Cholesterol oxidase sensor based on au-nd nanoszyme and its application in intestinal cancer

Abstract

The application constructs a cholesterol oxidase (CHOX) sensor based on Au-Nd nanoscale enzyme, and explores its application in the research of intestinal cancer. The Au-Nd nanoscale enzyme with peroxidase activity is synthesized by a one-step hydrothermal method, can catalyze the reduction of KMnO4 by H2O2, and make it change from purple red to yellow, and present absorption change at 526 nm, so as to realize the rapid visual detection of H2O2 (<20 s), which is simple and low in cost. The CHOX oxidizes cholesterol (CHOL) to generate H2O2, which further affects the color change of KMnO4, realizes the sensitive and quantitative detection of CHOX activity. With the increase of CHOX activity, the absorption peak at 526 nm decreases, and the color of KMnO4 changes from deep purple to yellow. Based on the RGB color mode, the relationship between R / G value and H2O2, CHOX, CHOL, Hg 2+ is established, and the miniaturization and intelligence potential are shown. The sensor is used for the analysis of E. coli CHOX activity for the first time, and the intestinal cancer environment is simulated to explore the change of CHOX, which provides a new perspective for the inflammation-cancer chain research. At present, there is no related report, and it has scientific and social value.

Description

Technical Field

[0001] This invention relates to a cholesterol oxidase sensor based on Au-Nd nanozymes, and particularly to a sensor constructed using this nanozyme and its application in colorectal cancer. This sensor can detect hydrogen peroxide (H2O2), cholesterol (CHOL), cholesterol oxidase (CHOX), and its inhibitor Hg. 2+ This invention enables efficient and rapid analysis and detection. Ultimately, it established an analytical model for cholesterol oxidase activity in *E. coli* and explored the relationship between cholesterol oxidase activity and the occurrence and development of colorectal cancer. This invention combines analytical chemistry and chemibiosensing technologies, providing an innovative breakthrough in the field of bioanalytical detection and possessing significant application value in colorectal cancer research and early diagnosis. Background Technology

[0002] Colorectal cancer is one of the leading causes of cancer death worldwide, and its incidence is increasing year by year. Early diagnosis is crucial for the prevention and treatment of colorectal cancer because it often presents with no obvious symptoms in its early stages, leading to most patients being diagnosed only when the disease has progressed to an advanced stage, at which point the effectiveness of treatment and the patient's survival rate are significantly reduced. Therefore, the key to early diagnosis of colorectal cancer lies in improving the accuracy and timeliness of diagnosis. Early diagnosis can significantly improve the cure rate of colorectal cancer. According to research data, the five-year survival rate of patients with colorectal cancer detected in the early stages is as high as 90%, while the survival rate of patients with advanced stages is much lower. Through timely screening and diagnosis, precancerous lesions or early tumors can be detected, and methods such as endoscopic surgical resection can be used to greatly reduce the incidence of colorectal cancer. In addition, early diagnosis also helps to reduce treatment costs and patient suffering. Treatment of advanced colorectal cancer usually requires complex surgery, chemotherapy, and radiotherapy, which are costly and have significant side effects. Early detection and treatment can avoid these expensive and burdensome treatments, reducing the economic and physical burden on patients. Therefore, early diagnosis of colorectal cancer is not only related to the patient's quality of life and chance of survival, but also of great significance to public health and the rational use of medical resources.

[0003] Escherichia coli (E. coli) is one of the most common bacteria in the gut. Under normal circumstances, it maintains a symbiotic relationship with the host. However, when the gut microbiota is imbalanced, certain types of E. coli may transform into pathogenic strains, which are closely related to the occurrence of various intestinal diseases. In recent years, studies have found a potential link between E. coli and the development of colorectal cancer. Pathogenic E. coli in the gut may play an important role in the development of colorectal cancer through their metabolites, the immune responses they trigger, and the oxidative stress they promote. Among them, cholesterol oxidase (CHOX) in certain specific E. coli species plays a key role in the development of colorectal cancer. Cholesterol oxidase catalyzes the oxidation of cholesterol, generating cholesterol oxidation products. These products may directly or indirectly affect the intestinal environment, causing damage to intestinal cells and promoting carcinogenesis. Increased cholesterol oxidase activity may lead to oxidative stress and inflammatory responses in intestinal cells, thus creating favorable conditions for the development of colorectal cancer. Studies have shown that cholesterol oxidase activity is closely related to the level of inflammation in the gut, the activation of immune responses, and the accumulation of cell mutations, especially under conditions of gut microbiota imbalance, where this enzyme activity may be significantly enhanced. Therefore, detecting cholesterol oxidase activity in *E. coli* has become an important method for the early diagnosis of colorectal cancer. Detecting cholesterol oxidase activity in *E. coli* can reveal early signs of gut microbiota dysbiosis and the potential risk of colorectal cancer. This detection method not only has high sensitivity and specificity but also helps identify high-risk groups, providing strong support for early colorectal cancer screening. Timely early diagnosis allows for intervention in the curable stage of colorectal cancer, significantly improving patient survival rates and reducing treatment complexity and costs. Therefore, cholesterol oxidase-based detection has significant clinical application value in the early diagnosis of colorectal cancer.

[0004] Au-lanthanide nanozymes, as emerging artificial enzyme materials, have gradually gained attention in the fields of catalysis and sensing in recent years. However, existing research mainly focuses on combinations of certain lanthanide elements such as Au-La and Au-Ce, and their applications are primarily limited to environmental remediation and basic catalytic reactions, with research in biomedicine and biosensing remaining relatively limited. In contrast, Au-Nd nanozymes, as a novel material that has not yet been widely reported and applied, have received almost no systematic research and development in domestic and international literature. Au-Nd nanozymes combine the excellent electronic conductivity and biocompatibility of Au nanoparticles with the unique coordination ability and redox catalytic properties of Nd (neodymium) ions, showing great potential in enhancing enzyme simulation activity, reaction selectivity, and biosensing sensitivity. In particular, in the cholesterol oxidation reaction system, Au-Nd nanozymes can achieve highly efficient hydrogen peroxide generation activity, providing a novel functional platform for constructing cholesterol oxidase sensors. Therefore, Au-Nd nanozymes, as a novel catalytic material that has not yet been fully utilized, have broad application prospects in cholesterol oxidase sensing, disease biomarker detection, and early diagnosis of colorectal cancer.

[0005] This invention designs a cholesterol oxidase sensor based on Au-Nd nanozymes and its application in colorectal cancer. The method employs a simple hydrothermal one-step synthesis of Au-Nd nanozymes with peroxidase activity. These nanozymes can one-step modify bovine serum albumin (BSA) with gold and neodymium to form Au-Nd nanoclusters. Experimental data show that the synthesized Au-Nd nanozymes can catalyze the reduction of hydrogen peroxide (H₂O₂). KMnO₄ itself produces two significant UV absorption peaks at 526 nm and 546 nm, respectively, and exhibits a purplish-red color. This is due to the presence of permanganate ions (MnO₄⁻) in the potassium permanganate solution. - Due to the presence of ), based on the principle of signal significance, this patent selects the 526nm peak for signal quantization calculation. When KMnO4 reacts with the reducing agent (H2O2), the permanganate ions (MnO4) are produced. - Mn was reduced to a pale yellow color. 2+The absorbance of the ions at 526 nm decreased significantly. Most importantly, this system enables rapid, visual, and quantitative analysis of H2O2 concentration, with quick operation, low cost, and completion in less than 20 seconds. Furthermore, CHOX oxidizes cholesterol (CHOL) and generates a certain amount of H2O2. Therefore, a cholesterol oxidase sensor based on Au-Nd nanozymes was used to measure the color change produced by the reaction of CHOX and CHOL, achieving rapid, sensitive, and quantitative detection of CHOX activity. With increasing CHOX activity, the amount of H2O2 produced increases, the intensity of the UV absorption peak at 526 nm decreases, and the color of KMnO4 gradually changes from deep purple to yellow, providing a simple method for rapid and visual determination of CHOX. Finally, based on the Red-Green-Blue (RGB) color mode in a mobile phone, R / G was established to correlate with H2O2, CHOX, CHOL, and Hg. 2+ The relationship between these factors suggests that this sensor has the potential for miniaturization and intelligentization, and promising application prospects. Most importantly, this patent applies it to the analysis of cholesterol oxidase activity in *E. coli*, and compares the activity of cholesterol oxidase under normal and simulated colorectal cancer environments by culturing *E. coli* under different culture conditions. Currently, there are no reports on cholesterol oxidase sensors based on Au-Nd nanozymes and their applications in colorectal cancer. Summary of the Invention

[0006] The technical problem to be solved by the present invention is to provide a cholesterol oxidase sensor based on Au-Nd nanozyme with high sensitivity, good specificity, rapid and simple operation, and its application in colorectal cancer.

[0007] The technical solution adopted by this invention to solve the above-mentioned technical problems is as follows: a cholesterol oxidase sensor based on Au-Nd nanozyme, particularly a sensor established using this nanozyme and its application in colorectal cancer, with the specific steps as follows:

[0008] I. Preparation of Au-Nd Nanozymes

[0009] Under vigorous stirring, HAuCl4 (1–5 mmol) and NdCl3 (1–5 mmol) were added to 1 mL of BSA (10–50 mg / mL) solution. After stirring for 5 min, 100 μL of NaCl solution (1–3 M) was added dropwise. The reaction was carried out at 37 °C for 12 h under continuous stirring to finally obtain Au-Nd nanoparticles.

[0010] II. Fabrication of Au-Nd Nanozyme-Driven Cholesterol Oxidase Sensor

[0011] A. Au-Nd nanozyme-catalyzed H2O2-KMnO4 system

[0012] UV absorption curve assay: 60 μL of phosphate buffer (10 mM, pH 7.4, prepared from NaH₂PO₄ and Na₂HPO₄), 24 μL of H₂O, 3 μL of Au-Nd nanozyme, 3 μL of 20 mM H₂O₂, and 10 μL of 0.01 mM KMnO₄ were added sequentially to a 96-well microplate. After incubation at room temperature with stirring for 20 s, a photograph was taken at a fixed height using a mobile phone, and the UV absorption change of the H₂O₂-KMnO₄ system was measured using a microplate reader. The scanning wavelength range was set to 300–600 nm.

[0013] UV kinetics assay: 60 μL of phosphate buffer (10 mM, pH 7.4, prepared from NaH₂PO₄ and Na₂HPO₄), 24 μL of H₂O, 3 μL of Au-Nd nanozyme, 3 μL of 20 mM H₂O₂, and 10 μL of 0.01 mM KMnO₄ were added sequentially to a 96-well microplate. The mixture was then immediately placed in a microplate reader, and the absorbance was measured at 526 nm over time to analyze the reaction kinetics of the H₂O₂-KMnO₄ system. Microplate reader parameters were set as follows: temperature 37℃, measurement time 300 s, and vibration enabled.

[0014] B. Cholesterol Oxidase CHOX Sensor

[0015] (1) Add 60 μL of phosphate buffer solution (10 mM, pH 7.4), 5 μL of H2O, 2 μL of 40 U / mL CHOX and 20 μL of 10 mM CHOL to a 200 μL centrifuge tube in sequence. Incubate the reaction solution in a 37°C water bath for 30 min.

[0016] (2) Add 3 μL of Au-Nd nanozyme and 10 μL of 0.01 mM KMnO4 to the reaction solution in (1), mix thoroughly, and incubate at 37°C with stirring for 20 s. Take a picture using a mobile phone at a fixed height. Then transfer the mixture to an ELISA plate and test the change in UV absorption of the reaction solution, with the wavelength range set to 300–600 nm. Alternatively, immediately transfer the above solution to an ELISA reader and measure the change in UV absorption of the reaction solution over time. The ELISA reader kinetic parameters are set as follows: 37°C, 300 s, and vibration.

[0017] III. H2O2, CHOL and CHOX and their inhibitor Hg 2+ Analysis and detection

[0018] By varying the concentration of H2O2 in step 2-A (final concentration: 0–1600 μM) while keeping other experimental conditions constant, the ultraviolet absorbance in the wavelength range of 300–600 nm was measured. A standard curve for H2O2 was plotted using the maximum ultraviolet absorbance at 526 nm, thus enabling the ultraviolet detection of H2O2 at different concentrations.

[0019] By varying the concentration of CHOL in step 2-B (final concentration: 0–6000 μM) while keeping other experimental conditions constant, the ultraviolet absorbance in the wavelength range of 300–600 nm was measured. A standard curve for CHOL was plotted using the maximum ultraviolet absorbance at 526 nm, thus enabling the ultraviolet detection of different concentrations of CHOL.

[0020] By varying the concentration of CHOX in step 2-B (final concentration: 0–4500 U / L) while keeping other experimental conditions constant, the ultraviolet absorbance in the wavelength range of 300–600 nm was measured. A standard curve of CHOX was plotted using the maximum ultraviolet absorbance at 526 nm, thereby enabling the ultraviolet detection of different concentrations of CHOX.

[0021] In step 2-B, first mix 2 μL of 40 U / mL CHOX with 1 μL of 100 μM Hg. 2+ The solution was incubated at 37°C for 5 minutes, and then the original experimental procedure was followed for detection to investigate Hg. 2+ Inhibitory effect on CHOX. Further alteration of Hg. 2+ The concentration (final concentration: 0–2.5 μM) was determined to measure its inhibitory effect on CHOX, and the IC50 was calculated. 50 value.

[0022] IV. Smartphone Sensing Method Design

[0023] This method utilizes the iPhone 15's camera function to achieve real-time monitoring of target analyte concentration changes. The device consists of a dark box, LED lights, and white paper. A small hole at the top of the dark box connects to the smartphone camera, limiting ambient light interference and ensuring image accuracy. The LED light provides a stable light source, ensuring uniform image brightness. The white paper serves as a background, making the target analyte stand out and facilitating subsequent image processing. Using this device, the smartphone can capture real-time color changes in KMnO4 caused by variations in the concentrations of H2O2, CHOL, and CHOX. The camera records these color changes, which are then analyzed using image processing algorithms. In operation, the sample is placed in a 96-well UV colorimetric plate and placed in the dark box. The LED light is turned on, and the phone's camera captures the image. After acquisition, the R (red) and G (green) values ​​in the image are analyzed using ColorColl software. The R / G ratio is compared with the concentrations of H2O2, CHOL, CHOX, and Hg. 2+ The concentration relationship was investigated, and standard curves were established to monitor changes in target analyte concentration in real time. This smartphone-based system is portable, easy to use, and low-cost, making it suitable for rapid and accurate monitoring in both field and laboratory settings.

[0024] Invention Principle: This invention proposes a cholesterol oxidase (CHOX) sensor based on Au-Nd nanozymes and explores its application in colorectal cancer research. The core mechanism utilizes Au-Nd nanozymes to catalyze the gradual reduction of KMnO4 induced by H2O2, causing the color to change from purplish-red to pink, then brownish-yellow, and finally light yellow. During this process, the UV absorption peak of KMnO4 at 526 nm significantly decreases, and the intensity of this absorption peak gradually weakens with increasing H2O2 concentration, thus achieving rapid visual detection of H2O2 (<20 s). When CHOL and CHOX are added to the system, CHOX selectively oxidizes CHOL to generate H2O2, further promoting the reduction of KMnO4, causing the solution color to gradually change from purplish-red to dark brown, and finally to light yellow. Within a certain concentration range, the higher the H2O2 concentration, the stronger its reducing ability for KMnO4, the more obvious the color change of KMnO4, and the weaker the intensity of the absorption peak at 526 nm. Similarly, higher concentrations of CHOL or CHOX result in more H2O2 generation, leading to a further weakening of the absorption peak at 526 nm and a gradual fading of the purple-red color of KMnO4. Experimental results show that, within a certain range, the absorption peak intensity at 526 nm exhibits a good linear relationship with the concentrations of H2O2, CHOL, and CHOX, enabling visual analysis and detection of these three components. Furthermore, the experiment verified the presence of CHOX in *E. coli* and constructed a method for detecting CHOX activity, providing a new approach for studying the inflammation-cancer chain transformation mechanism in colorectal cancer. Based on this, this invention achieves quantitative analysis of H2O2, CHOL, and CHOX, constructing a simple, rapid, and highly selective detection method.

[0025] Compared with the prior art, the advantages of the present invention are as follows:

[0026] (1) The method is simple and the detection is rapid. The reaction between KMnO4 and H2O2 is usually slow (color development time > 1 min). In this system, the introduction of Au-Nd nanozymes significantly improved the reaction rate (color development time < 20 s), enhanced the signal sensitivity, and enabled rapid and convenient visualization detection of the target analyte. Au-Nd nanozymes may function through the following mechanisms: mimicking natural peroxidase to catalyze the decomposition of H2O2 and accelerate the reaction; providing electron transfer channels to further improve the reaction rate; increasing a large number of active surface sites, allowing the decomposition of H2O2 to take place in a more efficient environment; and stabilizing H2O2 to prevent its non-specific decomposition and allow it to participate more in the target reaction.

[0027] (2) High sensitivity. This study is the first to construct a CHOX UV colorimetric sensor based on Au-Nd nanozymes, which exhibits a significant absorption peak at 526 nm, and the results were analyzed by changes in the R / G value. The following linear equations were obtained:

[0028] A526 The linear equation between the change in H2O2 concentration and the logarithmic value is: y = -0.088x + 0.41, R 2 =0.9884, detection limit is 0.041 μM;

[0029] A 526 The linear equation between the change in CHOL concentration and the logarithmic value is: y = -0.036x + 0.42, R0 2 =0.9842, detection limit is 0.035 μM;

[0030] A 526 The linear equation between the change in CHOX concentration and the logarithmic value is: y = -0.04x + 0.43, R 2 =0.9856, detection limit is 0.033 U / L.

[0031] The constructed sensor exhibits high sensitivity in detecting H2O2, CHOL, and CHOX. Furthermore, the smartphone-based sensing system detects the target analyte by leveraging the linear relationship between R / G value changes and analyte concentrations. The results show a high degree of agreement with those obtained from large-scale instrument analysis, laying the foundation for sensor miniaturization and on-site detection applications.

[0032] (3) High specificity. For H2O2 detection: other control substances such as sucrose (SUC), cholesterol (CHOL), tryptophan (Trp), maltose (MLT), xanthine (XA), and lactate (HL) do not interfere with H2O2 detection; for CHOL detection: other control substances such as reduced coenzyme I (NADH), adenosine diphosphate (ADP), xanthine (XA), glucose (GLU), lactate (HL), and uric acid (UA) do not interfere with the system; for CHOX detection: other control substances such as xanthine oxidase (XOD), terminal transferase (TdT), glucose oxidase (GOx), acetyltransferase (Tip60), papain, and alcohol dehydrogenase (ADH) do not interfere with the system.

[0033] (4) High accuracy. Recovery rates are all between 95% and 105%.

[0034] (5) Strong applicability. The smartphone sensor developed in this study is suitable for rapid on-site analysis and detection, requiring minimal reagent consumption, meeting the low-cost requirements of industrialization, and is easy to operate. The sensor features mild reaction conditions and a fast reaction speed, exhibiting stable performance, making it suitable for practical applications such as biosensing and disease screening. Furthermore, this patent simulates the colorectal cancer environment and develops a method for detecting CHOX activity in E. coli suitable for different environments, providing a new perspective for translational research on the inflammation-cancer chain.

[0035] In summary, this invention constructs a sensor for the rapid detection of cholesterol oxidase in *E. coli* and applies it to the visual analysis and detection of H2O2, CHOL, and CHOX. This method has advantages such as high sensitivity, strong specificity, simple operation, accurate results, and rapid analysis, enabling visual quantitative analysis of H2O2, CHOL, and CHOX in *E. coli* samples. Currently, no similar studies have been found, indicating that this technology has promising application prospects and significant scientific value in exploring the relationship between cholesterol oxidase activity and the development of colorectal cancer, as well as in monitoring *E. coli* growth. Attached Figure Description

[0036] Figure 1 Feasibility analysis of the Au-Nd nanozyme catalyzing the H2O2-KMnO4 system in this invention;

[0037] Figure 2 The sensor of this invention can detect different concentrations of H2O2. 526 Absorbance standard curve and kinetic analysis;

[0038] Figure 3 The sensor of this invention can detect different concentrations of CHOL. 526 Absorbance standard curve and kinetic analysis;

[0039] Figure 4 The sensor of this invention can detect different concentrations of CHOX in A. 526 Absorbance standard curve and kinetic analysis;

[0040] Figure 5 The sensor of this invention is for detecting different concentrations of Hg. 2+ A 526 Absorbance standard curve and kinetic analysis;

[0041] Figure 6 This invention relates to the sensor for the detection and analysis of CHOX activity in lysates of different bacteria and Escherichia coli.

[0042] Figure 7 This invention relates to the detection and analysis of CHOX activity in lysates of different bacteria and Escherichia coli using a mobile phone sensor. Detailed Implementation

[0043] The present invention will be further described in detail below with reference to the accompanying drawings and embodiments.

[0044] Example 1: Au-Nd nanozyme-catalyzed H2O2-KMnO4 system

[0045] UV absorption curve assay: 60 μL of phosphate buffer (10 mM, pH 7.4, prepared from NaH₂PO₄ and Na₂HPO₄), 24 μL of H₂O, 3 μL of Au-Nd nanozyme, 3 μL of 20 mM H₂O₂, and 10 μL of 0.01 mM KMnO₄ were added sequentially to a 96-well microplate. After incubation at room temperature with stirring for 20 s, a photograph was taken at a fixed height using a mobile phone, and the UV absorption change of the H₂O₂-KMnO₄ system was measured using a microplate reader. The scanning wavelength range was set to 300–600 nm.

[0046] UV kinetics assay: 60 μL of phosphate buffer (10 mM, pH 7.4, prepared from NaH₂PO₄ and Na₂HPO₄), 24 μL of H₂O, 3 μL of Au-Nd nanozyme, 3 μL of 20 mM H₂O₂, and 10 μL of 0.01 mM KMnO₄ were added sequentially to a 96-well microplate. The mixture was then immediately placed in a microplate reader, and the absorbance was measured at 526 nm over time to analyze the reaction kinetics of the H₂O₂-KMnO₄ system. Microplate reader parameters were set as follows: temperature 37℃, measurement time 300 s, and vibration enabled.

[0047] The ultraviolet absorption spectrum of the above sensor was measured in the range of 300–600 nm, such as... Figure 1 As shown in Figure A, the KMnO4 solution (final concentration: 1 μM) is purplish-red and shows two obvious UV absorption peaks at 526 nm and 546 nm. When H2O2 (final concentration: 600 μM) is added to the KMnO4 solution, the color of solution (6) becomes lighter and the absorption peak at 526 nm decreases, but the reaction rate is slow and the signal change is small. In contrast, when 3 μL of Au-Nd nanozyme and H2O2 (final concentration: 600 μM) are added to the KMnO4 solution together, the color of solution (1) turns yellow, the absorption peak at 526 nm decreases further, and the signal change is more significant. However, no obvious phenomena were observed in H2O2 solution (3), Au-Nd nanozyme (4), and Au-Nd nanozyme + H2O2 (7) alone, indicating that the sensing method has good response performance to H2O2 and can be used for the analysis and detection of H2O2. Meanwhile, Au-Nd nanozymes not only accelerated the reaction rate, but also, by adding 3 μL of Au-Nd nanozymes and 10 μL of 0.01 mM KMnO4 to the reaction solution in (1), mixing thoroughly, and stirring and incubating at 37°C for 20 s, a mobile phone was used to take a picture at a fixed height. The mixture was then transferred to an ELISA plate, and the change in UV absorption of the reaction solution was tested, with the wavelength range set to 300–600 nm. Alternatively, the above solution was immediately transferred to an ELISA reader, and the change in UV absorption of the reaction solution over time was measured. The ELISA reader kinetic parameters were set as follows: 37°C, 300 s, and vibration was set.

[0048] By varying the CHOL concentration (final concentrations of 0, 0.1, 0.2, 0.5, 1.25, 4, 8, 20, 50, 125, 400, 1000, 2000, 3500, 4500, and 6000 μM) while keeping other experimental steps constant, the ultraviolet spectra were measured using a microplate reader in the range of 300–600 nm, and the absorbance at 526 nm (A) was recorded. 526 A CHOL standard curve was established to enable UV detection of different concentrations of CHOL. Experimental results ( Figure 3 A) show that A 526 The value showed a good linear relationship with the CHOL concentration, and as the CHOL concentration increased, the solution color gradually changed from purplish-red to yellow. 526 The linear regression equation with respect to CHOL concentration is: y = -0.036x + 0.42 (where x is the logarithm of CHOL concentration), R0 2 =0.9842, with a linear detection range of 0.1–3500 μM and a limit of detection (LOD) of 0.035 μM, indicating that the sensor has high sensitivity and can effectively detect changes in CHOL concentration. Under the same CHOL concentration gradient conditions, the absorbance at a wavelength of 526 nm was monitored over time using a microplate reader to study the kinetic process of CHOL catalyzing the formation of H2O2 in the CHOX system and its further reaction with KMnO4 (final concentration: 1 μM). Experimental results ( Figure 3 B) shows that with increasing CHOL concentration, the yield of H2O2 produced by its catalysis increases, leading to a more significant change in the color of KMnO4, further demonstrating that the sensor can achieve rapid and efficient detection of CHOL. To further expand the application of this sensor, a smartphone camera combined with colorimetric analysis was used to detect CHOL concentration. During the experiment, the sample was placed in a 96-well UV colorimetric plate and placed in a dark chamber, with LED lights turned on for illumination, and images of the sample were captured using the smartphone camera. Subsequently, the R (red) and G (green) values ​​in the photos were extracted using ColorColl software, and the R / G ratio was calculated. A standard curve was established using the R / G values ​​of different CHOL concentrations. Experimental results ( Figure 3 C) shows that as the concentration of CHOL increases, the color of the KMnO4 solution gradually changes from purplish-red to light yellow, and the R / G value shows a decreasing trend. The linear regression equation is: y = -0.50x + 3.28 (x is the logarithm of the CHOL concentration), with a linear range of 0.1–3500 μM. 2 =0.9865. This indicates that the colorimetric detection method based on smartphones is feasible, and that the sensor has good potential for practical application, providing a simple and rapid analytical method for portable detection of CHOLs.

[0049] By varying the CHOX concentration (final concentrations of 0, 0.1, 0.2, 0.4, 1, 2.5, 5, 10, 20, 60, 120, 400, 800, 1500, 3000, and 4500 U / L), while keeping other experimental steps unchanged, the ultraviolet spectrum in the range of 300–600 nm was measured using a microplate reader, and the absorbance at 526 nm (A) was recorded. 526 A CHOX standard curve was established to enable UV detection of different concentrations of CHOX. Experimental results ( Figure 4 A) show that A 526 The value showed a good linear relationship with the CHOX concentration, and the solution color gradually changed from purple to yellow as the CHOX concentration increased. 526 The linear regression equation with CHOX concentration is: y = -0.04x + 0.43 (where x is the logarithm of CHOX concentration), R0 2 =0.9856, linear range of 0.1–1500 U / L, and detection limit of 0.033 U / L, indicating that the sensor has high sensitivity and can effectively detect changes in CHOX concentration. Furthermore, under the same CHOX concentration gradient conditions, the absorbance at 526 nm was monitored over time using a microplate reader to study the kinetics of CHOL's reaction with CHOX to produce H₂O₂ and subsequently react with KMnO₄. Experimental results ( Figure 4 B) shows that with increasing CHOX concentration, the yield of H2O2 catalyzed by CHOL increases, thereby accelerating the reaction of KMnO4 and resulting in a more significant change in solution color. This result further proves that the sensor can achieve rapid and efficient detection of CHOX. To expand the application of this sensor, a smartphone camera combined with colorimetric analysis was used to detect the CHOX concentration. During the experiment, the sample was placed in a 96-well UV colorimetric plate and placed in a dark chamber, with LED lights turned on for illumination, and the sample image was captured using the smartphone camera. Subsequently, the R (red) and G (green) values ​​in the image were extracted using ColorColl software, and the R / G ratio was calculated. A standard curve was established using the R / G values ​​of different CHOX concentrations. Experimental results ( Figure 4 C) shows that as the CHOX concentration increases, the color of the KMnO4 solution gradually changes from purplish-red to light yellow, and the R / G value decreases. The linear regression equation is: y = -0.77x + 3.92 (where x is the logarithm of the CHOX concentration), R... 2 =0.9869, with a linear range of 0.1 to 1500 U / L. This indicates that the colorimetric detection method based on smartphones is feasible and that the sensor has good application prospects, providing a simple and rapid analytical method for portable detection of CHOX concentration.

[0050] Example 4: Analysis and Detection of CHOX Inhibitors

[0051] Based on Example 3, firstly, 2 μL of 40 U / mL CHOX and 1 μL of 100 μM Hg were added. 2+ The solution was incubated at 37°C for 5 minutes, and then the original experimental procedure was followed for detection to investigate Hg. 2+ Inhibitory effect on CHOX. To investigate the inhibitory effect of different concentrations of Hg. 2 + Effects on CHOX activity, adjusted Hg 2+ Final concentrations (0, 0.2, 0.3, 0.4, 0.5, 0.55, 0.6, 0.8, 1, 1.4, 1.6, 1.8, 2, 2.5 μM) were used, keeping other experimental procedures unchanged, and the inhibitory effect on CHOX was measured. The IC50 was also calculated. 50 Value. The ultraviolet spectrum in the range of 300–600 nm was measured using an ELISA reader, and the result was expressed as A. 526 Establish Hg 2+ Standard curves are used to measure different concentrations of Hg. 2+ Ultraviolet detection. Furthermore, Hg can be detected using colorimetric analysis. 2+ Visual detection of concentration. Experimental results ( Figure 5 A) indicates that, with Hg 2+ As the concentration increases, the solution color gradually changes from yellow to purplish-red. 526 An increase in the value indicates a decrease in H2O2 production, suggesting that CHOX activity is affected by Hg. 2+ Suppression, IC 50 The value was 0.55 μM. Furthermore, keeping other experimental conditions constant, the absorbance at 526 nm was measured over time using a microplate reader to investigate the CHOL and CHOX systems under different Hg levels. 2+ Reaction kinetics at different concentrations. Experimental results ( Figure 5 B) shows that, with Hg 2+ With increasing concentration, the reaction of CHOL under CHOX catalysis is inhibited, the amount of H2O2 generated decreases, and the reaction rate with KMnO4 slows down, further verifying that this sensor can detect Hg. 2+ Rapid and efficient detection. Furthermore, analysis of small molecule inhibitors can be performed using smartphone sensors. Figure 5 C). Experimental results show that the ultraviolet absorption intensity gradually increases with increasing inhibitor concentration. This result is basically consistent with the measurement results of traditional large-scale instruments, indicating that the data are accurate and reliable. This suggests that the smartphone-based colorimetric detection method can be used for Hg. 2+ It can be used for detection and has good application prospects.

[0052] Example 5 Specificity Detection

[0053] The results are as follows Figure 6 As shown in A, 6B, and 6C, selective tests were performed on the target molecules H2O2, CHOL, and CHOX, respectively.

[0054] H2O2 detection: Other control substances (such as glucose (GLU), sucrose (SUC), cholesterol (CHOL), tryptophan (Trp), maltose (MLT), xanthine (XA) and lactic acid (HL)) did not interfere with the detection of H2O2, and the concentration of all substances was 600 μM.

[0055] CHOL assay: Other control substances (such as reduced coenzyme I (NADH), adenosine diphosphate (ADP), xanthine (XA), glucose (GLU), lactate (HL) and uric acid (UA)) did not interfere with the system, and the concentration of all substances was 2000 μM.

[0056] CHOX assay: Other control substances (such as xanthine oxidase (XOD), terminal transferase (TdT), glucose oxidase (GOx), acetyltransferase (Tip60), papain, and alcohol dehydrogenase (ADH)) did not interfere with the system, and the concentration of all substances was 800 U / L.

[0057] To further expand the application of this sensor, smartphone analysis methods will be employed. Figure 6 Comparison of results (D, 6E, 6F) with those from large-scale instruments revealed a consistent trend. The results indicate that the prepared UV colorimetric sensor and smartphone sensor exhibit good specificity for H2O2, CHOL, and CHOX.

[0058] Example 6: Analysis and Detection of CHOX Activity in Escherichia coli Lysate

[0059] 1. Extraction and cleavage treatment of cholesterol oxidase

[0060] BL21(DE3) Escherichia coli purchased from Kezhan Biotechnology Co., Ltd. was cultured in LB medium using standard methods. Induction with 0.25 mM IPTG was performed at 15°C, and the cells were collected by centrifugation at 6000 rpm for 10 min at 4°C. The bacterial cells were resuspended in an appropriate amount of lysis buffer (such as 50 mM phosphate buffer or Tris-HCl buffer, pH 7.4, containing protease inhibitors) to adjust the concentration to approximately 5 × 10⁻⁶. 4CFU / mL. Add lysozyme (1 mg / mL), incubate on ice for 10–15 min, then add SDS (0.1–1%) to lyse the cell membrane, and continue incubation on ice for 5–10 min. Subsequently, lyse the cells using an ultrasonic homogenizer at 4°C (e.g., 300 W, 3 s on / 5 s off, for 10–15 min). Centrifuge the lysate at 14000 rpm for 20 min, and collect the supernatant for cholesterol oxidase activity assay.

[0061] 2. Analysis of changes in cholesterol oxidase activity in Escherichia coli under simulated colorectal cancer environment

[0062] To investigate the effects of the colorectal cancer microenvironment on cholesterol oxidase activity in *Escherichia coli*, an experimental group was set up as a control group (normal environment) and an experimental group (simulated colorectal cancer environment), as shown in the table below. Identical pure cultures were inoculated into two different culture media formulations, with the initial inoculum size controlled at OD0.05. 600 =0.1, with 3 replicates per group. After incubation at 37℃ and 200rpm for 12–16 h (simulating anaerobic conditions can be set as needed), the bacterial culture was collected, and the above lysis process was repeated to obtain the supernatant for cholesterol oxidase activity detection. The differences in enzyme activity between the two groups were compared and analyzed to assess the impact of environmental changes on the expression of this metabolic enzyme.

[0063] condition normal group Colorectal cancer simulation group pH 7.0 6.2 cholesterol concentration none 0.01, 0.1 mg / mL Bile acids (ox bile salts) none 0.2％ oxygen conditions Routine ventilation Sealed bottle <![CDATA[Low H2O2 concentration (simulated oxidative stress)]]> none 10, 50, 100 μM

[0064] To verify whether CHOX naturally exists in Escherichia coli and to explore its activity trends, this study first constructed simulated Escherichia coli groups with different concentration gradients, with final concentrations of 0(1) and 1×10⁻⁶. 3 (2) 1×10 4 (3) 1×10 5 (4) CFU / mL, with 800 U / L CHOX as a reference point, CHOX was then extracted from each group of bacteria and its enzyme activity was measured. Figure 7 As shown in Figure A, the absorbance of the reaction system gradually decreased with increasing E. coli concentration, indicating that the substrate was transformed more rapidly, suggesting that the activity of CHOX increases with increasing bacterial count. This result indicates that the catalytic activity of CHOX is positively correlated with the number of bacteria, and its activity depends on the bacterial concentration.

[0065] To further confirm that the aforementioned absorbance changes indeed originated from CHOX activity, rather than interference from other non-specific factors, we used simulated E. coli lysate (1×10⁻⁶) for colorectal cancer. 5 Different concentrations of the CHOX inhibitor Hg were added to the CFU / mL solution. 2 + Perform verification. For example... Figure 7 As shown in B, with Hg 2+As the concentration increased, the absorbance of the reaction system gradually increased, indicating that enzyme activity was significantly inhibited. (Hg) 2+ As a metal ion inhibitor, CHOX can bind to thiol groups or other key functional groups in enzyme molecules, thereby inhibiting their catalytic function. This result further confirms that CHOX has actual catalytic activity in the system, and that the signal detected in the experiment did indeed originate from changes in CHOX activity.

[0066] Furthermore, to investigate the effect of a simulated colorectal cancer microenvironment (i.e., moderate oxidative stress) on CHOX activity in *E. coli*, we set up three control groups: one group under normal conditions (0 μM H2O2) and two groups under simulated pathological environments with 50 μM H2O2. In the latter two groups, cholesterol (0.01 and 0.1 mg / mL) was added as a substrate, and CHOX activity was measured. The results are as follows: Figure 7 As shown in Figure C, under normal conditions, CHOX activity remained at 2–3 U / L; however, in the 50 μM H2O2 treatment group, CHOX activity was significantly enhanced, increasing to approximately 10 (under 0.01 mg / mL cholesterol conditions) and 20 U / L (under 0.1 mg / mL cholesterol conditions), respectively, with P < 0.05, indicating statistical significance. This suggests that moderate oxidative stress can stimulate CHOX expression or activate its enzymatic function in *E. coli*. H2O2, as a typical oxidative stress factor, may act as a signaling molecule to initiate the bacterial stress response system, thereby upregulating enzymes such as CHOX related to lipid metabolism and antioxidant activity to enhance adaptability to adverse environments. On the other hand, increased substrate concentration may also further enhance the catalytic efficiency of CHOX by increasing the binding probability between the enzyme and the substrate.

[0067] To further clarify the response of CHOX activity to changes in H2O2 dosage, we set up four different H2O2 concentration treatment groups (0, 10, 50, and 100 μM) with a fixed cholesterol concentration of 0.1 mg / mL to detect CHOX activity. Figure 7 As shown in Figure D, enzyme activity gradually increased with increasing H2O2 concentration: CHOX activity was lowest at 0 μM, slightly increased at 10 μM, significantly increased at 50 μM, and reached its peak at 100 μM (P < 0.05), indicating statistical significance. This trend suggests that moderate H2O2 stimulation can activate the transcription of CHOX-related genes or enhance the conformational stability of the enzyme, thereby improving its catalytic performance. This process may represent a self-protective or metabolic adaptation mechanism of *E. coli* to oxidative stress. Furthermore, since CHOX itself generates H2O2 during cholesterol degradation, the addition of exogenous H2O2 can further form a positive feedback loop, enhancing CHOX expression and activity. To further expand the application of this sensor, detection was performed using a smartphone camera combined with colorimetric analysis. Figure 7 E, 7F, 7G, 7H) are consistent with the results of large instruments, showing great potential.

[0068] In summary, the experimental results systematically validated the presence of CHOX in *E. coli* and its correlation with bacterial load, oxidative stress level, and substrate concentration. In particular, the significant increase in CHOX activity under simulated colon cancer oxidative conditions reveals its potential key role in microbial oxidative stress response and lipid metabolism regulation. This finding not only deepens our understanding of the dynamic changes in the microbial ecosystem under disease backgrounds but also provides a theoretical basis for the application research of CHOX in disease surveillance, biosensing, and microbial ecological intervention.

[0069] It should also be noted that the specific embodiments described above are not intended to limit the present invention, nor is the present invention limited to the examples given. Any changes, modifications, additions, or substitutions made by those skilled in the art within the scope of the present invention should also fall within the protection scope of the claims of this invention.

Claims

1. A cholesterol oxidase sensor based on Au-Nd nanozymes and its application in colorectal cancer, the mechanism of which is as follows: This invention constructs a cholesterol oxidase (CHOX) sensor based on Au-Nd nanozymes for the rapid detection of H2O2, CHOL, and CHOX. The Au-Nd nanozymes catalyze the reduction of KMnO4 by H2O2, causing the solution color to lighten from purplish-red and significantly reducing the 526nm absorption peak; the detection time is only about 20 seconds. CHOX can oxidize CHOL to generate H2O2, further enhancing the reduction reaction of KMnO4, exhibiting a concentration-dependent linear change. Experiments verified the presence of CHOX in *E. coli* and established a method for its activity detection, providing a new approach for studying the inflammation-cancer chain transformation mechanism in colorectal cancer. This method is simple to operate, highly sensitive, and selective, and is suitable for biosensing and disease screening.

2. The cholesterol oxidase sensor based on Au-Nd nanozyme according to claim 1 and its application in colorectal cancer, characterized in that: This method is simple, efficient, and fast. The traditional reaction between KMnO4 and H2O2 is slow (>1 min), but the introduction of Au-Nd nanozymes shortens the color development time to <20 s, significantly improving the reaction rate and signal sensitivity, enabling rapid and convenient visual detection of the target analyte. The mechanism of action of Au-Nd nanozymes may include: mimicking natural peroxidases to catalyze the decomposition of H2O2 to accelerate the reaction; providing electron transport channels to further improve reaction efficiency; increasing active surface sites to optimize the H2O2 decomposition environment; and stabilizing H2O2 to reduce non-specific decomposition, allowing it to participate more effectively in the target reaction.

3. The cholesterol oxidase sensor based on Au-Nd nanozymes according to claims 1-2 and its application in colorectal cancer, characterized in that: A UV colorimetric and smartphone-based sensing method constructed using Au-Nd nanozymes and KMnO4 can be used to analyze different concentrations of H2O2, CHOL, and CHOX. Specific detection limits are as follows: H2O2 0.041 μM, CHOL 0.035 μM, and CHOX 0.033 U / L. Furthermore, this method can also be used for Hg... 2+ Inhibitor screening, IC 50 It is 0.55 μM.

4. The cholesterol oxidase sensor based on Au-Nd nanozymes according to claims 1-3 and its application in colorectal cancer, characterized in that: This invention validated the expression of CHOX in *E. coli*. Under simulated colorectal cancer microenvironment conditions (such as decreased pH, increased cholesterol and H2O2 concentrations, and hypoxia), CHOX activity was significantly enhanced, indicating that this enzyme may play an important physiological role in bacterial response to oxidative stress and lipid metabolism regulation. This study not only deepens our understanding of the dynamic changes in gut microbiota function under disease states such as colorectal cancer, but also provides a theoretical basis and technical support for the application of CHOX as a potential biomarker in the early diagnosis, disease monitoring, and microbiota intervention of colorectal cancer, demonstrating its potential for practical application in complex biological environments.

Images