claims 4 to 8 for detecting hydrogen peroxide and/or sarcosine content.
10. A method of using the hydrogel kit for rapid quantitative determination of hydrogen peroxide and sarcosine as claimed in any one of claims 4 to 8, wherein the sample to be measured is diluted, dripped into the kit, reacted at room temperature for 80 to 100 minutes, the color after the reaction is photographed, and the picture is imported into Image J software to be converted into data information, thereby obtaining a content determination result; the regression equation of sarcosine is y=0.0015x+0.2435, the linear range is 1 mu mol/L-500 mu mol/L, and the detection limit is 0.40 mu mol/L; regression equation of hydrogen peroxide is y=0.00099x+0.2912, linear range is 1. Mu. Mol/L-800. Mu. Mol/L, and detection limit is 0.61. Mu. Mol/L.
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