A metal-organic coordination gel-protected whole-cell biosensor for monitoring nitrosamine compounds in the digestive tract

Abstract

This invention discloses a metal-organic coordination gel-protected whole-cell biosensor for monitoring nitrosamine compounds in the digestive tract. It utilizes Fe... 3+ Using a metal-organic coordination gel (MOC) formed through coordination with 2,2'-thiodiacetic acid as a carrier, a coordination gel biosensor formed from *Saccharomyces cerevisiae* bacteria expressing GFP in response to nitrosamines is encapsulated within the MOC. When nitrosamines cause DNA damage, the DNA damage repair pathway in the yeast is activated, leading to enhanced expression of the GFP reporter gene. The intensity of the fluorescence signal reflects the genotoxicity of nitrosamines. This invention also discloses the application of a whole-cell biosensor protected by an MOC for monitoring nitrosamines in the digestive tract. The coordination gel biosensor of this invention can be used for the detection of nitrosamines, exhibiting high specificity and ease of operation.

Description

Technical Field

[0001] This invention relates to coordination gel-protected biofluorescent sensors, their preparation methods, and applications, specifically to a metal-organic coordination gel-protected whole-cell biosensor for monitoring nitrosamine compounds in the digestive tract, its preparation method, and applications. Background Technology

[0002] The widespread use of food additives in our daily diet can easily lead to the intake of genotoxic compounds, causing DNA damage and potentially inducing cancer. Nitrosamines, a common genotoxic impurity, are recognized by the International Agency for Research on Cancer as carcinogenic and teratogenic, posing a significant threat to human health and thus receiving close attention from regulatory agencies. The precursor to nitrosamines, nitrite, is widely found in common foods such as meat, pickles, dairy products, and vegetables. When the human body ingests excessive amounts of nitrite, it is metabolized and activated by cytochrome P450 enzymes, producing α-hydroxynitrosamines. These α-hydroxynitrosamines are further decomposed to form highly reactive positive ions or diazoium, leading to alkylation of DNA bases and causing gene mutations, posing a potential hazard. Therefore, accurate detection of nitrosamine compounds is extremely important.

[0003] Conventional methods for detecting nitrosamines mainly include gas chromatography-mass spectrometry (GC-MS), liquid chromatography-mass spectrometry (LC-MS), and liquid-liquid microextraction. However, these methods still have some limitations. For example, while GC-MS provides good qualitative results and is less affected by the matrix, it is difficult to detect N-nitrosamines, which have poor thermal stability. Derivatization methods may lead to incomplete reactions and inaccurate quantification. Although HPLC-MS is simple to operate, has high recovery rates, and good reproducibility, it is expensive and difficult to popularize. Therefore, it is essential to explore a novel, low-cost, and universally applicable method that does not rely on traditional instruments.

[0004] Microbial whole-cell biosensors (MWCBs) utilize the viability, metabolic activity, and cell viability of microorganisms to qualitatively and quantitatively identify target analytes. Typically, in microbial gene regulation, DNA sequence elements and regulatory protein elements work together to regulate gene expression in the microbial genome. The binding of RNA polymerase to the promoter region is crucial for initiating gene expression; the promoter is the starting point of gene expression. Some regulatory proteins promote / inhibit the binding of RNA polymerase to the promoter, thereby regulating gene expression. Therefore, genetically engineered genes or plasmids can be introduced into microorganisms or encoded into their genomes, and the presence of the target analyte can be reflected by the expression status and signal intensity of the reporter gene. Compared to traditional detection methods, MWCBs offer advantages such as simplicity, low cost, and the ability to adjust the composition of the introduced gene according to the target analyte to respond to different / multiple target analytes. However, the detection performance of microbial-based biosensors is based on the survival of the microorganism. The chemical environment in the human digestive tract is complex, and MWCBs cannot maintain detection capability while exposed to gastric acid, which limits their application in in situ detection in vivo. Summary of the Invention

[0005] The purpose of this invention is to overcome the shortcomings of existing nitrosamine compound detection technologies, such as cumbersome operation, complexity, and time consumption. This invention applies a detection platform using metal-organic gel (MOG) encapsulated with metal-organic ligands (MWCBs) to the field of nitrosamine compound analysis. By utilizing the gastric acid protection capability of MOG and the flexibility and in-situ analysis capabilities of MWCBs, this invention provides a rapid, simple, low-cost, and interference-resistant method for detecting nitrosamine compounds.

[0006] The objective of this invention is achieved through the following technical solution:

[0007] A metal-organic coordination gel-protected whole-cell biosensor for monitoring nitrosamine compounds in the digestive tract, it is based on Fe 3+ Using a metal-organic coordination gel formed by coordination with 2,2'-thiodiacetic acid (TDA) as a carrier, a coordination gel biosensor formed by Saccharomyces cerevisiae expressing GFP (Green Fluorescent Protein) in response to nitrosamines was encapsulated in the metal-organic coordination gel.

[0008] Another object of the present invention is to provide a method for preparing the whole-cell biosensor protected by the metal-organic coordination gel described above, comprising the following steps:

[0009] Step (1): Mix ferric chloride hexahydrate (FeCl3·6H2O) and 2,2'-thiodiacetic acid with a polar solvent, and sonicate to completely dissolve ferric chloride hexahydrate and 2,2'-thiodiacetic acid in the polar solvent to obtain a metal-organic coordination gel solution.

[0010] Step (2), OD 600 Yeast culture of 0.2-0.4 g was added to the organometallic coordination gel solution obtained in step (1), and allowed to stand at room temperature until gel formation was achieved, thus obtaining the coordination gel biosensor.

[0011] In step (1), the molar ratio of ferric chloride hexahydrate and 2,2'-thiodiacetic acid is (0.01-0.10):(0.01-0.10), preferably 1:1.

[0012] In the aforementioned organometallic coordination gel solution, the molar concentration of ferric chloride hexahydrate is 0.006–0.08 mmol / mL, preferably 0.05 mmol / mL.

[0013] The polar solvent is selected from water, ethanol, methanol, or aqueous solutions of methanol or ethanol.

[0014] The duration of the ultrasound is 1 to 10 minutes, preferably 5 minutes.

[0015] In step (2), the volume ratio of the organometallic coordination gel solution (based on the volume of polar solvent) to the yeast culture is 20:1 to 1500:1, preferably 20:1 to 100:1.

[0016] The GFP-expressing Saccharomyces cerevisiae strain is either a genome-fusion type DNA damage-responsive fluorescent Saccharomyces cerevisiae strain with an introduced promoter or a plasmid-responsive Saccharomyces cerevisiae strain with an introduced enhanced green fluorescent protein reporter gene plasmid.

[0017] The aforementioned genome-fusion DNA damage-responsive fluorescent yeast strain is a Saccharomyces cerevisiae strain capable of expressing GFP fusion protein. It is constructed by inserting a gene for green fluorescent protein (GFP) after the promoter of a protein gene corresponding to the DNA damage repair pathway in the genome.

[0018] Specifically, the promoter and corresponding DNA damage repair pathway of the genome fusion-type DNA damage-responsive fluorescent yeast strain are as follows:

[0019]

[0020] The culture of the genome-fusion DNA damage-responsive fluorescent yeast strain was prepared by the following method: the genome-fusion DNA damage-responsive fluorescent yeast strain was inoculated into SD-HIS liquid medium and cultured at 30°C with shaking at 220 rpm until the OD of the culture was reached. 600 The value is 0.2–0.4, preferably the OD value of the culture solution. 600 The value is 0.4.

[0021] The plasmid-responsive yeast strains described above were constructed by transfecting plasmids pMAG1-yEGFP and pPHR1-yEGFP into GFP-expressing Saccharomyces cerevisiae strains using the LiAc / PEG / ssDNA method.

[0022] The culture of the genome fusion-type DNA damage-responsive fluorescent yeast strain was prepared by the following method: A plasmid-responsive Saccharomyces cerevisiae strain was inoculated into SD-URA-HIS-LEU liquid medium and cultured at 30°C with shaking at 220 rpm until the OD of the culture reached [missing value]. 600 When the value is 0.2 to 0.4, it is preferable to culture the bacterial culture to the point where the OD value is 0.2 to 0.4. 600 The value is 0.4.

[0023] like Figure 1 The present invention relates to a metal-organic coordination gel-protected whole-cell biosensor for monitoring nitrosamine compounds in the digestive tract, which is based on Fe... 3+ A coordination gel biosensor (MWCB@Fe-TDA) was developed by encapsulating *Saccharomyces cerevisiae* bacteria (Fe-TDA) with different promoters or enhanced green fluorescent protein reporter gene plasmids. Fe-TDA is acid-resistant in the stomach and dissolves in the intestine, leading to its release by the yeast. When nitrosamines cause DNA damage, the DNA damage repair pathway in the yeast is activated, resulting in plasmid expression and fluorescence signal generation. By monitoring the fluorescence intensity in situ, the concentration of nitrosamines in the intestine can be monitored.

[0024] Another object of the present invention is to provide the application of the metal-organic coordination gel-protected whole-cell biosensor for monitoring nitrosamine compounds in the digestive tract in the detection of nitrosamine compounds.

[0025] The application described is to detect nitrosamine compounds in the digestive tract of a living organism by detecting nitrosamines carried in feces.

[0026] The nitrosamine compounds mentioned are N-nitrosodiethylamine (NDEA), N-nitrosomorpholine (NMOR), N-nitroso-N-methylaniline (NMPhA), N-nitrosodimethylamine (NDMA), N-nitrosodi-n-propylamine (NDPA), N-nitrosodi-n-butylamine (NDBA), and 1-nitrosopyrrolidine (NPYR).

[0027] Another object of the present invention is to provide a method for detecting nitrosamine compounds, comprising:

[0028] Step (1): The whole-cell biosensor protected with the metal-organic coordination gel for monitoring nitrosamines in the digestive tract was administered, and fecal samples were collected every other day. The fecal samples were dispersed in PBS buffer, filtered through a 75-mesh cell filter, centrifuged, and resuspended in SD-HIS liquid medium or SD-URA-HIS-LEU liquid medium to obtain OD. 600 The bacterial solution had a concentration of approximately 0.8.

[0029] Step (2): Set up test group, control group and blank group. Test group: Take 200 μL of bacterial solution obtained in step (1). Control group: Add 10 μL of PBS buffer to 190 μL of bacterial solution obtained in step (1). Blank group: Add 10 μL of PBS buffer to 190 μL of SD-HIS liquid medium or SD-URA-HIS-LEU liquid medium.

[0030] Step (3): Place the solution obtained in step (2) in a 30°C constant temperature incubator and incubate at 100 rpm for 30 min. Then, measure the OD using a microplate reader. 600 The total number of yeast cells was measured, and the fluorescence response intensity (denoted as GFP) was measured at an excitation wavelength of 485 nm and an emission wavelength of 535 nm.

[0031] The total protein expression level (TEL) of proteins related to DNA damage repair pathways was calculated using the following method:

[0032] (1) OD 600 Correction of raw GFP data:

[0033] GFP 校正 =GFP 测试 -GFP 空白 ;

[0034] OD 校正 =OD 测试 -OD 空白 ;

[0035] (2) Calculation of the average relative fluorescence intensity (AF) per unit cell:

[0036] AF=GFP 校正 / OD 校正 ;

[0037] (3) Calculation of TEL:

[0038] TEL=AF 测试 / AF 对照 ;

[0039] When TEL > 1.5, it is considered that nitrosamine compounds are present in the digestive tract.

[0040] The metal-organic coordination gel-protected whole-cell biosensor for monitoring nitrosamine compounds in the digestive tract can be administered orally or by gavage.

[0041] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0042] The coordination gel biosensor of this invention has simple preparation conditions and is easy to operate, requiring no complex pretreatment. It can be used for the detection of nitrosamine compounds and features low cost, high specificity, and simple operation. Specifically:

[0043] (1) The whole-cell biosensor based on metal-organic coordination gel of the present invention is simple to prepare, does not require strict synthesis conditions, and is low in cost.

[0044] (2) The whole-cell biosensor based on metal-organic coordination gel of the present invention avoids the problem of impaired metabolic activity of microbial sensors in the gastric acid environment and inaccurate detection results.

[0045] (3) The metal-organic coordination gel is resistant to gastric acid. The whole-cell biosensor based on the metal-organic coordination gel of this invention can be used to transfer the yeast sensor to the intestine without affecting the performance of the yeast sensor in situ detection of nitrosamines, thus realizing in situ monitoring of nitrosamine genotoxic substances in the digestive tract.

[0046] (4) The whole-cell biosensor based on metal-organic coordination gel of the present invention has the ability to specifically detect nitrosamine compounds. Attached Figure Description

[0047] Figure 1 This invention describes the principle behind the metal-organic coordination gel-protected whole-cell biosensor for detecting nitrosamine compounds in the intestine.

[0048] Figure 2 Transmission electron microscopy image of metal-organic coordination gel (Fe-TDA).

[0049] Figure 3 For plasmid pMAG1-yEGFP ( Figure 3A) and the spectrum of pPHR1-yEGFP ( Figure 3 B).

[0050] Figure 4 The response diagram of yeast biosensors to nitrosamine compounds ( Figure 4 A) and the detection limit exploration diagram ( Figure 4 B).

[0051] Figure 5 This demonstrates the specificity of yeast pMAG1 for detecting nitrosamine compounds.

[0052] Figure 6 Figure showing the protective effect of Fe-TDA on the sensing performance of yeast pMAG1.

[0053] Figure 7 This is a graph showing the detection of nitrosamines in the mouse intestine by pMAG1@Fe-TDA. Detailed Implementation

[0054] The technical solution of the present invention will be described in detail below.

[0055] Unless otherwise specified, the experimental methods described in the following examples are conventional methods.

[0056] Unless otherwise specified, all test materials used in the following examples were purchased from regular biochemical reagent stores.

[0057] Example 1

[0058] Fabrication of whole-cell biosensors protected by metal-organic coordination gels:

[0059] 1.1. Preparation of organometallic coordination gel solutions

[0060] Add 0.05 mmol FeCl3·6H2O and 0.05 mmol TDA to the culture bottle, then add 1 mL of water and sonicate for 5 min to completely dissolve FeCl3·6H2O in water, thus obtaining a metal-organic coordination gel solution.

[0061] The metal-organic coordination gel solution was allowed to stand at room temperature to form a metal-organic coordination gel (Fe-TDA), the transmission electron microscope image of which is shown below. Figure 2 It can be seen that the morphology of Fe-TDA is long fibrous.

[0062] 1.2. Strains

[0063] The genome-fusion-type DNA damage-responsive fluorescent yeast strain (No. 95702, ATCC 201388) was purchased from Invitrogen, USA, as shown in Table 1. The selected yeast cell set consisted of four DNA damage-responsive fluorescent yeast strains, covering four different DNA damage repair pathways. When nitrosamine compounds induced different DNA damage, the corresponding repair pathways were activated, and specific GFP fusion proteins were expressed through endogenous promoters.

[0064] Table 1. Genome-fusion DNA damage-responsive fluorescent yeast strains

[0065]

[0066] The plasmids of plasmid-responsive Saccharomyces cerevisiae strains contain GFP genes regulated by DNA damage repair promoters. When nitrosamine compounds cause DNA damage in cells, the corresponding damage pathway is activated, and while proteins are expressed in the yeast, the expression of GFP on the plasmid is also activated. As shown in Table 2, the plasmid-responsive yeast strains are developed by separately applying the plasmid pMAG1-yEGFP (plasmid map shown in Table 2). Figure 3 A) Plasmid pPHR1-yEGFP (plasmid map see...) Figure 3 B) Using the LiAc / PEG / ssDNA method [1] Saccharomyces cerevisiae BY4741, transformed into double-knockout Saccharomyces cerevisiae genes pdr5 and snq2, respectively. [2] The strains obtained from the process are designated as pMAG1 and pPHR1, respectively.

[0067] Table 2. Plasmid-introduced DNA damage-responsive fluorescent yeast strains

[0068]

[0069] The specific conversion method is as follows:

[0070] Step (1): Take an appropriate amount of yeast culture, centrifuge to collect the cells, wash several times with sterile water, and resuspend in 1 mL of 0.1 mol / L LiAc solution;

[0071] Step (2): Incubate in a 30℃ water bath for 5 min, centrifuge and discard the supernatant; add 200 ng plasmid pMAG1-yEGFP or plasmid pPHR1-yEGFP, 36 μL of 1 mol / L LiAc solution, 15 μL of 10 mg / mL salmon sperm ssDNA solution and 240 μL of 50% PEG4000, and mix well;

[0072] Step (3): Incubate in a 42℃ water bath for 45 min, centrifuge and discard the supernatant, resuspend in SD-URA-HIS-LEU liquid medium, and revive at 30℃ and 200 rpm for 2 h.

[0073] Step (4): Spread the obtained bacterial solution onto SD-URA-HIS-LEU plates using a spreader. After standing upright for 30 minutes, incubate upside down at 30°C until single colonies grow. Verify by PCR. Inoculate the verified colonies into SD-URA-HIS-LEU liquid medium and incubate until the OD of the bacterial solution reaches a certain value. 600 The value is approximately 0.4;

[0074] Step (5): Prepare several sterile centrifuge tubes, add 50% (v / v) glycerol solution, add the yeast culture obtained in step (4) to each centrifuge tube, mix well, seal the tubes, and the final concentration of glycerol is 12.5%. Take the glycerol tubes and let them stand at 4℃ for several hours, then let them stand at -20℃ for several hours, and finally freeze them in a -80℃ freezer.

[0075] 1.3. Resuscitation of bacterial strains and preparation of bacterial culture

[0076] 1. Recovery of genome-fusion DNA damage-responsive fluorescent yeast strains

[0077] SD-HIS liquid culture medium: Weigh 5.58g of SD-HIS culture medium solid powder, dissolve it in 200mL of ultrapure water, sterilize at 121℃ for 15min to prepare SD-HIS liquid culture medium for later use.

[0078] SD-HIS solid medium: Add 4g of agar powder to SD-HIS liquid medium before sterilization.

[0079] The four genome-fusion DNA damage-responsive fluorescent yeast strains described in "1.2" were inoculated onto SD-HIS solid plates and incubated upside down at 30°C for 48 hours. SD-HIS liquid culture medium was added to sterilized test tubes, and single colonies were picked from the plate using an inoculation loop and submerged below the liquid surface in the test tubes. The test tubes were then incubated at 30°C with shaking at 220 rpm until the OD of the bacterial culture reached [missing value]. 600 When the value is 0.4, it is reserved.

[0080] 2. Resuscitation of plasmid-responsive Saccharomyces cerevisiae strains

[0081] SD-URA-HIS-LEU liquid medium: Weigh 5.58g of SD-URA-HIS-LEU solid powder, dissolve it in 200mL of ultrapure water, sterilize at 121℃ for 15min to prepare SD-URA-HIS-LEU liquid medium for later use.

[0082] SD-URA-HIS-LEU solid medium: Add 4g of agar powder to SD-URA-HIS-LEU liquid medium before sterilization.

[0083] The two plasmid-responsive *Saccharomyces cerevisiae* strains described in "1.2" were inoculated onto SD-URA-HIS-LEU solid plates and incubated upside down at 30°C for 48 hours. SD-URA-HIS-LEU liquid medium was added to sterilized test tubes, and a single colony was picked from the plate using an inoculation loop and submerged below the liquid surface in the test tube. The test tubes were then incubated at 30°C and 220 rpm with shaking until the OD of the bacterial culture reached [missing value]. 600 When the value is 0.4, it is reserved.

[0084] 1.4. Fabrication of Coordination Gel Biosensors

[0085] 10 μL of OD prepared by "1.4" 600 The bacterial solution with a concentration of 0.4 was added to the metal-organic coordination gel solution obtained in step 1.1, and allowed to stand at room temperature until gel formation, thus obtaining the coordination gel biosensor.

[0086] Example 2

[0087] Screening of yeast cell sensors and their response to nitrosamines:

[0088] Step (1): Weigh out 4 mg each of the negative control drug salicylic acid (SA), the positive control drug methyl mesylate (MMS), and four nitrosamine compounds (NEDA, NMOR, NMPhA, NDMA), dissolve them in 10 μL DMSO, and dilute with PBS buffer (pH 7.4) to a concentration of 2 × 10⁻⁶. -2 g / L;

[0089] Step (2): Prepare the test group, control group, and blank group solutions. Test group: Take the OD solution prepared in "1.3" of Example 1. 600 =0.4 190 μL of each of the six yeast bacterial cultures, 10 μL of the solution prepared in step (1) were added respectively; control group: 190 μL of yeast bacterial culture prepared in step (1) was added to 10 μL of PBS buffer; blank group: 190 μL of SD-HIS liquid culture medium or SD-URA-HIS-LEU liquid culture medium was added to 10 μL of PBS buffer;

[0090] Step (3): Place the solution obtained in step (2) in a 30°C constant temperature incubator and incubate at 100 rpm for 30 min. Then, measure the OD using a microplate reader. 600 The total number of yeast cells was measured, and the fluorescence response intensity (denoted as GFP) was measured at an excitation wavelength of 485 nm and an emission wavelength of 535 nm.

[0091] Calculate the total protein expression level (TEL) of proteins related to DNA damage repair pathways. The calculation method is as follows:

[0092] (1) OD 600 Correction of raw GFP data:

[0093] GFP 校正 =GFP 测试 -GFP 空白 ;

[0094] OD 校正 =OD 测试 -OD 空白 ;

[0095] (2) Calculation of the average relative fluorescence intensity (AF) per unit cell:

[0096] AF=GFP 校正 / OD 校正 ;

[0097] (3) Calculation of TEL:

[0098] TEL=AF 测试 / AF 对照 ;

[0099] Figure 4 A represents the response of six yeast strains to four nitrosamine compounds. The results show that yeast strain pMAG1 exhibits the highest response to NMOR.

[0100] The NMOR solution from step (1) was diluted sequentially with PBS to 1×10⁻⁶. -3 1×10 -4 1×10 -5 1×10 -6 1×10 -7 1×10 -8 1×10 -9 1×10 -10 g / L, treated according to steps (2)-(3), to investigate the detection limit of yeast pMAG1 for NMOR, the results are as follows Figure 4 B, it was found that when the NMOR concentration was 1×10 -10 It still exhibits good response at g / L.

[0101] Example 3

[0102] The specific response of yeast pMAG1 to nitrosamines:

[0103] Weigh out 4 mg each of SA, MMS, NEDA, NMOR, NMPhA, NDMA, NaHCO3, monosodium glutamate (L-C5H8NO4Na), glucose, citric acid (CA), Brilliant Blue, and acesulfame K (AK), dissolve them in 10 μL LDMSO, and dilute with PBS buffer (pH 7.4) to a concentration of 2 × 10⁻⁶. -2 g / L. The fluorescence response of yeast pMAG1 to the above compound was detected according to steps (2)-(3) of Example 2.

[0104] Depend on Figure 5 It can be seen that yeast pMAG1 showed no response (TEL < 1.5) to NaHCO3, monosodium glutamate (L-C5H8NO4Na), glucose, citric acid (CA), brilliant blue, and acesulfame K (AK), indicating that yeast pMAG1 has the ability to specifically detect nitrosamine compounds.

[0105] Example 4

[0106] An investigation into the protective capabilities of Fe-TDA for pMAG1 sensor performance:

[0107] Take the plasmid-responsive Saccharomyces cerevisiae strain pMAG1 from Example 1 "1.2", and synthesize a coordination gel biosensor (denoted as pMAG1@Fe-TDA) according to the method in Example 1. Then, combine pMAG1@Fe-TDA with the yeast strain pMAG1 (OD... 600 =0.4) were placed in sterile artificial gastric fluid, and after 2 hours, the following steps were performed to obtain bacterial cultures:

[0108] pMAG1@Fe-TDA was extracted from artificial gastric fluid, mixed with 1 mL of SD-URA-HIS-LEU liquid medium, and the gel was broken by pipetting. The mixture was then spread onto SD-URA-HIS-LEU solid plates and incubated for 72 h. Single colonies were picked from the plates using an inoculation loop and inoculated into test tubes containing SD-URA-HIS-LEU liquid medium. The test tubes were incubated at 30°C and 220 rpm with shaking until the OD600 value reached 0.4. For yeast pMAG1 exposed to artificial gastric fluid for 2 h, the cells were collected by centrifugation, resuspended in 1 mL of SD-URA-HIS-LEU, spread onto SD-URA-HIS-LEU solid plates, and incubated for 72 h. Single colonies were picked from the plates using an inoculation loop and inoculated into test tubes containing SD-URA-HIS-LEU liquid medium. The test tubes were incubated at 30°C and 220 rpm with shaking until the OD600 value reached 0.4. Take 190 μL of each of the two bacterial cultures and add them to a solution prepared according to the method in Example 3 at a concentration of 2 × 10⁻⁶. -2Fluorescence intensity was detected by measuring 10 μL of a solution of SA, MMS, NEDA, NMOR, NMPhA, NDMA, NaHCO3, monosodium glutamate, glucose, citric acid, brilliant blue and acesulfame potassium according to steps (2)-(3) of Example 2.

[0109] Figure 6 A represents yeast pMAG1 exposed to gastric acid. It was found that pMAG1 did not respond to any of the four nitrosamine compounds (TEL < 1.5), indicating that pMAG1 loses its detection activity after exposure to gastric acid.

[0110] Figure 6 B indicates that the yeast pMAG1, under the protection of Fe-TDA, maintained cell viability and did not lose its ability to respond to nitrosamine compounds when exposed to gastric acid.

[0111] Example 5

[0112] Oral administration of pMAG1@Fe-TDA for the detection of nitrosamines in the intestine:

[0113] Step (1): Take 1 mL of the organometallic coordination gel solution from Example 1 "1.1" and 50 μL of the pMAG1 yeast culture from Example 1 "1.3" (OD2000). 600 =0.4), the bacterial solution was added to the organometallic coordination gel solution, and allowed to stand at room temperature until gel formation, thus obtaining the coordination gel biosensor, denoted as pMAG1@Fe-TDA;

[0114] Mice were administered pMAG1@Fe-TDA by gavage at a dose of 0.3 mL per mouse. One day after administration of pMAG1@Fe-TDA, the mice were divided into a treatment group and a control group. The treatment group was given Balb / c mice with NMOR mixed in drinking water (concentration of 0.68125 mg / L), while the control group was given Balb / c mice with glucose mixed in drinking water (concentration of 0.68125 mg / L).

[0115] Step (2): After administering NMOR or glucose for three days, sacrifice and dissect the mice, remove and process the ileum, jejunum and colon, wash with PBS buffer, and cut into pieces in PBS buffer.

[0116] Step (3): Filter using a 75-mesh cell filter to remove non-cellular debris, centrifuge at 10000g for 2 min, and resuspend in 1 mL of SD-URA-HIS-LEU liquid culture medium to obtain OD. 600 The resuspension of the treatment group and the resuspension of the control group were approximately 0.8.

[0117] Step (4): Take 200 μL of the resuspension of the treatment group and the control group into a 96-well plate with a black wall and transparent bottom, and calculate the total expression level (TEL) of the relevant protein according to the method of steps (2)-(3) in Example 2.

[0118] See results Figure 7 This indicates that yeast pMAG1 did not proliferate in the ileum or jejunum, but multiplied in the colon, proving that Fe-TDA can protect pMAG1 in the stomach and release it in the colon, and successfully detect nitrosamines in the intestine.

[0119] References

[0120] [1]Gietz, RD, & Schiestl, RHHigh-efficiency yeast transformation using the LiAc / SS carrier DNA / PEG method[J]. Nature Protocols, 2007, 2(1):31-34.

[0121] [2]He Y, Xia XY, Wei JL, et al. A highly sensitive yeast cell sensor constructed by overlap PCR was used to evaluate genotoxic compounds[J].J ChinaPharm Univ,2021,52(2):236-471.

Claims

1. A metal-organic coordination gel-protected whole-cell biosensor for monitoring nitrosamine compounds in the digestive tract, characterized in that: It is made of Fe 3+ Using a metal-organic coordination gel formed by coordination with 2,2'-thiodiacetic acid as a carrier, a coordination gel biosensor formed by Saccharomyces cerevisiae that can respond to nitrosamine compounds and express GFP was encapsulated in the metal-organic coordination gel. The metal-organic coordination gel-protected whole-cell biosensor is prepared by the following method, including the following steps: Step (1): Ferric chloride hexahydrate and 2,2'-thiodiacetic acid are mixed with a polar solvent and sonicated to completely dissolve ferric chloride hexahydrate and 2,2'-thiodiacetic acid in the polar solvent to obtain a metal-organic coordination gel solution; the molar ratio of ferric chloride hexahydrate and 2,2'-thiodiacetic acid is 1:1; the molar concentration of ferric chloride hexahydrate in the metal-organic coordination gel solution is 0.006-0.08 mmol / mL; Step (2), OD 600 Yeast culture of 0.2-0.4 g was added to the organometallic coordination gel solution obtained in step (1), and allowed to stand at room temperature until gel formation was achieved, thus obtaining the coordination gel biosensor.

2. The whole-cell biosensor protected by a metal-organic coordination gel for monitoring nitrosamine compounds in the digestive tract according to claim 1, characterized in that: The Saccharomyces cerevisiae strain that can respond to nitrosamine compounds by expressing GFP is either a genome fusion-type DNA damage-responsive fluorescent Saccharomyces cerevisiae strain with an introduced promoter or a plasmid-responsive Saccharomyces cerevisiae strain with an introduced enhanced green fluorescent protein reporter gene plasmid. The aforementioned genome-fusion DNA damage-responsive fluorescent yeast strain was constructed by inserting a green fluorescent protein (GFP) gene after the promoter of a protein gene corresponding to the DNA damage repair pathway in the genome; the promoter and its corresponding DNA damage repair pathway are as follows: The plasmid-responsive yeast strains described above were constructed by transfecting plasmids pMAG1-yEGFP and pPHR1-yEGFP into GFP-expressing Saccharomyces cerevisiae strains using the LiAc / PEG / ssDNA method.

3. A method for preparing a metal-organic coordination gel-protected whole-cell biosensor for monitoring nitrosamine compounds in the digestive tract, as described in claim 1, characterized in that: Includes the following steps: Step (1): Ferric chloride hexahydrate and 2,2'-thiodiacetic acid are mixed with a polar solvent and sonicated to obtain a metal-organic coordination gel solution; the molar ratio of ferric chloride hexahydrate to 2,2'-thiodiacetic acid is 1:1; the molar concentration of ferric chloride hexahydrate in the metal-organic coordination gel solution is 0.006-0.08 mmol / mL; Step (2), OD 600 Yeast culture of 0.2-0.4 g was added to the metal-organic coordination gel solution obtained in step (1) and allowed to stand at room temperature to obtain a coordination gel biosensor.

4. The method for preparing the whole-cell biosensor protected by the metal-organic coordination gel according to claim 3, characterized in that: In step (1), the molar concentration of ferric chloride hexahydrate in the organometallic coordination gel solution is 0.05 mmol / mL.

5. The method for preparing the whole-cell biosensor protected by the metal-organic coordination gel according to claim 3, characterized in that: In step (1), the polar solvent is selected from water, ethanol, methanol, or aqueous solutions of methanol and ethanol.

6. The method for preparing the whole-cell biosensor protected by the metal-organic coordination gel according to claim 3, characterized in that: In step (2), the volume ratio of the metal-organic coordination gel solution to the yeast culture is 20:1 to 1500:1, based on the polar solvent.

7. The method for preparing the whole-cell biosensor protected by the metal-organic coordination gel according to claim 6, characterized in that: In step (2), the volume ratio of the metal-organic coordination gel solution to the yeast culture is 20:1 to 100:1, based on the polar solvent.

8. The application of the metal-organic coordination gel-protected whole-cell biosensor for monitoring nitrosamine compounds in the digestive tract as described in claim 1 in the detection of nitrosamine compounds.

9. The application according to claim 8, characterized in that: The nitrosamine compounds mentioned are N-nitrosodiethylamine, N-nitrosomorpholine, N-nitroso-N-methylaniline, N-nitrosodimethylamine, N-nitrosodi-n-propylamine, N-nitrosodi-n-butylamine, and 1-nitrosopyrrolidine.

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