Application of whole-cell biosensor in in-situ detection of phenolic substances
By using Citrate Bacillus malonic acid PS01 transferred to the plasmid pCM-dmpR-lux as a whole-cell biosensor, rapid and non-invasive phenolic substance detection in the intestines of living mice was achieved, and the detection in the prior art was solved The problem of cumbersome and inability to meet in situ testing has achieved efficient and low-cost assessment of intestinal phenolic substances.
Patent Information
- Application Number
- CN202410317349.1
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-03-19
- Publication Date
- 2024-12-31
AI Technical Summary
The existing technology cannot achieve rapid, non-invasive, low-cost and in-situ detection of phenol and p-toluene in the intestine. The traditional method samples are cumbersome and cannot meet the in-situ detection requirements, and the detection of volatile substances is poor.
Citrate malonic acid PS01 transferred with the plasmid pCM-dmpR-lux was used as a whole-cell biosensor. After gavage, a small animal live imager was used to detect phenolic substances in the intestines to achieve rapid and non-invasive In-situ detection.
It has achieved rapid and preliminary evaluation of the concentration of phenol and p-toluen in the living intestine, which is easy to operate, reduces the detection time and cost, and can better evaluate the production of phenol substances in the living colon.
Smart Images
Figure CN119223940A_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of molecular detection, and specifically relates to an application of a whole-cell biosensor in in-situ detection of phenolic substances in living animals. The whole-cell biosensor is a malonate-free Citrobacter PS01 into which a plasmid pCM-dmpR-lux is transferred, and the phenolic substance is phenol or p-cresol. Background Art
[0002] Phenol and p-cresol are the most commonly used industrial intermediates, and they play a very important role in the production of medicine, food, and agriculture. Phenol and p-cresol have been proven to be two simple phenolic substances with genetic toxicity, which may cause serious effects on the respiratory and digestive systems after being ingested by the human body. In environmental science, phenol and p-cresol are also considered to be the primary pollutants and have received worldwide attention.
[0003] Traditionally, it is believed that the amount of phenol and p-cresol ingested by the human body is most closely related to occupational exposure and environmental exposure. However, there is evidence that tyrosine in the diet can also be converted into phenol and p-cresol under the metabolism of bacterial TyrB enzymes and TPL enzymes in the intestine. These simple phenolic substances produced directly in the intestine can be absorbed by intestinal epithelial cells and enter the blood. Long-term exposure to low concentrations of phenol and p-cresol in the human body may cause diseases including intestinal cancer and chronic nephritis, posing a hidden danger to human health. Therefore, rapid detection of phenol and p-cresol in the intestine is of great clinical significance.
[0004] Existing technologies are unable to detect small chemical molecules in situ in the intestine. Targeted metabolomics is a more feasible and commonly used method. After collecting and extracting the fecal samples, researchers use gas chromatography-mass spectrometry, liquid chromatography-mass spectrometry or nuclear magnetic resonance spectroscopy to detect the chemical substances present in them one by one. Testing colon contents is another way to test fecal samples. Except for the different locations where the samples are obtained, there is no difference in technical means.
[0005] Targeted metabolomics is a technology for detecting target metabolites in samples, but its steps are cumbersome and the sample requirements are high. A lot of sample pretreatment is required before detection. During processing, targeted metabolomics relies on expensive high-performance liquid chromatography, mass spectrometry, gas chromatography and nuclear magnetic resonance instruments, all of which require long-term training for operators and have a high time cost.
[0006] In addition, the processing time of a single sample is long, and it cannot meet the requirements of in-situ detection under any conditions. For humans, samples are generally collected by feces or anal swabs. Phenol and p-cresol are mainly produced in the colon. After being absorbed by the intestinal epithelium, it is difficult for phenolic substances to remain intact in fecal samples, resulting in distortion of test data. At the same time, fecal samples will be stored in the rectum for a certain period of time before being discharged. For the detection of volatile phenolic substances, feces is not the best detection method. Compared with feces, colon contents are better test samples, but considering that the pre-step of colonoscopy is intestinal emptying, it is not realistic to collect human colon content samples. For experimental animals, the collection of colon contents requires euthanasia and laparotomy, and it is not possible to track the changes in the concentrations of phenol and p-cresol in the individual intestine for a long time. It is still not a rapid in-situ detection method. Summary of the invention
[0007] In order to solve the above problems, the present invention provides an application of a whole-cell biosensor for in-situ detection of phenolic substances, wherein the whole-cell biosensor is a non-malonic acid Citrobacter rodentium PS01 into which the plasmid pCM-dmpR-lux is introduced, and the phenolic substance is phenol or p-cresol. The purpose of the present invention is to provide an oral whole-cell biosensor, which can quickly, efficiently, non-invasively, in-situ, and at low cost detect the production of phenol and p-cresol in the intestine, thereby enabling a rapid and preliminary evaluation of the concentration of phenolic compounds in the intestine of a living body.
[0008] The technical solution of the present invention is as follows:
[0009] The present invention discloses an application of a whole-cell biosensor for in-situ detection of phenolic substances. The whole-cell biosensor is a malonateless Citrobacter PS01 (hereinafter referred to as "PS01[dmpR]") into which a plasmid pCM-dmpR-lux is introduced. The construction method of the whole-cell biosensor is shown in application number CN202211226178.9. The invention name is a malonateless Citrobacter and its phenol whole-cell biosensor and application. The phenolic substance is phenol or p-cresol.
[0010] The present invention also discloses a method for in-situ detection of phenolic substances by the whole-cell biosensor, comprising the following steps: taking the whole-cell biosensor and gavaging it into mice, and then detecting it with a small animal living imaging device after the whole-cell biosensor enters the colon of the mice.
[0011] In one embodiment of the present invention, before the whole-cell biosensor is gavaged into mice, the time when the biosensor enters the mouse colon and the time it stays in the colon are determined, and the steps are: the biosensor is incubated with phenol in vitro, then gavaged into mice, and detected using a small animal in vivo imaging device.
[0012] In one embodiment of the present invention, 7-8 week old C57BL / 6 female mice were taken, and 400 μl of a whole-cell biosensor with an OD600 value of 0.8 was incubated with phenol in a horizontal shaker at 37°C and 180 rpm in vitro for 2 hours, and then the mice were gavaged; the mice were killed at regular intervals, and the terminal small intestine, cecum and colorectum were taken and detected using a small animal in vivo imaging device.
[0013] In one embodiment of the present invention, the time of entering the mouse colon is 2 hours after oral administration and staying for at least 2 hours.
[0014] Compared with the prior art, the present invention has the following beneficial effects:
[0015] The whole-cell biosensor of the present invention is used for in-situ detection of phenolic substances. The whole-cell biosensor PS01 can detect the levels of phenol and p-cresol in the intestinal tract of mice in situ and rapidly. Compared with the traditional detection method, it can better evaluate the production of simple phenolic substances in the colon of living bodies. The detection method is easy to operate, does not require complicated sample processing and instrument operation, and can complete qualitative detection in a short time.
[0016] Of course, any product implementing the present invention does not necessarily need to achieve all of the advantages described above at the same time. BRIEF DESCRIPTION OF THE DRAWINGS
[0017] Figure 1 This is a schematic diagram of the movement and retention of the whole-cell biosensor incubated with phenol in Example 1 of the present invention in the intestine of mice;
[0018] Figure 2 This is a schematic diagram of the response of the whole-cell biosensor of Example 2 of the present invention to different concentrations of phenol in the intestine of mice;
[0019] Figure 3 This is a schematic diagram of the response of the whole-cell biosensor of Example 2 of the present invention to different concentrations of p-cresol in the intestine of mice;
[0020] Figure 4 Schematic diagram of using whole-cell biosensor to detect mice fed high-tyrosine diet and normal diet.
[0021] Figure 5Schematic diagram of the whole-cell biosensor constructed for the chassis cells of Citrobacter amalonicic acid (PS01) and Gram-negative bacteria Escherichia coli Nissle 1917 (ECN1917) to detect phenol luminescence;
[0022] Figure 6 The survival rates of whole-cell biosensors constructed with chassis cells of non-malonic acid Citrobacter rodentium and Gram-negative bacterium Escherichia coli Nissle1917 under artificial gastric acid and artificial bile acid stress. Figure A: the survival rates of PS01[dmpR] and ECN1917[dmpR] under artificial gastric acid treatment; Figure B: the survival rates of PS01[dmpR] and ECN1917[dmpR] under 0.3%, 0.6%, and 0.9% artificial bile acid stress. DETAILED DESCRIPTION
[0023] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, the specific embodiments of the present invention are described in detail below with reference to the accompanying drawings.
[0024] Many specific details are set forth in the following description to facilitate a full understanding of the present invention. However, the present invention can be implemented in many other ways than those described herein, and those skilled in the art can make similar generalizations without violating the connotation of the present invention, so the present invention is not limited to the specific implementation disclosed below.
[0025] Example 1
[0026] 7-8 week old C57BL / 6 female mice were gavaged with 400 μl of whole cell biosensors with an OD600 value of 0.8 in vitro using phenol, incubated for 2 h at 37° C. and 180 rpm in a horizontal shaker, and then gavage was performed on the mice.
[0027] A mouse was euthanized every 30 minutes, and the terminal small intestine, cecum, and colorectum were obtained for bioluminescence signal acquisition using a small animal in vivo imager.
[0028] The residence and movement of the whole-cell biosensor in the mouse body were observed, and it was determined that the whole-cell biosensor entered the mouse colon 2 hours after oral administration and could stay there for at least 2 hours. Figure 1 shown.
[0029] Example 2
[0030] After gavage of 400 μl of whole-cell biosensor with an OD600 value of 0.8 into mice, wait for about 2 hours based on the data determined above. After the whole-cell biosensor completely enters the colon of the mice, gavage of different concentrations of phenol and p-cresol solutions is performed. After waiting for 2 hours, the mice are detected using the bioluminescence detection function of the small animal in vivo imager.
[0031] Considering that the concentrations of phenol and p-cresol in the intestine are not the same, the concentrations of phenol administered intragastrically were 100 μM, 50 μM, 25 μM, 10 μM and 0 μM, a total of five gradients, with 4 mice in each group, and the concentrations of p-cresol administered intragastrically were 500 μM, 250 μM, 100 μM, 50 μM and 0 μM, a total of five gradients, with 4 mice in each group. The configurations were all phenol and p-cresol crystals dissolved in sterile aqueous solution.
[0032] The imaging results of the mouse live imaging instrument showed that the whole-cell biosensor responded to different concentrations of phenol (such as Figure 2 as shown) and p-cresol (as Figure 3 The response of the samples was good and there were significant differences between the corresponding values at different concentrations.
[0033] Example 3
[0034] Two groups of mice, 5 in each group, with the same or similar weight and gender, were fed with a normal diet and a high-tyrosine diet, respectively, for 15 consecutive days. The normal diet group was fed with a purified breeding feed based on the AIN-93G standard, and the high-tyrosine diet group was fed with a customized purified feed with a protein content of 60% and an additional 5% tyrosine based on the AIN-93G standard. The feed consumption of the two groups of mice was recorded to confirm that the daily feed consumption was basically the same.
[0035] The whole-cell biosensor was gavaged into the two groups of mice, and after waiting for 2 hours, the two groups of mice were imaged one by one using a small animal in vivo imaging device. It was detected that the mice fed with high-tyrosine diet could clearly detect the bioluminescent signal value, while the mice in the normal diet group could not detect the signal within the measurement range. Figure 4 shown.
[0036] Example 4
[0037] The chassis cells used in the whole-cell biosensor of the present invention are Citrobacter amalonicacidophilus. Compared with the commonly used Gram-negative bacteria Escherichia coli Nissle 1917 engineered bacteria in vivo, the chassis cells have the following advantages:
[0038] In addition to the different chassis cells, the Gram-negative bacteria Escherichia coli Nissle 1917 also uses "application number CN202211226178.9, invention name is a kind of malonate-free Citrobacter and its phenol whole-cell biosensor and application" to construct a whole-cell biosensor.
[0039] Phenol solution was added to the culture medium of PS01 and Escherichia coli ECN1917, both carrying the pCM-dmpR-lux plasmid, and the final concentrations were adjusted to 0.05μM, 0.075μM, 0.1μM, 0.25μM and 0.5μM, respectively. After incubation for 2 hours at 37°C and 180rpm in a horizontal shaker, the bioluminescence values were measured and their responses to different concentrations of phenol were observed by exposing them to light in a dark field for 30s.
[0040] Based on the same plasmid pCM-dmpR-lux, the whole-cell biosensor constructed based on PS01 had a higher response value under the same concentration of phenol induction, such as Figure 5 In the case of the body, a higher value means it is easier to be detected by the instrument.
[0041] At the same time, artificial gastric acid and different concentrations of ox bile salts were used to evaluate and compare the tolerance of non-malonic acid Citrobacter rodentium PS01 and Gram-negative bacteria Escherichia coli ECN1917 in the digestive tract. 9 1mL of PS01 and ECN1917 with CFU was mixed with artificial gastric juice, 0.3% bile salt solution, 0.6% bile salt solution and 0.9% bile salt solution in a volume ratio of 1:10. After incubation in a constant temperature horizontal shaker (37°C, 180rpm) for 1h, the bacteria in the mixture were collected for gradient dilution and plated for overnight culture. The number of monoclonal clones grown on the plate was counted the next day, and the survival rate of bacteria in artificial gastric juice and bile acid solutions of different concentrations was calculated. Compared with E. coli Nissle 1917, PS01 has basically the same tolerance to gastric acid, but PS01 has a stronger ability to tolerate intestinal bile salts, such as Figure 6 As shown, after entering the intestinal environment, it can better exist in the intestine under the stress of bile salts and thus exert its function.
[0042] The preferred embodiments of the present invention disclosed above are only used to help illustrate the present invention. The preferred embodiments do not describe all the details in detail, nor do they limit the invention to the specific implementation methods described. Obviously, many modifications and changes can be made according to the content of this specification. This specification selects and specifically describes these embodiments in order to better explain the principles and practical applications of the present invention, so that those skilled in the art can understand and use the present invention well. The present invention is limited only by the claims and their full scope and equivalents.
Claims
1. Application of a whole-cell biosensor for in-situ detection of phenolic substances, characterized in that: The whole-cell biosensor is a malonate-free Citrobacter PS01 into which a plasmid pCM-dmpR-lux is introduced, and the phenolic substance is phenol or p-cresol.
2. The method for in situ detection of phenolic substances by a whole-cell biosensor according to claim 1, characterized in that: The method comprises the following steps: taking the whole-cell biosensor according to claim 1 and gavaging mice, and then detecting with a small animal living imaging device after the whole-cell biosensor enters the colon of the mice.
3. The method for in situ detection of phenolic substances by whole cell biosensor according to claim 2, characterized in that: Before the whole-cell biosensor is gavaged into mice, the time when the biosensor enters the mouse colon and the time it stays in the colon are determined. The steps are: the biosensor is incubated with phenol in vitro, then gavaged into mice, and detected using a small animal in vivo imaging device.
4. The method for in situ detection of phenolic substances by whole cell biosensor according to claim 3, characterized in that: Take 7-8 week old C57BL / 6 female mice and use 400 μl, OD 600 The whole-cell biosensor with a value of 0.8 was incubated with phenol in vitro at 37°C and 180 rpm in a horizontal shaker, and then gavage was performed on mice. The mice were killed at regular intervals, and the terminal small intestine, cecum and colorectum were removed and detected using a small animal in vivo imaging device.
5. The method for in situ detection of phenolic substances by whole cell biosensor according to claim 4, characterized in that: The time of entry into the mouse colon was 2 hours after oral administration and the cells remained there for at least 2 hours.