MOF@Os nanoszyme for non-invasive screening of prostate cancer and preparation method and application thereof

Abstract

The present application relates to a kind of MOF for prostate cancer noninvasive screening @Os nanoenzyme and its preparation method, application.First, zirconium source, 2,2-pyridine-5,5-dicarboxylic acid is dissolved in N,N-dimethylformamide and the like organic solvent, then the two solutions obtained are mixed in proportion, then pH regulator is added and shaken, the obtained mixture is heated to carry out solvothermal reaction, reaction is separated and purified to obtain MOF;MOF prepared is dispersed in solvent, then osmium source, reducing agent is added and heated to carry out reduction reaction, reaction is separated and purified to obtain MOF @Os nanoenzyme.The MOF @Os nanoenzyme prepared by the method of the present application has a polyhedral structure, and the particle size is 500-1200nm, which has excellent peroxidase-like catalytic activity and almost no oxidase-like activity, and has important application value in early noninvasive diagnosis and bedside real-time detection of prostate cancer.

Description

Technical Field

[0001] This invention relates to the fields of nanocomposite materials, biomedicine, and detection and diagnostics, specifically to a MOF@Os nanozyme for non-invasive screening of prostate cancer, its preparation method, and its application. Background Technology

[0002] Prostate cancer is a highly prevalent malignant tumor among men worldwide and one of the leading causes of cancer death in men. Its incidence and mortality rates are rising annually, seriously threatening men's lives and health. Early-stage prostate cancer often presents insidiously with no typical or specific clinical symptoms, and most patients are diagnosed at a locally advanced stage or with distant metastases, resulting in extremely poor prognosis. Therefore, early, rapid, non-invasive, and accurate screening and diagnosis are of crucial clinical significance in reducing prostate cancer mortality and improving patient prognosis.

[0003] Prostate-specific antigen (PSA) is currently the most widely used biomarker for prostate cancer in clinical practice. However, it has a clinical "gray zone" (4.0-10.0 ng / mL), which significantly reduces its diagnostic specificity and easily leads to diagnostic confusion. Furthermore, traditional PSA testing suffers from drawbacks such as invasive sampling and cumbersome procedures, making it unsuitable for large-scale rapid screening. Creatine, an N-methyl derivative of glycine, is another promising early metabolic biomarker for prostate cancer. Although creatine possesses excellent diagnostic potential, existing detection methods are prone to false positives and false negatives, limiting the accuracy of quantification. Therefore, there is an urgent clinical need to develop a simple, rapid, and standardized method for quantifying urinary creatine.

[0004] Nanozymes are a class of artificial nanomaterials that mimic the catalytic function of natural enzymes. They can catalyze biochemical reactions under mild conditions and, compared to natural enzymes, offer multiple advantages such as low production cost, strong structural stability, simple storage conditions, and tolerance to extreme environments. Noble metal-based nanozymes can drive a variety of chemical reactions; their peroxidase-like activity can directly catalyze the oxidation and colorimetric reaction of hydrogen peroxide, requiring no complex specialized equipment and perfectly suited for point-of-care rapid detection. They are ideal materials for constructing stable and accurate sarcosine catalytic sensing systems. However, existing nanozyme materials generally suffer from high interference from oxidase-like activity, limited catalytic efficiency, and weak resistance to matrix interference, severely limiting their practical application in complex urine samples. Furthermore, existing detection systems also suffer from limitations such as single detection modes, poor scenario adaptability, and the inability to simultaneously achieve high-precision and point-of-care detection. Summary of the Invention

[0005] The main objective of this invention is to overcome the aforementioned problems and limitations in existing prostate cancer screening methods, and to provide a universally applicable MOF@Os nanozyme, its preparation method, and its application in prostate cancer screening. The MOF@Os nanozyme not only possesses advantages such as high peroxidase catalytic specificity, high stability, strong anti-interference, simple and easy preparation method, and low cost, but also has creatine detection capabilities. It can be combined with mobile terminals (such as smartphones) and ResNet18 machine learning models for visual detection or to build a non-invasive, rapid, and intelligent prostate cancer screening platform, effectively compensating for the shortcomings of existing detection technologies.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows: One objective of this invention is to provide a method for preparing MOF@Os nanozymes, comprising: S1, Metal-organic frameworks (MOFs) were prepared using a zirconium source and 2,2-bipyridine-5,5-dicarboxylic acid; S2, MOF@Os nanozymes were prepared using MOF, osmium source, and reducing agent.

[0007] In the above scheme, the specific process of S1 includes: dissolving the zirconium source and 2,2-bipyridine-5,5-dicarboxylic acid in an organic solvent to obtain a zirconium source solution and a 2,2-bipyridine-5,5-dicarboxylic acid solution; mixing the zirconium source solution and the 2,2-bipyridine-5,5-dicarboxylic acid solution evenly, adding a pH adjuster and heating to react, and separating and purifying to obtain MOF.

[0008] In the above scheme, the zirconium source mentioned in S1 is selected from at least one of zirconium tetrachloride (ZrCl4), zirconium oxychloride or its hydrate (such as ZrOCl2·8H2O), preferably zirconium tetrachloride.

[0009] In the above scheme, the organic solvent mentioned in S1 is specifically N,N-dimethylformamide (DMF).

[0010] In the above scheme, the pH adjuster mentioned in S1 is specifically an organic acid, selected from at least one of formic acid, acetic acid, and benzoic acid, preferably acetic acid.

[0011] In the above scheme, the concentration of the zirconium source solution in S1 is 1-4 g / L, the concentration of the 2,2-bipyridine-5,5-dicarboxylic acid solution is 1-4 g / L, and the concentrations of the two solutions are equal or unequal.

[0012] In the above scheme, the mass ratio of zirconium source to 2,2-bipyridine-5,5-dicarboxylic acid in S1 is 100-300:200-400, the total amount of organic solvent used is 50-300 mL / 100-300 mg zirconium source, and the amount of pH adjuster used is 2000-5000 μL / 100-300 mg zirconium source.

[0013] In the above scheme, the mixture obtained in S1 is heated to 100-200℃ for a solvothermal reaction for 10-20h. After the reaction is complete, it is naturally cooled to room temperature, and the solid product is collected by centrifugation and washed to obtain MOF.

[0014] In the above scheme, the specific process of S2 includes: dispersing MOF in a solvent (such as ethanol or other alcohol solvents) to obtain MOF dispersion; adding osmium source and reducing agent to MOF dispersion and heating reaction, and separating and purifying to obtain MOF@Os nanozyme.

[0015] In the above scheme, the osmium source in S2 is selected from at least one of potassium hexachloroosmium, sodium hexachloroosmium, and ammonium hexachloroosmium, preferably potassium hexachloroosmium.

[0016] In the above scheme, the reducing agent in S2 is selected from at least one of sodium borohydride, ascorbic acid, and citric acid.

[0017] In the above scheme, the concentration of the MOF dispersion in S2 is 0.5-15 g / L.

[0018] In the above scheme, the amount of osmium source in S2 is 10-200 mol / 5-15 g MOF, and the amount of reducing agent is 0.5-20 mmol / 5-15 g MOF.

[0019] In the above scheme, the S2 reduction reaction temperature is 50-100℃, the reaction time is 1-30min, the reaction is allowed to cool naturally to room temperature after completion, the solid product is collected by centrifugation and washed to obtain MOF@Os nanozyme.

[0020] In the above scheme, the product in S1 or S2 is repeatedly washed multiple times with at least one of N,N-dimethylformamide, tetrahydrofuran, ethanol, and deionized water to remove residual impurities.

[0021] The second objective of this invention is to provide a MOF@Os nanozyme with a polyhedral structure and a particle size of 500-1200 nm.

[0022] In the above scheme, MOF serves as the framework in the MOF@Os nanozyme, and Os reduction modification is performed inside or on the surface of the MOF.

[0023] The third objective of this invention is to provide the application of the above-mentioned MOF@Os nanozyme in the quantitative detection of sarcosine content in liquid samples (such as urine). This application can be extended to non-invasive screening test reagents or kits for prostate cancer, realizing early non-invasive diagnosis and bedside immediate detection of prostate cancer. Combined with mobile terminals (such as smartphones) and ResNet18 machine learning models, it can also perform visual detection or build a non-invasive, rapid, and intelligent prostate cancer screening platform.

[0024] This invention preferentially uses osmium (Os) as the modified active component of MOF. Compared with other traditional noble metal nanozymes, osmium-based catalytic materials possess specific enzymatic advantages and biosensor compatibility advantages. They not only endow the materials with highly selective peroxidase-like catalytic properties, but also exhibit extremely low oxidase-like activity, effectively reducing background interference in biological systems. Therefore, this invention introduces osmium to modify the MOF framework, selectively amplifying POD catalytic activity and inhibiting OXD side reactions, achieving highly specific, low-background, and highly interference-resistant colorimetric detection of sarcosine. This invention can also be combined with mobile terminals (such as smartphones) and ResNet18 machine learning models for visual detection or to build a non-invasive, rapid, and intelligent prostate cancer screening platform, effectively overcoming the shortcomings of existing detection technologies.

[0025] This invention fully utilizes the advantages of metal-organic framework (MOF) materials, such as their porous and ordered structure, large specific surface area, tunable structure, and excellent stability, to serve as an ideal carrier for Os nanozymes. This achieves uniform dispersion and immobilization of the noble metal osmium active sites, effectively enhancing catalytic activity and specificity. Furthermore, this invention also develops a simple, high-performance detection method and platform based on MOF@Os nanozymes, suitable for non-invasive clinical screening, which has significant application value for early non-invasive diagnosis and point-of-care testing of prostate cancer.

[0026] Compared with existing similar products or technologies, the beneficial effects of this invention are mainly reflected in the following aspects: (1) The process for preparing MOF@Os nanozymes in this invention is relatively simple and reproducible. The product can be used without complex post-modification treatment, making it suitable for large-scale batch preparation and clinical industrialization.

[0027] (2) The MOF@Os nanozyme prepared in this invention possesses excellent peroxidase-like catalytic activity while exhibiting almost no oxidase-like activity, effectively eliminating non-specific catalytic interference. During the detection process, sarcosinase catalyzes sarcosine to produce hydrogen peroxide, and the MOF@Os nanozyme catalyzes hydrogen peroxide to produce hydroxyl radicals, thereby oxidizing TMB for color development, achieving quantitative detection of sarcosine content in urine, and thus realizing early non-invasive diagnosis of prostate cancer.

[0028] (3) The MOF@Os nanozyme prepared by this invention has excellent anti-interference ability, can tolerate a variety of endogenous interfering substances in urine, and has high detection stability, repeatability and accuracy, making it suitable for complex clinical urine sample detection and other application scenarios.

[0029] (4) This invention constructs a triple complementary detection mode, all of which realize high-precision spectrophotometric quantitative detection in the laboratory; among them, the naked-eye visualization detection of smartphones eliminates the dependence on large instruments, is low-cost and easy to operate, and is suitable for rapid initial screening in primary medical institutions and at home; the ResNet-18 machine learning model further eliminates human error and greatly improves the level of intelligence and accuracy of detection. These detection modes have the advantages of simple operation, non-invasiveness and high safety, and can effectively distinguish between healthy people and prostate cancer patients, providing a new and efficient solution for early non-invasive screening of prostate cancer, and are suitable for non-invasive screening in multiple scenarios such as primary care and home. Attached Figure Description

[0030] Figure 1 SEM and TEM images of MOF@Os nanozymes.

[0031] Figure 2 The results of the TMB oxidation experiment and the mechanism diagram of MOF@Os nanozyme are shown.

[0032] Figure 3 Figure showing the results of an experiment to verify the types of free radicals generated by MOF@Os nanozymes.

[0033] Figure 4 Figure showing the feasibility experiment results of constructing a sarcosine detection system by combining MOF@Os nanozymes with sarcosine oxidase.

[0034] Figure 5 Figure shows the results of the experimental optimization of conditions for the MOF@Os nanozyme system to detect sarcosine.

[0035] Figure 6 Figure showing the results of the specificity and anti-interference tests of MOF@Os nanozymes.

[0036] Figure 7 The figure shows the difference in absorbance of sarcosine detected by MOF@Os nanozymes in different solutions.

[0037] Figure 8 The image shows the detection results of MOF@Os nanozymes on a smartphone.

[0038] Figure 9 This is a schematic diagram of the ResNet18-sarcosine model training. Detailed Implementation

[0039] To enable those skilled in the art to fully understand the technical solution and beneficial effects of the present invention, further detailed description is provided below in conjunction with specific embodiments and accompanying drawings. It should be emphasized that the embodiments listed below are merely preferred embodiments of the present invention and do not constitute a limitation thereof. Any simple improvements made based on these embodiments will fall within the protection scope of the present invention.

[0040] To avoid obscuring the invention with unnecessary details, the following embodiments only show the raw materials, methods, and steps closely related to the technical solutions of the invention, while omitting some other conventional details. The term "comprising / including" in the invention indicates the presence of features, elements, steps, etc., but does not exclude the presence or addition of one or more other features, elements, steps.

[0041] Unless otherwise specified, all raw materials mentioned in this invention are commercially available, and all units of measurement are international units.

[0042] All experiments in this invention strictly comply with relevant domestic and international laws and regulations.

[0043] Example 1 (1) Using zirconium tetrachloride as the zirconium source and 2,2-bipyridine-5,5-dicarboxylic acid as the ligand, UiO-67MOF was synthesized.

[0044] 244 mg ZrCl4 and 233 mg 2,2-bipyridine-5,5-dicarboxylic acid were dissolved in 100 mL N,N-dimethylformamide and sonicated for 5 min until completely dissolved. The two solutions were then added to a glass bottle and mixed thoroughly. 4650 μL of acetic acid was added and shaken well. The glass bottle was then heated to 120 °C and reacted at this temperature for 18 h.

[0045] After the reaction was completed and allowed to cool naturally to room temperature, the mixture was transferred to a centrifuge and centrifuged at 9500 rpm for 8 min. The white solid product was collected and washed repeatedly with DMF, tetrahydrofuran, ethanol, and deionized water in sequence to obtain purified MOF. The obtained MOF was dispersed in ethanol to obtain a MOF ethanol dispersion with a concentration of 5 mg / mL for later use.

[0046] (2) MOF@Os nanozymes were prepared using potassium hexachloroosmium tetroxide as the osmium source and ascorbic acid as the reducing agent.

[0047] Take the above dispersion containing 8 mg MOF, add 100 mmol K2OsCl6, and sonicate thoroughly to mix evenly. Heat the mixture to 80°C, then quickly add 10 μL of 100 mM ascorbic acid solution, and react at this temperature for 10 min. After the reaction, centrifuge to collect the product and wash it several times with deionized water. Then resuspend the product in deionized water to obtain a MOF@Os nanozyme dispersion with a concentration of 10 mg / mL, which should be stored at low temperature and protected from light for later use.

[0048] Example 2 (1) Using zirconium tetrachloride as the zirconium source and 2,2-bipyridine-5,5-dicarboxylic acid as the ligand, UiO-67MOF was synthesized.

[0049] 110 mg ZrCl4 and 300 mg 2,2-bipyridine-5,5-dicarboxylic acid were dissolved in 30 mL of N,N-dimethylformamide and sonicated for 10 min until completely dissolved. The two solutions were added to a glass bottle and mixed thoroughly. Then 2000 μL of acetic acid was added and shaken well. The glass bottle was then heated to 160 °C and reacted at this temperature for 10 h.

[0050] After the reaction was complete, the mixture was allowed to cool naturally to room temperature. It was then transferred to a centrifuge and centrifuged at 10,000 rpm for 5 minutes. The white solid product was collected and washed sequentially with DMF, tetrahydrofuran, ethanol, and deionized water to obtain purified MOF. The obtained MOF was dispersed in ethanol to obtain an ethanol dispersion with a concentration of approximately 5 mg / mL for later use.

[0051] (2) MOF@Os nanozymes were prepared using potassium hexachloroosmium tetroxide as the osmium source and sodium borohydride as the reducing agent.

[0052] Take the above dispersion containing 5 mg MOF, add 10 mmol K2OsCl6, and sonicate thoroughly to mix evenly. Heat the mixture to 60°C, then quickly add 1 μL of 100 mM sodium borohydride solution, and react at this temperature for 5 min. After the reaction, centrifuge to collect the product and wash it several times with deionized water. Then resuspend the product in deionized water to obtain a MOF@Os nanozyme dispersion with a concentration of approximately 5 mg / mL, which should be stored in the dark at low temperature for later use.

[0053] Example 3 (1) Using zirconium tetrachloride as the zirconium source and 2,2-bipyridine-5,5-dicarboxylic acid as the ligand, UiO-67MOF was synthesized.

[0054] 110 mg ZrCl4 and 400 mg 2,2-bipyridine-5,5-dicarboxylic acid were dissolved in 200 mL N,N-dimethylformamide and sonicated for 10 min until completely dissolved. The two solutions were added to a glass bottle and mixed thoroughly. Then 2000 μL of acetic acid was added and shaken well. The glass bottle was then heated to 100 °C and reacted at this temperature for 18 h.

[0055] After the reaction was completed and allowed to cool naturally to room temperature, the mixture was transferred to a centrifuge and centrifuged at 8000 rpm for 10 min. The white solid product was collected and washed repeatedly with DMF, tetrahydrofuran, ethanol, and deionized water in sequence to obtain purified MOF. The obtained MOF was dispersed in ethanol to obtain an ethanol dispersion with a concentration of approximately 15 mg / mL for later use.

[0056] (2) MOF@Os nanozymes were prepared using potassium hexachloroosmium tetroxide as the osmium source and citric acid as the reducing agent.

[0057] Take the above dispersion containing 15 mg MOF, add 150 mmol K2OsCl6, and sonicate thoroughly to mix evenly. Heat the mixture to 95 °C, then quickly add 60 μL of 50 mM citric acid solution, and react at this temperature for 20 min. After the reaction, centrifuge to collect the product and wash it several times with deionized water. Then resuspend the product in deionized water to obtain a MOF@Os nanozyme dispersion with a concentration of approximately 15 mg / mL, which should be stored at low temperature and protected from light for later use.

[0058] To fully understand the performance of the MOF@Os prepared in the various embodiments of the present invention, the following tests were conducted using the product of Example 1 as raw material: I. Scanning Electron Microscopy (SEM) and Transmission Electron Microscopy (TEM) Testing.

[0059] The MOF@Os nanozyme powder prepared in Example 1 was used as a dispersion medium and ultrasonically treated to obtain a uniform suspension. A small amount of the suspension was dropped onto a silicon wafer (SEM sample preparation) and a carbon-supported copper mesh (TEM sample preparation), respectively. After thorough drying at room temperature, the samples were tested. The results are as follows: Figure 1 As shown.

[0060] As can be seen from the figure, the MOF@Os nanozyme exhibits a well-dispersed polyhedral structure with a particle size range of approximately 500-1200 nm.

[0061] II. POD enzyme activity test.

[0062] Using the MOF@Os nanozyme dispersion prepared in Example 1 as raw material, its POD enzyme activity was investigated according to the following method: (1) The enzyme-like activity of MOF@Os nanozymes was verified using the TMB-hydrogen peroxide colorimetric system. Control groups (TMB system only, TMB+H2O2 system, MOF@Os+TMB system, and complete MOF@Os+TMB+H2O2 system) were set up simultaneously. Samples from each group were placed in an acetate buffer solution at pH 3.5 for reaction, and the absorbance change at 652 nm was monitored. The results are as follows: Figure 2 As shown.

[0063] The catalytic reaction behavior of TMB solutions under different reaction conditions was monitored by UV-Vis absorption spectroscopy. The results showed that when H2O2 and MOF@Os nanozymes were present simultaneously, TMB was oxidized to oxidized TMB (TMBox), and the solution changed from colorless to blue, exhibiting a characteristic absorption peak at 652 nm, demonstrating that the MOF@Os nanozymes possess peroxidase-like activity. Meanwhile, under the same conditions, the MOF@Os+TMB system showed no color reaction, indicating that it lacked peroxidase-like activity.

[0064] A schematic diagram of the mechanism of MOF@Os-mediated H2O2 catalytic reaction is shown below. Figure 2 As shown in the figure, after confirming the peroxidase-like activity of the MOF@Os nanozyme, its catalytic performance in the presence of H2O2 was further investigated. With increasing H2O2 and MOF@Os concentrations, the absorbance at 652 nm continuously increased, indicating a gradual improvement in the catalytic activity of the MOF@Os nanozyme. Increasing the TMB substrate concentration and extending the reaction time also showed the same trend, with a corresponding increase in absorbance at 652 nm.

[0065] To quantitatively analyze the catalytic kinetics, the steady-state oxidation process of TMB catalyzed by MOF@Os nanozymes under different H2O2 concentrations was measured. The experimental results showed that the Michaelis-Menten equation model was well fitted, and the apparent Michaelis constant K was obtained from the fitting. m =1.20×10 -4 M, maximum reaction rate V max =2.00×10 -6 M·s -1 .

[0066] III. Reactive Oxygen Probe Experiment.

[0067] (1) The superoxide anion in the system was detected by the dihydroethidium fluorescent probe. A control group was set up (dihydroethidium system only and complete system of dihydroethidium co-incubated with MOF@Os). The excitation wavelength was 535nm and the fluorescence emission spectrum range was 550-650nm.

[0068] (2) Use methylene blue colorimetric probe to identify hydroxyl radicals. Set up control groups (methylene blue system only and methylene blue + H2O2 + MOF@Os co-incubation system with different concentrations). Collect ultraviolet-visible absorption spectra in the range of 500-700 nm. Characterize the level of hydroxyl radical generation based on the degree of methylene blue absorbance decay.

[0069] (3) The catalytic mechanism was verified by electron paramagnetic resonance free radical capture experiment. The free radical capture test used 5,5-dimethyl-1-pyrrolline-N-oxide (DMPO) as the capture agent and the experiment was carried out in accordance with the general experimental procedure of electron paramagnetic resonance free radical capture experiment.

[0070] To further elucidate the catalytic mechanism of MOF@Os nanozymes, the types of reactive oxygen species generated in the reaction system were investigated, and the results are shown in Figure 3. Dihydroethidium and methylene blue probes were used to detect superoxide anions and hydroxyl radicals, respectively. Superoxide anions can dehydrogenate dihydroethidium to produce the red product ethidium bromide, resulting in an excitation wavelength λ. ex The fluorescence signal of dihydroethidium decreases at λ = 370 nm. exThe fluorescence signal of dihydroethidium increased at 535 nm. During the test, MOF@Os nanozyme dispersions with concentrations of 0 μg / mL, 1 μg / mL, and 10 μg / mL were added to the dihydroethidium solution, respectively. No significant change in fluorescence intensity was observed, indicating that MOF@Os nanozymes do not generate superoxide anions, further confirming the absence of oxidase-like activity in the material. Conversely, hydroxyl radicals can lead to the degradation of methylene blue. The results showed that in the presence of H2O2, the absorbance of methylene blue at 664 nm continuously decreased with increasing MOF@Os nanozyme concentration, proving the generation of hydroxyl radicals in the system. This confirms that MOF@Os nanozymes possess excellent peroxidase-like activity, and the amount of hydroxyl radicals generated increases with increasing MOF@Os concentration.

[0071] Further detection of hydroxyl radicals generated during the catalytic process using electron paramagnetic resonance spectroscopy revealed a quadruple characteristic signal with an intensity ratio of 1:2:2:1 when H2O2 and MOF@Os nanozymes were present in the system. This confirmed that 5,5-dimethyl-1-pyrroline-N-oxide successfully captured hydroxyl radicals. This result directly demonstrates that MOF@Os nanozymes can effectively catalyze the decomposition of H2O2 to generate hydroxyl radicals, further supporting their peroxidase-like catalytic properties.

[0072] IV. Creatine detection test.

[0073] A sarcosine detection system was constructed using the MOF@Os nanozyme prepared in Example 1 in conjunction with sarcosine oxidase. The detection principle is as follows: sarcosine oxidase specifically oxidizes sarcosine to generate hydrogen peroxide; the MOF@Os nanozyme catalyzes the hydrogen peroxide reaction, mediating TMB color development; and sarcosine quantification is achieved through absorbance changes. Specific testing methods are as follows. (1) System construction method: A gradient concentration of sarcosine standard solution was prepared and incubated with sarcosine oxidase in phosphate buffer (pH 7.40, 100 mM). After the reaction was completed, acetate buffer (pH 3.50) was added to terminate the enzymatic reaction. Then, a colorimetric system containing TMB and MOF@Os nanozyme was added, and the absorbance at 652 nm was measured to establish a sarcosine concentration-absorbance standard curve. The spiked recovery rate was calculated as: measured sarcosine concentration in the nanozyme system / theoretical spiked concentration.

[0074] (2) Specificity verification experiment: Tyrosine (Tyr), tryptophan (Trp), histidine (His), uric acid (UA), glutamate (Glu), alanine (Ala), and sarcosine were selected as parallel controls. Interference verification experiment used K... + Mg 2+ Ca 2+ Cu 2+ Zn 2+ Na+ Common metal ions were added to the detection system for investigation. The experimental results are as follows: Figure 4 As shown in the figure, the following conclusions can be drawn: when only MOF@Os nanozyme, TMB, sarcosine oxidase, and sarcosine are present, the reaction system exhibits obvious color changes.

[0075] The detection parameters were optimized using a single-factor variable method. In the sarcosine detection system, the MOF@Os concentration was varied to 40, 60, 80, 100, and 120 μg / mL; the sarcosine oxidase concentration was varied to 20, 30, 50, 80, 100, and 200 μg / mL; the reaction time was varied to 1, 2, 5, 10, and 15 min; and the reaction temperature was varied to 25℃, 30℃, 35℃, 37℃, 40℃, and 45℃. The absorbance at 652 nm was measured for different systems, and the results are as follows: Figure 5 As shown. The final optimal detection conditions were: MOF@Os nanozyme concentration of 100 μg / mL, sarcosine oxidase concentration of 80 μg / mL, enzyme reaction time of 10 min, and reaction temperature of 37℃.

[0076] V. Specificity test.

[0077] The specificity and anti-interference ability of the sarcosine biosensor based on MOF@Os nanozymes were evaluated by adding potential interfering substances to the detection system. The results are as follows: Figure 6 As shown. Interfering substances included tyrosine (Tyr), tryptophan (Trp), histidine (His), uric acid (UA), glutamate (Glu), and alanine (Ala). The results showed that the experimental group with only sarcosine added exhibited a significant difference in absorbance. This result confirms that, compared to other common metabolites in urine, the MOF@Os nanoenzyme sensing platform has preferential recognition and specific response capabilities for sarcosine.

[0078] Further interference resistance experiments were conducted using typical inorganic ions and endogenous organic compounds at physiological concentration levels. The concentrations of major metal ions in urine were adjusted to physiological levels: Na+... + (150mM), K + (100mM), Ca 2+ (5mM), Mg 2+ (4mM) was used to investigate several potential interfering components: glucose (Glu, 0.06mM), uric acid (UA, 3mM), urea (Ur, 400mM), and bovine serum albumin (BSA, 0.03g / L). The results showed that, compared with the signal response generated by creatine, none of the above-mentioned interfering substances caused significant changes in absorbance, indicating that the interference effect was weak under the experimental conditions.

[0079] The above series of experiments further confirm that the MOF@Os nanoenzyme colorimetric sensing platform has excellent selectivity and anti-interference performance in complex urine-like environments.

[0080] VI. Non-invasive screening test for prostate cancer in clinical practice.

[0081] Urine spiked test: The pH of the urine was adjusted to 7-8 using sodium hydroxide. Then, 10 μM, 50 μM, 80 μM, 100 μM, 300 μM and 500 μM sarcosine standards were added to the urine to establish a spiked standard curve.

[0082] Actual urine sample testing: The urine sample was concentrated by heating, and the supernatant was collected after centrifugation at 3500 rpm for 5 minutes for testing.

[0083] Under the aforementioned optimal conditions, a standard curve was established, showing a good linear relationship between creatine concentration and absorbance in the range of 5–500 μM. R0 2 =0.997, with a detection limit as low as 3.77μM, which can meet the clinical needs for detecting trace amounts of creatine in urine.

[0084] After optimizing the reaction conditions of the MOF@Os nanozyme detection system, colorimetric methods were used for the quantitative detection of sarcosine (Sar). Figure 6 As shown by the red curve, within the 5–500 μM creatine concentration range, the absorbance difference ΔA exhibits a good linear relationship with the creatine concentration. The linear regression equation is as follows:

[0085] The coefficient of determination R of the curve 2 =0.997, and the detection limit of this method is calculated to be 3.77 μM, which is sufficient to meet the detection requirements of creatine in actual samples.

[0086] To further verify its practical application potential, a urine matrix spiking experiment was conducted. The MOF@Os nanozyme detection system was validated using spiked real urine samples, achieving specific recognition and quantitative detection of sarcosine. Figure 6 (Black curve), the corresponding linear regression equation is as follows:

[0087] The coefficient of determination R of the curve 2 =0.995, showing excellent linearity, proving that reliable quantification of sarcosine can be achieved within the test concentration range.

[0088] The above results demonstrate that the MOF@Os nanozyme biosensor described in this invention can efficiently and accurately assist in the diagnosis of prostate cancer by detecting sarcosine.

[0089] The differences in urinary creatine test results between 7 healthy individuals and 7 prostate cancer patients were compared. The test results are as follows: Figure 7 As shown in the figure, the urinary creatine concentration in prostate cancer patients was significantly higher than that in healthy individuals, exhibiting a highly statistically significant difference. The urine spiked recovery rate experiment results showed that the creatine spiked recovery rate was 105.78%–116.15%, with RSDs all less than 13%, indicating good accuracy and precision, and making it stable for clinical non-invasive prostate cancer screening.

[0090] VII. Visualized smartphone prostate cancer screening.

[0091] To achieve portable on-site testing, this invention establishes a smartphone-based visual colorimetric scheme: Rhodamine is introduced as an internal control dye to improve color differentiation between different concentrations. Sarcosine solutions with concentrations of 50 μg / mL, 100 μg / mL, 200 μg / mL, 300 μg / mL, 500 μg / mL, and 800 μg / mL were prepared and incubated with sarcosine oxidase at a concentration of 80 μg / mL for 10 min. Then, a colorimetric system (0.5 mg / mL TMB, 100 μg / mL MOF@Os nanozyme, and 15 μL Rhodamine) was added, and the solution was incubated in the dark for 20 min. Images of the colorimetric solutions were captured using a smartphone, and the RGD color channel values ​​were extracted using the Color Collect APP from Fuzhou Micro Color Technology.

[0092] Test results are as follows Figure 8 As shown in the figure, it can be seen that as the concentration of creatine increases, the solution color gradually transitions from pink to purple, and finally to dark blue. The red (R) channel values ​​of the image were extracted using a mobile APP, and the linear equation was obtained by linear fitting of the R channel values ​​with the creatine concentration. The detection limit of this visualization method is 13.35 μM.

[0093] The color signal of the chromogenic solution showed no significant change after two months of storage at room temperature, indicating the excellent long-term stability of the visualization platform. Compared with traditional quantitative methods that rely on large spectrophotometers, the smartphone colorimetric method is simple to operate, portable, fast, and inexpensive, making it ideal for rapid on-site screening.

[0094] 8. Building the ResNet18-Sar machine learning prediction model.

[0095] Each concentration gradient colorimetric image was split into 50 sub-images. The entire dataset was imported into a ResNet18-Sar model built using Python for training and validation. The model's concentration prediction accuracy was evaluated using multiple metrics, and the results are as follows: Figure 9 As shown.

[0096] ResNet18 Machine Learning-Assisted Intelligent Quantification of Creatine: This invention employs an improved ResNet18 convolutional neural network model to identify subtle color differences in chromogenic images with different creatine concentrations, establishing a quantitative correlation between color features and creatine concentration, and accurately predicting the concentration of the analyte. Each concentration's chromogenic image is split into 50 sub-images as the dataset input to the model, automatically extracting image color features; overfitting is suppressed by stratified random sampling to divide the training / validation sets, ensuring balanced samples across concentrations, improving color feature recognition capabilities, and achieving accurate mapping from visual features to creatine concentration. Model training employs a two-stage training strategy, comprehensively evaluated through multi-dimensional indicators: training loss continuously decreases, model convergence; mean absolute error (MAE) = 25.49, mean squared error (MSE) = 1155.34, and coefficient of determination (R²)... 2 =0.986; the overall accuracy of the model concentration prediction was 93.30%.

[0097] The ResNet18-Sar model provided by this invention can automatically predict creatine concentration in a very short time, and can achieve high-precision intelligent quantification on site with only a small number of samples, which has great application prospects in non-invasive screening of prostate cancer.

[0098] Those skilled in the art will readily understand that the above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for preparing MOF@Os nanozymes, characterized in that... The method includes: S1, MOF was prepared using a zirconium source and 2,2-bipyridine-5,5-dicarboxylic acid; S2, MOF@Os nanozymes were prepared using MOF, osmium source, and reducing agent.

2. The method as described in claim 1, characterized in that... The specific process of S1 includes: dissolving a zirconium source and 2,2-bipyridine-5,5-dicarboxylic acid in an organic solvent to obtain a zirconium source solution and a 2,2-bipyridine-5,5-dicarboxylic acid solution; mixing the zirconium source solution and the 2,2-bipyridine-5,5-dicarboxylic acid solution evenly, adding a pH adjuster, and carrying out a solvothermal reaction at 100-200℃; after the reaction is complete, naturally cooling to room temperature, centrifuging to collect the solid product and washing it to obtain MOF.

3. The method as described in claim 2, characterized in that: The zirconium source in S1 is selected from at least one of zirconium tetrachloride, zirconium oxychloride, or their hydrates; the organic solvent is specifically N,N-dimethylformamide; and the pH adjuster is specifically an organic acid selected from at least one of formic acid, acetic acid, and benzoic acid.

4. The method as described in claim 2, characterized in that: The concentration of the zirconium source solution in S1 is 1-4 g / L, and the concentration of the 2,2-bipyridine-5,5-dicarboxylic acid solution is 1-4 g / L, and the two concentrations may be equal or unequal; when mixing, the mass ratio of zirconium source to 2,2-bipyridine-5,5-dicarboxylic acid is 100-300:200-400, the total amount of organic solvent used is 50-300 mL / 100-300 mg zirconium source, and the amount of pH adjuster used is 2000-5000 μL / 100-300 mg zirconium source.

5. The method as described in claim 1, characterized in that... The specific process of S2 includes: dispersing MOF in a solvent to obtain MOF dispersion; adding osmium source and reducing agent to MOF dispersion and heating to 50-100℃ for reaction; after the reaction is completed, naturally cooling to room temperature; centrifuging to collect solid product and washing to obtain MOF@Os nanozyme.

6. The method as described in claim 5, characterized in that: The osmium source in S2 is selected from at least one of potassium hexachloroosmium tetroxide, sodium hexachloroosmium tetroxide, and ammonium hexachloroosmium tetroxide, and the reducing agent is selected from at least one of sodium borohydride, ascorbic acid, and citric acid.

7. The method as described in claim 5, characterized in that: The concentration of the MOF dispersion in S2 is 0.5-15 g / L, the amount of osmium source is 10-200 mol / 5-15 g MOF, and the amount of reducing agent is 0.5-20 mmol / 5-15 g MOF.

8. The method as described in claim 2 or 5, characterized in that: The obtained product is repeatedly washed multiple times with at least one of N,N-dimethylformamide, tetrahydrofuran, ethanol, and deionized water.

9. A MOF@Os nanozyme, characterized in that: The MOF@Os nanozyme has a polyhedral structure, in which MOF serves as the framework and Os reduction modification is performed inside or on the surface of MOF. The particle size of the MOF@Os nanozyme is 500-1200 nm.

10. The application of the MOF@Os nanozyme of claim 9 in quantitative detection of sarcosine and in non-invasive screening reagents or kits for prostate cancer, characterized in that: The testing methods include laboratory testing, bedside point-of-care testing, on-site testing using a visual portable terminal, and large-scale intelligent testing.

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