Preparation method and device of intelligent biosensor
By combining fluorescence colorimetry with an intelligent detection platform, LDH activity can be detected using a smartphone, which solves the problems of insufficient sensitivity and portability of existing detection methods. This achieves highly sensitive and convenient LDH activity detection, which is suitable for cancer diagnosis and chemotherapy response assessment.
Patent Information
- Application Number
- CN202411312385.5
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-09-20
- Publication Date
- 2026-03-20
AI Technical Summary
Existing methods for detecting LDH activity lack sensitivity and specificity, are complex to operate, and are not portable, making it difficult to meet the needs of point-of-care and portable detection.
A fluorescence colorimetric method combined with an intelligent detection platform was adopted. A smartphone was used as a colorimetric device, and the RGB algorithm was used to extract the chromaticity value. A linear relationship between the fluorescence emission peak and the chromaticity value corresponding to different concentrations was established to achieve the visual detection of LDH activity.
It significantly improves the sensitivity and convenience of detection, enabling rapid and portable LDH activity detection, suitable for accurate analysis of complex biological samples, and particularly suitable for cancer diagnosis and chemotherapy response assessment.
Smart Images
Figure CN121703056A_ABST
Abstract
Description
TECHNICAL FIELD
[0001] The present application relates to the field of analytical chemistry and chemical biosensing, and in particular to a method for preparing an intelligent biosensor and a device thereof. BACKGROUND
[0002] Lactate dehydrogenase (LDH) is a key metabolic enzyme widely present in tissues and cells of organisms, participating in important biological processes such as glycolysis and gluconeogenesis. LDH can catalyze the interconversion between pyruvate and lactate, and in the process, it regulates the redox balance of NAD + / NADH in the body. LDH activity is an important indicator of cell metabolic status, especially in rapidly proliferating cancer cells, and abnormal elevation of LDH activity often indicates vigorous cell metabolism. Therefore, LDH activity detection can not only be used as a biomarker for evaluating cell health and function, but also play an important role in the diagnosis, monitoring and prognosis evaluation of various diseases such as cancer. In particular, in gastric cancer, abnormal elevation of LDH activity is often closely related to tumor progression, invasion and metastasis, and becomes an important indicator for the diagnosis and treatment of gastric cancer. Therefore, the development of efficient and sensitive LDH activity detection methods is of great significance for the early diagnosis and subsequent treatment of gastric cancer.
[0003] Currently, there are various methods for detecting LDH activity, including enzyme reaction colorimetric method, fluorescence analysis method and electrochemical sensor method, etc. Although these methods have been applied in clinical practice, they still have some limitations: 1) Insufficient sensitivity and specificity: Traditional methods usually rely on in vitro kit detection, which is difficult to achieve efficient detection of complex biological samples, and the sensitivity and specificity are not ideal.
[0004] 2) Complex operation and time-consuming: Many detection methods require complex experimental steps and precise instruments, which cannot meet the needs of instant detection or on-site detection.
[0005] 3) Lack of portability: Most existing detection methods rely on laboratory environment, which is difficult to perform portable and rapid in vitro diagnosis, and cannot be applied to mobile medical or intelligent medical devices. These deficiencies prompted us to develop a lactate dehydrogenase detection method based on intelligent technology to improve detection sensitivity, simplify operation process, and achieve portable and rapid detection. By introducing fluorescence colorimetric method combined with intelligent detection platform, visual detection of LDH activity can be achieved, breaking through the bottleneck of traditional detection technology and meeting the needs of modern medical technology for rapid and intelligent detection. SUMMARY
[0006] The technical problem to be solved by the present application is to provide a smart biosensor preparation method with simple operation, high sensitivity, good specificity, low cost and visual detection.
[0007] The technical scheme adopted by the present application to solve the above technical problem is: A smart biosensor preparation method comprises the following steps: preparing a mixed solution with different concentrations, transferring the mixed solution to a cuvette of a fluorescence spectrophotometer, first establishing a linear relationship: analyzing the fluorescence intensity of the mixed solution in a wavelength range of 350 nm to 500 nm; establishing a linear relationship between different concentrations and maximum fluorescence emission peak values; and establishing a linear relationship between different concentrations and chroma values; secondly, setting a smart biosensor: taking a photo and extracting chroma values by using an RGB algorithm; and comparing the linear relationship to obtain a corresponding concentration.
[0008] The method can also be used for screening of LDH small molecule inhibitors, and the half-inhibitory concentration (IC50) is 0.41 mM.
[0009] In addition, another technical problem to be solved by the present application is to provide a smart biosensor preparation device with simple operation, high sensitivity, good specificity, low cost and visual detection.
[0010] The technical scheme adopted by the present application to solve the above technical problem is: A smart biosensor preparation device comprises a biosensor prepared according to the preparation method of the present application and a dark box, and further comprises: A light source, which is a fluorescence excitation light source, is located at the top of the dark box and is used for exciting fluorescence signals in a sample; The biosensor is located at the bottom of the dark box, so that the light source can irradiate a covered position; A camera is located directly above the biosensor and is used for capturing fluorescence signals or colorimetric changes generated after reaction of the biosensor; An RGB value calculation device obtains a photo taken by the camera and calculates a red-blue color value ratio (R / B); An RGB value comparison device obtains the red-blue color value ratio (R / B) obtained by the RGB value calculation device and compares the red-blue color value ratio (R / B) with the linear relationship between different concentrations and maximum fluorescence emission peak values to obtain NADH, LA and LDH concentrations.
[0011] Compared with the prior art, the present application has the following advantages: (1) Novel target object; The present application is the first time to prepare a smart biosensor based on LDH catalyzing oxidation of LA to NADH +The combined detection of NADH, LA and LDH is carried out, and a smartphone is introduced as a colorimetric device, which significantly improves the convenience and practicality of detection. The change of LDH activity is closely related to various diseases, such as gastric cancer, nervous system diseases and metabolic disorders. Under the pathological conditions, the activity of LDH usually changes significantly. By accurately detecting the activity of LDH, the present application can be used for early detection, diagnosis and disease monitoring of these diseases, filling the gap in the field of LDH activity detection, and providing new research opportunities and challenges for in-depth understanding of the role of LDH in physiological and pathological processes and development of related treatment strategies.
[0012] (2) The method is simple, easy to use, fast in analysis and low in cost; The present application adopts a simplified operation process, combines real-time monitoring of fluorescence signals, and realizes rapid and efficient detection of LDH activity. Compared with the traditional method, the present application avoids complex experimental steps and expensive equipment dependence, and users only need conventional fluorescence equipment and a smartphone to perform detection, which greatly reduces the cost and is particularly suitable for laboratory and on-site detection. At the same time, the method has strong applicability and is suitable for instant analysis in resource-limited environments.
[0013] (3) High sensitivity; The present application significantly improves the sensitivity of detection by optimizing the reaction conditions and using the camera of a smartphone to capture fluorescence signals. The detection limits of NADH, LA and LDH are 0.033 μM, 3.3 nM and 0.033 U / mL respectively, which shows high accuracy and detection capability for target substances. The linear correlation equations of fluorescence intensity and NADH, LA and LDH concentration are as follows: NADH concentration: y = 1276.66x + 1875.80, R² = 0.9914, detection limit is 0.033 μM.
[0014] LA concentration: y = 702.80x + 1411.75, R² = 0.9891, detection limit is 3.3 nM.
[0015] LDH concentration: y = 806.43x + 952.35, R² = 0.9960, detection limit is 0.033 U / mL.
[0016] This shows that the sensor of the present application realizes high sensitivity detection of NADH, LA and LDH, and is suitable for accurate analysis of complex biological samples.
[0017] (4) Strong specificity; The present application has carried on the extensive specific verification when detecting NADH, LA and LDH respectively. By comparing a series of possible interfering substances, including glucose (GLU), dopamine (DA), citric acid (CA), ascorbic acid (AA) and the like, the results show that these substances have no interference to the system. Similarly, in the detection of LA and LDH, other biological molecules such as tyrosine (Tyr), adenosine triphosphate (ATP), acetylcholine esterase (AChE), papain (Papain) and the like are replaced, and no significant interference is found. This verifies that the present application has high specificity and selectivity for the target in various complex samples, ensuring the accuracy of the detection.
[0018] (5) Strong application; The sensor of the present application is suitable for various experimental environments, and especially shows great application potential in the detection of LDH in cancer cells and circulating tumor cells (CTCs). By comparing the LDH release amount of normal cells and gastric cancer cells, gastric cancer CTCs under different treatment conditions, the metabolic reaction differences of different cells under chemotherapy drugs and hypoxic environment are revealed, specifically as follows: 1) normal cells and cancer cells - the LDH activity of gastric cancer cells is significantly higher than that of normal cells and CTCs, and after treatment with cisplatin and paclitaxel, the LDH release amount of gastric cancer cells significantly increases, especially under cisplatin conditions, showing a strong cell damage response; 2) circulating tumor cells (CTCs) - the LDH release amount of CTCs is lower than that of gastric cancer cells, but under cisplatin, paclitaxel or hypoxic conditions, the LDH activity increases, reflecting its adaptability and tolerance to drug and environmental changes; 3) hypoxic conditions - the hypoxic environment significantly enhances the LDH activity of gastric cancer cells and CTCs, indicating the strong influence of hypoxic environment on tumor cell metabolism, and the LDH activity change of normal cells is relatively small. The present application provides a new sensing platform for the detection of LDH activity and related biomarkers, especially suitable for cancer diagnosis, chemotherapy response evaluation, drug screening and the like, with wide application prospect and great scientific and clinical significance, due to its high sensitivity, strong specificity, simple use and low cost. BRIEF DESCRIPTION OF DRAWINGS
[0019] Figure 1 A feasibility analysis diagram of NADH and LDH of the present application; Figure 2 A fluorescence absorption intensity of different concentrations of NADH of the present application; Figure 3 A linear relationship diagram of different concentrations of NADH and RGB value of the present application; Figure 4 A fluorescence absorption intensity of different concentrations of LA of the present application; Figure 5 A fluorescence absorption intensity of different concentrations of LDH of the present application; Figure 6 Linear relationship diagram of different concentrations of LA and LDH with RGB value for the present application; Figure 7 Linear relationship diagram of different concentrations of GF with fluorescence absorption intensity for the present application; Figure 8 Linear relationship diagram of different concentrations of GF with RGB value for the present application; Figure 9 Selective experiment diagram of NADH, LA and CHDH for the present application; Figure 10 Analysis and detection experiment diagram in gastric cancer cells for the present application; Figure 11 Analysis and detection experiment diagram in gastric cancer cells and gastric cancer CTCs for the present application. DETAILED DESCRIPTION
[0020] The present application will be further described in detail below with reference to the accompanying drawings. It should be noted that the specific embodiments are not a limitation on the present application, and the present application is not limited to the examples. Those skilled in the art can make changes, modifications, additions or substitutions within the spirit and scope of the present application, which shall also belong to the protection scope of the present application.
[0021] Example 1:
[0022] Construction of fluorescence colorimetric sensing method for detecting NADH, LA and LDH.
[0023] A. NADH-based LDH analysis and detection feasibility exploration: a. NADH analysis and detection: 14 μL of different concentrations of NADH solution and 86 μL of 0.1 M, pH=9 phosphate buffer solution prepared by NaH2PO4 and Na2HPO4 were added into 100 μL centrifuge tube and mixed thoroughly. Then, the mixture was transferred to the colorimetric cell of the fluorescence spectrophotometer for analysis and testing, and the high-definition camera was used to take pictures to record the experimental results.
[0024] b. LA analysis and detection: 5 μL of 10 μM LA solution (final concentration 80 μM) and 4 μL of 10 mM LDH solution (final concentration 50 U / mL) were pre-mixed at room temperature for 10 minutes. Subsequently, the mixture was quickly added into 100 μL centrifuge tube with 5 μL of NADH solution (final concentration 50 μM) and 86 μL of 0.1 M, pH=9 phosphate buffer solution, mixed thoroughly and continued to react at room temperature for 30 minutes. Then, the above mixture was transferred to the colorimetric cell of the fluorescence spectrophotometer for detection, and the high-definition camera was used to take pictures of the sample. + and 86 μL of 0.1 M, pH=9 phosphate buffer solution, mixed thoroughly and continued to react at room temperature for 30 minutes. Then, the above mixture was transferred to the colorimetric cell of the fluorescence spectrophotometer for detection, and the high-definition camera was used to take pictures of the sample.
[0025] c. Analysis of LDH: 5 μL of 10 μM LA solution (final concentration 80 μM) was pre-mixed with 4 μL of 10 mM LDH solution (final concentration 50 U / mL) for 10 minutes, then the mixture was quickly added to 5 μL of NAD+ and 86 μL of 0.1 M, pH 9 phosphate buffer solution in a 100 μL centrifuge tube, mixed well, and reacted at room temperature for 30 minutes. After the reaction, the mixture was transferred to the cuvette of the fluorescence spectrophotometer for analysis, and a high-definition camera was used to take pictures to record the experimental process.
[0026] Figure 1 The fluorescence intensity curve of the sensor in the wavelength range of 350-500 nm is shown. It can be seen that the fluorescence response under different processing conditions.
[0027] NAD + Alone (Curve 1): In the absence of lactate (LA) and lactate dehydrogenase (LDH), the sensor has almost no significant fluorescence emission peak, especially no obvious fluorescence signal is observed at 390 nm. This indicates that NAD + itself does not excite the sensor to produce fluorescence. At this time, NAD + is not reduced to NADH.
[0028] Only LDH is introduced (Curve 2): In the presence of only LDH, there is also no significant fluorescence emission peak, indicating that LDH alone cannot reduce NAD + , NADH is not generated, and thus no fluorescence signal is generated at 390 nm.
[0029] Only LA is introduced (Curve 3): In the presence of only LA, the fluorescence emission peak is still not obvious, indicating that LA alone cannot reduce NAD + , NADH is not generated, and thus there is no obvious fluorescence emission peak at 390 nm.
[0030] NAD + is introduced in the presence of LA and LDH (Curve 4): When LA and LDH are present at the same time, an obvious fluorescence emission peak appears at 390 nm. This phenomenon indicates that, under the joint action of LA and LDH, NAD + is reduced to NADH, and NADH produces a strong fluorescence signal at 390 nm. This further confirms that LA and LDH react to generate NADH, and the fluorescence signal intensity is closely related to the amount of NADH generated.
[0031] Figure 1 The results in Table 1 show that when LA and LDH are present at the same time, the sensor has a strong fluorescence response to NAD +The reduction reaction of NAD+ is very sensitive, and generates detectable NADH, which produces a clear fluorescence emission peak at 390 nm. In comparison with other treatment conditions (NAD + The comparison with other treatment conditions (NAD
[0032] B. Analysis and detection of different concentrations of NADH: In this experiment, first, according to the steps, different concentrations of NADH solution (final concentration range: 0, 0.1, 0.2, 0.4, 0.8, 1, 2, 4, 8, 10, 15, 20, 25, 30, 40, 50 μM) were added to the reaction system. The fluorescence intensity in the range of 350 nm to 500 nm was measured using a fluorescence spectrophotometer, and the fluorescence emission peak at 390 nm wavelength was monitored.
[0033] Figure 2 The fluorescence intensity at 390 nm wavelength showed a trend of increasing with the increase of NADH concentration. By constructing a linear fitting curve of fluorescence intensity and the logarithmic value of NADH concentration, it was found that there was a good linear relationship between them, and the linear equation was: y = 1276.66x + 1875.80, the correlation coefficient R² = 0.9914, which proved that the detection method had good accuracy and sensitivity. Through calculation, the detection limit was 0.033 μM, which indicated that this method could be used for the detection of low concentration of NADH.
[0034] In addition, by taking pictures of the samples with a smart phone and combining with RGB chroma value analysis technology, the average value of three RGB values was extracted from different positions of the sample picture, and the relationship between NADH concentration and chroma value was further established. As shown in Figure 3 With the increase of NADH concentration, the chroma value of the sample changed significantly, especially the red (R) component in RGB changed most significantly. This indicated that by taking pictures and analyzing the RGB values of the sample color with a smart phone, quantitative detection of NADH could be realized quickly.
[0035] This detection method based on fluorescence measurement combined with smart phone RGB analysis not only has high sensitivity and accuracy, but also can realize convenient and rapid on-site detection, providing a novel and efficient means for the analysis of NADH.
[0036] C. Analysis and detection of different concentrations of LA and LDH: Fluorescence detection of LA: Based on the aforementioned experimental procedures, different concentrations of LA solutions were set by changing the LA concentration (final concentration: 0, 0.01, 0.02, 0.04, 0.08, 0.2, 0.4, 0.8, 1, 2, 4, 8, 10, 20, 40, 80 μM), while keeping other experimental procedures unchanged. The fluorescence intensity was measured using a fluorescence spectrophotometer in the wavelength range of 355 nm to 500 nm, focusing on the maximum fluorescence emission peak at 390 nm. The experimental results showed that the maximum fluorescence intensity at 390 nm and the logarithmic value of LA concentration showed a good linear relationship. With the increase of LA concentration, the fluorescence intensity gradually increased. Based on the maximum fluorescence emission peak at 390 nm, the linear relationship curve of LA was established, and the equation was: y = 702.80x + 1411.75, the correlation coefficient R² = 0.9891, and the detection limit was 3.3 nM. This indicates that this method has high sensitivity and good linear response to different concentrations of LA, and is suitable for fluorescence detection of LA. 2 =0.9891, the detection limit was 3.3 nM. This indicates that this method has high sensitivity and good linear response to different concentrations of LA, and is suitable for fluorescence detection of LA.
[0037] Fluorescence detection of LDH: By a similar experimental method, the concentration of LDH was detected by fluorescence. By changing the concentration of LDH, different final concentration gradients were set (0, 0.1, 0.2, 0.4, 0.8, 1, 2, 4, 8, 10, 15, 20, 25, 30, 40, 50 U / mL). The fluorescence emission intensity was measured in the wavelength range of 355 nm to 500 nm, and the fluorescence emission peak at 390 nm was monitored. The experimental results showed that the maximum fluorescence emission intensity at 390 nm and the logarithmic value of LDH concentration showed a good linear relationship. With the increase of LDH concentration, the fluorescence intensity significantly increased. Based on this data, the linear relationship curve of LDH was established, and the equation was: y = 806.43x + 952.35, the correlation coefficient R² = 0.9960, and the detection limit was 0.033 U / mL. The experimental results proved that this detection method has very high sensitivity and reliability for the change of LDH concentration, and is suitable for accurate fluorescence detection of LDH.
[0038] Intelligent detection: In each experiment, the photos of the samples were taken using a camera, and the R, G, B values of each sample were analyzed by the RGB value calculation device. Three samples were taken from different positions for each sample, and the average values of R, G, B were recorded to ensure the accuracy and consistency of the data. By analyzing the RGB color values of different concentrations of LA samples, it was found that the RGB color values changed significantly with the increase of LA concentration. Especially the change of R / B ratio was most obvious, as shown in Figure 6(A and C) show. Based on the relationship between R / B ratio and LA concentration, a good linear curve was constructed. This indicates that the RGB colorimetric analysis of RGB value analysis device can be effectively used to construct the LA sensor to realize the rapid on-site detection of LA. RGB colorimetric value analysis was performed on samples of different concentrations of LDH. The change of LDH concentration also caused significant changes in RGB values. Figure 6 (B and D) show the linear relationship curve of R / B ratio and LDH concentration. The results show that the RGB colorimetric analysis technology can be used to construct the LDH sensor, which can realize the rapid on-site analysis and detection of LDH.
[0039] Example 2:
[0040] Analysis and detection of LDH inhibitor GF. In this experiment, the concentrations of LA and LDH were kept at 80 μM and 50 U / mL, respectively. By adding different concentrations of LDH inhibitor GF, the inhibitory effect of GF on the catalytic reaction of LDH was analyzed. The concentration range of GF was set as: 0, 0.01, 0.02, 0.04, 0.08, 0.1, 0.2, 0.4, 0.8, 1, 2, 4, 8, 10, 15, 25 μM. The effect of the inhibitor on the fluorescence emission peak intensity in the system was determined, and all other steps remained unchanged. To further verify the experimental results, the experiment took pictures of the fluorescence detection samples by camera and analyzed them in combination with the changes in RGB colorimetric values. Figure 8 The effects of different concentrations of GF on RGB colorimetric values are shown. With the increase of GF concentration, the color of the sample gradually darkened, especially the red (R) component decreased significantly. The RGB value calculation device was used to take three samples of RGB values and calculate their average values. The results showed that there was a good linear relationship between R / B ratio and GF concentration. This result indicates that the RGB colorimetric analysis of the smartphone can effectively reflect the change of GF concentration, proving that it can be used as a feasible means for GF inhibitor detection. Through the dual detection method of fluorescence emission intensity and RGB colorimetric value, the experiment successfully verified the effect of GF as an LDH inhibitor. The fluorescence method showed high sensitivity, while the RGB calculation and comparison device embedded in the smartphone provided a new idea for rapid on-site detection of inhibitors, which is suitable for detection applications under limited resources.
[0041] Example 3:
[0042] Specificity detection. In order to verify the selectivity of the sensor, the sensor preparation steps of Example 1 were referred to, and different control substances were used to replace the target substances in the detection of NADH, LA and LDH, respectively, to observe the influence of the interferents on the system. In the NADH detection system, various common biomolecules were used as interferents to replace NADH, including glucose (GLU), dopamine (DA), citric acid (CA), ascorbic acid (AA), and uric acid (UA). As shown in Figure 9 A, the influence of various interferents on the fluorescence intensity was not significant, indicating that the sensor had high selectivity for NADH, and other similar molecules would not cause false detection or interference. In the LA detection system, lactic acid was replaced by other biomolecules, including tyrosine (Tyr), glucose (GLU), xanthine (XA), adenosine diphosphate (ADP), and adenosine triphosphate (ATP) for detection. Figure 9 B shows the fluorescence intensity results, and it is found that these control substances do not significantly affect the fluorescence signal of the system, indicating that the sensor has very high selectivity for the detection of lactic acid. In the LDH detection, various common enzyme substances were used as interferents, including acetylcholinesterase (AChE), papain (Papain), terminal deoxynucleotidyl transferase (TdT), histone acetyltransferase (HAT), and alkaline phosphatase (ALP). As shown in Figure 9 C, all these interferents have no significant influence on the fluorescence signal of the system, proving that the sensor has high selectivity for LDH. By comparing the detection results of various interferents, it can be clearly concluded that the sensor shows good selectivity in the detection of NADH, LA and LDH, and is not affected by common biomolecules or enzymes. Therefore, the application of the sensor in complex biological samples has good specificity, and is suitable for precise biomolecule detection.
[0043] Example 4:
[0044] Analysis and detection of LDH in CTCs. This experiment used the gastric cancer cell line MGC-803 as a model. Cancer cells were obtained through passage culture and added to whole blood samples to form blood samples containing CTCs. CTCs were enriched from the blood using magnetic bead sorting, and the enriched CTCs were subjected to LDH fluorescence detection and RGB colorimetric analysis using a smartphone to evaluate the expression of LDH activity in CTCs. MGC-803 cell line was cultured in L-15 medium containing 10% fetal bovine serum and antibiotics, maintained at 37°C and 5% CO2. When the cell confluence reached 90%, the cells were digested and passaged using a digestion solution containing 0.25% trypsin and 0.02% EDTA, passaged every 2-3 days. MGC-803 cells were digested with Trypsin-EDTA solution, suspended in PBS solution, and whole blood samples were added to adjust the concentration of CTCs in whole blood to 1-5 cells per milliliter. Then, CTCs were enriched using magnetic bead sorting technology, re-inoculated into the culture medium, counted, and the concentration was adjusted to 500 × 10⁻⁶. 4 Cells / mL were used as experimental samples for subsequent LDH detection.
[0045] Figure 10 The results of LDH detection in CTCs are presented, including fluorescence analysis and RGB colorimetric analysis. Figure 10 A indicates that LDH activity in CTCs samples at different concentrations was detected by fluorescence spectrophotometry. With increasing CTC concentration, the fluorescence emission peak intensity significantly increased, indicating a positive correlation between LDH activity and CTC quantity. The gradual increase in fluorescence intensity reflects the high expression of LDH in CTCs. To further confirm successful extraction, the LDH inhibitor GF was also added for fluorescence detection. Figure 10 (B) The results showed that the fluorescence gradually weakened with increasing inhibitor concentration, indicating that the extract in CTCs was LDH.
[0046] Simultaneously, samples were photographed using a smartphone, and RGB colorimetric analysis software was used to detect the RGB values in the images. Figure 10 C). The results showed that the chromaticity of the samples changed significantly with increasing CTC concentration, especially the R / B ratio, which changed dramatically with increasing LDH activity. This result demonstrates that RGB chromaticity analysis can effectively reflect the expression level of LDH in CTCs. Figure 10 As shown in D, the RGB analysis of the control group indicates that the inhibitor GF has an inhibitory effect on LDH activity, further verifying the successful extraction of LDH.
[0047] This experiment shows that both the sensor and the smartphone can sensitively and specifically detect LDH activity in CTCs, demonstrating the potential application value of this method in cancer cell detection.
[0048] Example 5:
[0049] Analysis and detection of LDH released by cells in the culture medium. This experiment aims to evaluate the release of LDH in gastric cancer cells (MGC-803 cells) and normal cells under different treatment conditions by detecting the level of lactate dehydrogenase (LDH) released in the cell culture medium. As a marker of cell damage and apoptosis, the activity level of LDH can reflect the health status of cells. Multiple control and treatment groups were set up, and different types of chemotherapy drugs were added to observe their effect on the release of cell LDH. A total of 5 groups were set up, labeled ABCDE: Group A (normal control group): BJ cells (untreated); Group B (cancer cell untreated group): MGC-803 cells / CTCs (untreated); Group C (cancer cell treatment group 1): MGC-803 cells / CTCs + chemotherapy drug 1 (5 µM cisplatin); Group D (cancer cell treatment group 2): MGC-803 cells / CTCs + chemotherapy drug 2 (50 nM paclitaxel); Group E (other stimulation group): MGC-803 cells / CTCs + other stimulation (hypoxic conditions).
[0050] ABCD group experimental steps: trypsinize the cells, count, and then inoculate an appropriate amount of cells into a 96-well plate (about 20,000 cells per well), and let the cells adhere overnight. The next day, remove the supernatant and add RPMI-1640 medium containing different concentrations of drugs. Control group AB: add the same volume of drug-free medium. Treatment group C: add 5 µM cisplatin to the culture medium. Treatment group D: add 50 nM paclitaxel to the culture medium. After treatment, the cells were cultured in a 37°C, 5% CO2 incubator for 24 hours. After drug treatment, the supernatant of each well was collected for LDH activity detection. According to the instructions of the LDH detection kit, mix the collected supernatant with the reagent, incubate at the appropriate temperature, and then test the LDH release amount by the method established in this patent to reflect the cell damage or death. Group E (other stimulation group): MGC-803 cells / CTCs + other stimulation (hypoxic conditions), add the pre-configured oxygen consumption agent 1 mM sodium sulfite solution to the cell culture medium, control the volume ratio at 0.1% (V / V), and mix quickly by stirring or shaking to ensure uniform distribution of the oxygen consumption agent. The treated culture medium is added to the cell culture dish and the container is quickly sealed to prevent oxygen from re-dissolving. The cells are placed in a standard incubator for culture, and the cell response under hypoxic conditions is observed and recorded. The results are shown in Figure 11 1. Untreated cell group: In the untreated condition, the amount of LDH released from the culture medium of cancer cells (MGC-803 cells) is the highest, followed by the circulating tumor cells (CTCs), and the normal cells have the lowest LDH release. This is probably because normal cells rarely undergo cell membrane rupture or apoptosis in a healthy state, resulting in lower LDH release. In contrast, gastric cancer cells are more prone to spontaneous damage or apoptosis due to their unstable metabolic state, leading to higher LDH release. CTCs exhibit strong survival ability, and although their LDH release is lower than that of gastric cancer cells, it is still higher than that of normal cells, indicating that partial metabolic stress or microenvironmental stress of CTCs can cause slight LDH release.
[0051] 2. Cisplatin treatment group: Cisplatin is a DNA-damaging agent that forms crosslinks with DNA, preventing DNA replication and repair, ultimately leading to apoptosis or necrosis. Gastric cancer cells proliferate rapidly and are highly sensitive to cisplatin, resulting in a significant increase in LDH release. CTCs are also affected by cisplatin, but due to their strong environmental adaptability, they are slightly less sensitive to cisplatin, resulting in a relatively lower LDH release compared to gastric cancer cells. However, cisplatin has less toxicity to normal cells, although it can cause DNA damage, normal cells have stronger tolerance, so the LDH release is lower than that of cancer cells.
[0052] 3. Paclitaxel treatment group: Paclitaxel, as a microtubule stabilizer, can prevent cell division by inhibiting microtubule depolymerization, especially effective for rapidly proliferating cancer cells. Paclitaxel causes cell cycle arrest and induces apoptosis, so the LDH release of gastric cancer cells after treatment is significantly increased. Compared with gastric cancer cells, CTCs have lower sensitivity to paclitaxel, because CTCs have slower proliferation and stronger drug resistance, resulting in relatively less LDH release. For normal cells, the effect of paclitaxel is lighter, and the LDH release is significantly lower than that of cancer cells, indicating that paclitaxel has milder cytotoxicity to normal cells than cisplatin.
[0053] 4. Hypoxic environment treatment group: Under hypoxic conditions, normal cells are under metabolic stress due to insufficient oxygen supply, leading to some degree of damage and release of LDH. Gastric cancer cells have significantly increased LDH release under hypoxic conditions, because cancer cells experience stronger stress in a hypoxic environment. In contrast, CTCs have higher tolerance to hypoxia due to their frequent exposure to hypoxic environments in the circulatory system, resulting in relatively less LDH release. This indicates that CTCs can maintain better cell integrity and survival status when faced with hypoxic environments.
[0054] In summary, the experimental results of each treatment group showed that LDH release can effectively reflect the metabolic and damage status of different cell types under various stress conditions, especially in the application of cancer treatment has potential clinical significance.
Claims
1. A method for preparing an intelligent biosensor, comprising preparing a mixed solution of different concentrations, and transferring the mixed solution to a cuvette of a fluorescence spectrophotometer, characterized in that: Fluorescence intensity analysis was performed on the mixed solution in the wavelength range of 350 nm to 500 nm; a linear relationship was established between the maximum fluorescence emission peak corresponding to different concentrations; a linear relationship was established between the chromaticity values corresponding to different concentrations. Take a picture and extract the chromaticity value using the RGB algorithm; compare it with the linear relationship to obtain the corresponding concentration.
2. The method for fabricating an intelligent biosensor according to claim 1, characterized in that: The preparation of mixed solutions of different concentrations specifically involves: Preparation of NADH mixed solutions: 14 μL of NADH solutions of different concentrations and 86 μL of 0.1 M phosphate buffer solution (pH=9) prepared from NaH2PO4 and Na2HPO4 were added to a 100 μL centrifuge tube and mixed thoroughly. Preparation of LA mixed solution: 5 μL of LA at different concentrations was premixed with 4 μL of 10 mM LDH and reacted at room temperature for 10 minutes. The mixture was then rapidly mixed with 5 μL of NAD. + Mix with 86 μL of 0.1 M phosphate buffer solution (pH=9), stir thoroughly, and react at room temperature for 30 minutes; LDH mixed solution preparation: 5 μL of 10 μM LA was mixed with 4 μL of LDH of different concentrations, and the mixture was pre-reacted at room temperature for 10 minutes, followed by 5 μL of NAD. + Mix with 86 μL of 0.1 M phosphate buffer solution (pH=9), stir thoroughly, and react at room temperature for 30 minutes.
3. The method for fabricating an intelligent biosensor according to claim 2, characterized in that: The linear relationship between the maximum fluorescence emission peak corresponding to different concentrations of the NADH mixed solution is: y = 1276.66x +1875.80; The linear relationship between the maximum fluorescence emission peak corresponding to different concentrations of the LA mixed solution is: y = 702.80x + 1411.75; The linear relationship between the maximum fluorescence emission peak corresponding to different concentrations of the LDH mixed solution is: y = 806.43x + 952.
35.
4. The method for fabricating an intelligent biosensor according to claim 3, characterized in that: The different concentrations of NADH solutions are: 0, 0.1, 0.2, 0.4, 0.8, 1, 2, 4, 8, 10, 15, 20, 25, 30, 40, and 50 μM; The different concentrations of LA solutions are: 0, 0.01, 0.02, 0.04, 0.08, 0.2, 0.4, 0.8, 1, 2, 4, 8, 10, 20, 40, and 80 μM; The different concentrations of LDH solutions are: 0, 0.1, 0.2, 0.4, 0.8, 1, 2, 4, 8, 10, 15, 20, 25, 30, 40, and 50 U / mL.
5. The method for fabricating an intelligent biosensor according to claim 1, characterized in that: The wavelength corresponding to the maximum fluorescence emission peak is 390 nm.
6. The method for fabricating an intelligent biosensor according to claim 1, characterized in that: This method can also be used for screening small molecule inhibitors of LDH, and for determining the half-maximal inhibitory concentration (IC50). 50 The value is 0.41 mM.
7. The apparatus for fabricating a smart biosensor according to claim 1, comprising fabricating a biosensor according to claim 1 and a dark chamber, characterized in that: Also includes: The light source, which is a fluorescence excitation light source, is located at the top of the dark chamber and is used to excite the fluorescence signal in the sample; The biosensor is located at the bottom of the dark box, allowing the light source to illuminate and cover the area. A camera, located directly above the biosensor, is used to capture fluorescence signals or colorimetric changes generated after the sensor reacts. An RGB value calculation device acquires photos taken by the camera and calculates the red / blue value ratio (R / B). An RGB value comparison device is used to obtain the red-blue value ratio (R / B) obtained by the RGB value calculation device and compare it with the linear relationship of the maximum fluorescence emission peak corresponding to different concentrations to obtain the concentrations of NADH, LA and LDH.
8. A method for fabricating an intelligent biosensor according to claim 1 or 7, characterized in that: It can be applied to the analysis and detection of cancer cells, circulating tumor cells and their biomarkers.
9. The apparatus for fabricating an intelligent biosensor according to claim 7, characterized in that: The camera can be a mobile phone camera.
10. The apparatus for fabricating an intelligent biosensor according to claim 7, characterized in that: The RGB value calculation device and RGB value comparison device can be embedded in the mobile phone system.