Method for evaluating vibrio parahaemolyticus VBNC state in digestive tract in vitro

Abstract

The method for evaluating the vibrio parahaemolyticus VBNC state in the digestive tract in vitro comprises the following steps that simulated gastric juice and vibrio parahaemolyticus bacterial liquid are added into a centrifugal tube, test groups 1, 2 and 3 without glucose are set to be 2.5, 3.5 and 4.5 in pH value and test groups 4, 5 and 6 with the pH value being 2.5, 3.5 and 4.5 and 4 g of glucose are set to be 2.5, 3.5 and 4.5 respectively, after constant-temperature shaking bath culture is carried out for 120 min, simulated intestinal juice is added, then culture continues to be carried out for 120 min, and the vibrio parahaemolyticus VBNC state in the digestive tract is obtained. The simulated digestive juice is extracted at 30 min, 60 min, 120 min and 240 min, a plate counting method, PMAxx-qPCR, flow cytometry, ATP detection, SEM observation and cell membrane permeability detection are conducted on the simulated digestive juice, and the growth and metabolism conditions of the vibrio parahaemolyticus in the digestive tract and the condition that the vibrio parahaemolyticus enters the VBNC state are evaluated; therefore, rapid and systematic evaluation of the vibrio parahaemolyticus in the digestive tract is realized.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of microbial technology, in particular to a method for evaluating VBNC state of Vibrio parahaemolyticus in digestive tract in vitro. BACKGROUND

[0002] Vibrio parahaemolyticus is a common foodborne pathogen, usually exists in seafood, and can cause gastrointestinal diseases. The VBNC (Vibrio parahaemolyticus) state refers to the "viable but non-culturable state", which can destroy the cell membrane permeability and activity in extreme pH environment. After entering the VBNC state, which is a special survival form of some bacteria, the cell will undergo a series of changes in shape, cell wall and membrane composition, physiology and biochemistry, etc. In this state, although the bacteria are alive, they cannot be cultured, but they may still retain their metabolic activity and cause infection to the human body after resuscitation in the intestinal tract.

[0003] Studies have shown that Vibrio parahaemolyticus can enter the VBNC state under certain conditions such as low temperature and acidic environment, thereby escaping detection by conventional means. Therefore, under the influence of the acidic environment of human gastric juice, Vibrio parahaemolyticus may enter the VBNC state, on the one hand, it may re-enter the environment through excretion and other ways, posing a risk to the environment, and on the other hand, it may resuscitate in the intestinal environment and directly cause infection to the human body. At present, the performance evaluation of Vibrio parahaemolyticus mainly focuses on acid tolerance, bile salt tolerance and other indicators, but the conventional evaluation method cannot truly reflect the growth and metabolism of Vibrio parahaemolyticus in the gastrointestinal tract and the survival form. Therefore, it is necessary to explore the induction of simulated gastric juice to the VBNC state of Vibrio parahaemolyticus and the change of its biological characteristics, and to evaluate the resuscitation of VBNC state bacteria in intestinal juice, which will provide an important basis for the prevention and control strategy of Vibrio parahaemolyticus, so as to reduce the problems of missed detection and false detection, and reduce the public health risk brought by it. SUMMARY

[0004] Therefore, it is necessary to provide a method for evaluating VBNC state of Vibrio parahaemolyticus in digestive tract in vitro, which can truly evaluate the growth and metabolism of Vibrio parahaemolyticus in the digestive tract, whether it will enter the VBNC state, and the potential danger after resuscitation.

[0005] The present application adopts the following technical scheme:

[0006] The present application provides a method for evaluating VBNC state of Vibrio parahaemolyticus in digestive tract in vitro, comprising the following steps:

[0007] Prepare Vibrio parahaemolyticus sample diluent, simulated gastric juice and simulated intestinal juice, respectively;

[0008] Simulated gastric digestion: Add simulated gastric juice with pH of 2.5, 3.5, 4.5 to centrifuge tubes containing V. parahaemolyticus bacterial solution, respectively, and record as test group 1, test group 2, test group 3. Add simulated gastric juice with pH of 2.5, 3.5, 4.5 to centrifuge tubes containing V. parahaemolyticus bacterial solution and glucose, respectively, and record as test group 4, test group 5, test group 6. Place all samples in a 37°C constant temperature water bath shaker and shake for 30 min, 60 min, and 120 min to obtain gastric juice;

[0009] Simulated intestinal digestion: Take the gastric juice after 120 min of culture in the six groups of samples, add simulated intestinal juice, control the pH value of the mixed solution to be 7, and shake in a 37°C constant temperature water bath shaker for 2 h to obtain intestinal juice;

[0010] System evaluation:

[0011] At 30 min, 60 min, 120 min, and 240 min, extract the simulated digestion solution from the six groups of samples, and perform plate counting, flow cytometry detection, PMAxx-PCR detection, and ATP detection to evaluate the growth and metabolism of V. parahaemolyticus and the entering of V. parahaemolyticus into the VBNC state.

[0012] In some embodiments, the volume ratio of the simulated gastric juice to the V. parahaemolyticus bacterial solution is 49 mL: 1 mL.

[0013] In some embodiments, the volume ratio of the simulated gastric juice to the simulated intestinal juice is 20 mL: 12 mL.

[0014] In some embodiments, the preparation steps of the simulated gastric juice are as follows: weigh 2.075 g protease-peptone, 0.5125 g NaCl, 0.0925 g KCl, 0.15 g KH2PO4, and 0.0275 g CaCl2, respectively; add 250 mL of sterile water to a 250 mL conical flask, and prepare two 250 mL solutions in the above proportions, sterilize them in a 121°C high-pressure sterilizer for 15 min, and cool them for standby use; divide the 250 mL solution into three 80 mL portions, and adjust the pH to 2.5, 3.5, and 4.5 with HCl, and add 4 g of glucose to each of the three portions of one of the solutions; finally, weigh 0.08 g of bovine bile, 0.16 g of lysozyme, and 0.02128 g of pepsin from each group of solutions, and add them to obtain six groups of simulated gastric juice;

[0015] In some embodiments, the preparation step of the simulated intestinal fluid is: weighing 0.334 g of NaCl, 0.072 g of KCl, 0.106 g of KH2PO4, and 0.088 g of CaCl2, respectively; adding 200 mL of sterile water into a 250 mL conical flask, sterilizing in a 121℃ high-pressure sterilization pot for 15 min, and reserving after cooling; weighing 0.02 g of pancreatin in 50 mL of sterile water; weighing 0.2 g of cholate in 50 mL of sterile water; mixing 4 mL of pancreatin solution and 2.4 mL of cholate solution for each 12 mL of simulated intestinal fluid, and adjusting the pH value to 7 with NaOH solution.

[0016] The medium in the plate counting process is tryptone soy agar (TSA) medium.

[0017] In some embodiments, the preparation step of the Vibrio parahaemolyticus sample dilution liquid is: adding 20 mL of laboratory preserved Vibrio parahaemolyticus into 200 mL of LB broth medium, and culturing at 37℃ in a constant-temperature water bath shaker for 12 h.

[0018] In some embodiments, in the PMAxx-PCR detection, the PMAxx concentration is 20 μM / L in the process of PMAxx treatment of the bacterial solution, 10 μL of PMAxx is added in 1 mL of the sample, the light incubation time is 10 min, and the light crosslinking time is 15 min.

[0019] 1) The method for evaluating the VBNC state of Vibrio parahaemolyticus in the digestive tract in vitro can link the gastric and intestinal digestion processes together, simulate different pH values and nutritional systems in the gastrointestinal digestion environment, and more comprehensively evaluate the growth and metabolism of Vibrio parahaemolyticus in the digestive tract.

[0020] 2) The method comprehensively uses plate counting, PMAxx-PCR, flow cytometry, ATP detection, SEM observation, and cell membrane permeability detection, can most truly reflect the growth and metabolism of Vibrio parahaemolyticus in different nutritional environments in the simulated digestive tract, and thus determine whether the VBNC state Vibrio parahaemolyticus is produced. BRIEF DESCRIPTION OF DRAWINGS

[0021] Figure 1 The figure is the amount of cultivable bacteria of Vibrio parahaemolyticus obtained after different condition treatments according to the plate counting result;

[0022] Wherein (a) is without the addition of glucose, and (b) is with the addition of glucose;

[0023] Figure 2 The figure is the proportion of living bacteria obtained after flow cytometry detection;

[0024] Wherein (a) no glucose is added, (b) glucose is added;

[0025] Figure 3 The culturable bacteria amount and VBNC state bacteria amount of V. parahaemolyticus obtained under different pH gastric juice conditions are counted; wherein (a) no glucose is added, (b) glucose is added;

[0026] Figure 4 PMAxx-qPCR amplification curve;

[0027] Figure 5 Effect of different digestive fluids on ATP content of V. parahaemolyticus;

[0028] Wherein (a) no glucose is added, (b) glucose is added;

[0029] Figure 6 Scanning electron microscope observation of V. parahaemolyticus before and after gastric juice treatment;

[0030] Figure 7 Statistics of V. parahaemolyticus length observed by SEM under different gastric juice conditions;

[0031] Figure 8 CLSM micrograph under different gastric juice conditions. DETAILED DESCRIPTION

[0032] The present application will be further described in conjunction with specific examples, so that those skilled in the art can more clearly understand the present application. The following examples are used to illustrate the present application, but are not used to limit the scope of the present application. Based on the specific examples in the present application, all other examples obtained by those skilled in the art without creative labor are within the protection scope of the present application. In the examples of the present application, all raw material components are commercially available products well known to those skilled in the art, unless otherwise specified; in the examples of the present application, the technical means used are conventional means well known to those skilled in the art, unless otherwise specified.

[0033] Key test materials:

[0034] V. parahaemolyticus: laboratory preserved V. parahaemolyticus atcc17802.

[0035] TSA medium: Beijing Aobosheng Biotechnology Co., Ltd.

[0036] Bacterial genomic DNA extraction kit: Tiangen Bioscience Technologies (Beijing) Co., Ltd.

[0037] ATP content detection kit: Solabio Biochemical Reagent Kit Business Department

[0038] Vibrio parahaemolyticus nucleic acid detection kit: Jiangsu Shuo Shi Biological Technology Co., Ltd.

[0039] Example 1

[0040] The present embodiment provides a method for evaluating the VBNC state of Vibrio parahaemolyticus in the digestive tract in vitro, comprising the following steps: S1, preparing a Vibrio parahaemolyticus sample diluent:

[0041] The Vibrio parahaemolyticus is subcultured, 20 mL of Vibrio parahaemolyticus liquid is added into 200 mL of LB broth medium, and placed in a constant temperature water bath shaker at 37℃ for 12h.

[0042] S2, simulating gastric digestion:

[0043] After the prepared simulated gastric juice is filtered through a 0.22μm filter membrane, 49mL of simulated gastric juice with pH of 2.5, 3.5, and 4.5 is added into centrifuge tubes containing 1mL of Vibrio parahaemolyticus liquid, respectively, which are recorded as test group 1, test group 2, and test group 3. Then, 49mL of simulated gastric juice with pH of 2.5, 3.5, and 4.5 is added into centrifuge tubes containing 1mL of Vibrio parahaemolyticus liquid and 4g of glucose, respectively, which are recorded as test group 4, test group 5, and test group 6 after stirring with a glass rod until completely dissolved. All samples are placed in a 37℃ constant temperature water bath shaker for 30min, 60min, and 120min, respectively, and 5mL of gastric juice is taken for standby;

[0044] S3, simulating intestinal digestion:

[0045] In a clean bench, 20mL of gastric juice from each of the six groups is extracted, 12mL of simulated intestinal juice is added into 20mL of gastric juice, and the pH value is adjusted to 7 with NaOH after mixing. Then, 4mL of trypsin solution and 2.4mL of bile salt solution are added, and intestinal juice is obtained after 2h of vibration culture at 37℃ in a constant temperature water bath shaker.

[0046] S4, plate counting detection:

[0047] The growth and metabolism of Vibrio parahaemolyticus are evaluated: samples from test group 1, test group 2, and test group 3 are extracted at 30min, 60min, 120min, and 240min, respectively, for appearance observation and Vibrio parahaemolyticus counting. The specific steps are as follows: the sample liquid is diluted step by step, 100μL of diluted sample liquid is uniformly coated on the culture medium, three parallel samples are prepared for each dilution ratio, and the coated samples are placed in a 37℃ constant temperature incubator for 24h. Then, the growth of Vibrio parahaemolyticus is observed and counted.

[0048] The results showed that pH had an effect on the growth of bacteria. At pH 2.5, the number of cultivable bacteria was zero, whether or not glucose was added. At pH 3.5 and pH 4.5, the number of cultivable bacteria decreased to zero after a short increase, indicating that long-term exposure caused membrane damage and energy depletion. When glucose was added, the number of cultivable bacteria showed a growth trend at pH 4.5, indicating that glucose alleviated the stress of acidic conditions by providing energy. At pH 3.5, the number of cultivable bacteria fluctuated and decreased, which may be related to the accumulation of metabolic products or competitive inhibition. The above analysis showed that glucose as a carbon source greatly increased the number of cultivable bacteria. When glucose was added at pH 4.5, the number of cultivable bacteria peaked at 120 minutes, indicating that most of the bacteria recovered under near-neutral conditions, which suggested that most of the cells entered the VBNC state in the stomach.

[0049] S5, PMAxx-PCR method:

[0050] After extraction, 1 mL of the bacterial solution was added with 10 μL of PMAxx dye with a concentration of 20 μM / L, and incubated in the dark for 15 min, then cross-linked with light for 20 min before extracting DNA for PCR detection.

[0051] The results showed that the Ct value decreased gradually in the stomach juice as the treatment time increased, and increased in the intestinal juice. The decrease in Ct value indicated an increase in the number of viable bacteria, reflecting the potential resuscitation tendency of VBNC bacteria, which was consistent with the increase in the percentage of viable bacteria in the intestinal juice detected by flow cytometry.

[0052] S6, flow cytometry detection:

[0053] In a 1.5 mL microcentrifuge tube, 3 μL of dye mixture was added to 1 mL of neutralized sample, and incubated in the dark for 15 min at room temperature. After incubation, the sample was run in the flow cytometer for 2 minutes, and the green and red fluorescence was analyzed to determine the proportion of live and dead pathogens in the cell map.

[0054] Flow cytometry detection found that the proportion of viable bacteria at pH 2.5 without added glucose changed little over time, and the proportion of viable bacteria in the intestinal environment was close to 100%. Combined with the analysis of the number of cultivable bacteria, it was shown that most of the bacteria entered the VBNC state and had a certain resistance to artificial gastric juice, and still had strong cell activity under long-term treatment.

[0055] The viable bacteria ratio under each pH condition was significantly different from that without glucose addition. The viable bacteria ratio under pH 2.5 decreased significantly over time, the viable bacteria ratio under pH 3.5 was relatively stable, and the viable bacteria ratio under pH 4.5 continued to rise, and the viable bacteria ratio in the intestinal environment was close to 100%. This shows that under suitable conditions, cells in the VBNC state almost all recover, but the viable bacteria ratio is less and the dead bacteria increase compared with the state without glucose addition. This indicates that gastric juice has a certain bactericidal effect.

[0056] S7, VBNC state Vibrio parahaemolyticus bacterial quantity calculation:

[0057] The VBNC bacterial quantity (VBNC bacterial quantity = viable bacteria percentage x original bacterial liquid concentration - cultivable bacterial quantity) was calculated from the flow cytometry detection results. It can be concluded that whether glucose is added or not, the VBNC state bacteria under pH 2.5 and pH 3.5 and pH 4.5 are much higher than the cultivable bacteria. Under extreme pH conditions, the bacterial quantity in the intestinal juice is more than that in the gastric juice, indicating that the intestinal juice provides suitable conditions for most cells, and under this condition, the VBNC state bacteria will recover, posing a risk to the environment.

[0058] S8, ATP detection:

[0059] Under ice block preservation, the extracted bacterial liquid was broken using an ultrasonic molecular pulverizer, and then the corresponding reagent was added according to the ATP detection kit, and the sample was detected using a UV-visible spectrophotometer. The sample was detected immediately after adding the reagent and after incubation at 37°C for three minutes.

[0060] ATP concentration directly reflects the metabolic activity and survival state of cells. Under each pH condition without glucose addition, ATP fluctuated and decreased, and decreased to a low level at 120 minutes, indicating that the cells were damaged, and many bacteria entered an irreversible death or VBNC state. Under glucose addition conditions, ATP concentration showed a slow upward trend within 0-60 minutes, and the ATP content of all pH values was much higher than that without glucose addition. In the later period, it showed the same performance as without glucose addition, indicating that glucose was efficiently utilized under near-neutral pH. At 240 minutes, the ATP value under pH 3.5 was the highest, indicating that a large number of Vibrio parahaemolyticus recovered under intestinal conditions. The ATP concentration results were consistent with the flow cytometry and PMA-PCR results, which showed that most bacteria would enter the VBNC state under strong acid conditions and then recover in the intestinal juice.

[0061] S9, SEM observation:

[0062] Take 106 or more cells, scrape with a cell scraper, and collect by centrifugation. After pouring off the supernatant medium, rinse gently with PBS, discard the PBS, add 2.5% pre-cooled fixative, and gently blow with a pipette to fully disperse the cells in the fixative. Fix at room temperature for 30 min, and then place in a 4°C refrigerator overnight. The next day, pour off the fixative, and rinse the sample three times for 15 min each time with 0.1M, pH 7.0 phosphate buffer; fix the sample with 1% osmium acid solution for 1-2 h, carefully pour out the osmium acid waste liquid, and rinse the sample three times for 15 min each time with 0.1M, pH 7.0 phosphate buffer; dehydrate the sample with gradient concentrations (including 30%, 50%, 70%, 80%, 90%, and 95% five concentrations) of ethanol solution, 15 min for each concentration, and then treat twice with 100% ethanol, 20 min for each time. Treat the sample with a mixture of ethanol and isoamyl acetate (V / V = 1 / 1) for 30 min, and then treat the sample with pure isoamyl acetate and place overnight. After critical point drying, take a small amount of sample liquid dispersion and drop on a silicon wafer, spray gold for 60 s, and then start testing. This test uses a field emission scanning electron microscope (Hitachi SU8010), and the test mode is secondary electron mode with a test voltage of 5 kv.

[0063] The cell morphological structure of VBNC state of Vibrio parahaemolyticus changed to some extent; the surface roughness intensified, local areas appeared concave and microporous structure, the granular structure increased significantly, cell adhesion was common, and the volume of some individuals decreased to submicron level under the 5.00 μm scale, with fuzzy boundaries and cell aggregation.

[0064] S10, cell membrane permeability detection:

[0065] Centrifuge the test bacterial solution (4000 rpm for 5 min) to obtain the precipitate, and wash twice with PBS. Mix SYTO-9 and PI dyes according to the recommended concentration of the manufacturer. Generally, the final concentration of SYTO-9 is 5 μM, and the final concentration of PI is 20 μM. Take an appropriate amount of dye mixture and add it to the bacterial suspension to ensure that the dye is fully mixed with the bacteria. Incubate the mixture of bacteria and dye at room temperature for 30 min in the dark. After resuspending the cells by washing with PBS, take 10 uL and drop it onto a glass slide, cover it with a cover glass. Place it in a laser confocal microscope for direct observation.

[0066] The cell length of Vibrio parahaemolyticus in VBNC state changed to some extent, the bacterial cell structure was destroyed and divided into smaller cells, and in a higher pH environment, some cells significantly elongated due to the destruction of the cell membrane and re-fusion and aggregation into larger cells, which is consistent with the structural changes of Vibrio parahaemolyticus, indicating that extreme acidic ring stress can significantly induce Vibrio parahaemolyticus VBNC state.

[0067] The results of the above examples show that, except for the condition of pH 4.5, the amount of cultivable bacteria is relatively low, but flow cytometry and PMAxx-PCR detection confirm the presence of viable bacteria, suggesting their potential pathogenicity. The addition of glucose delays the entry of bacteria into the VBNC state, but cannot completely reverse the damage of acid stress. Studies have shown that strong acidic conditions can induce bacteria to enter the VBNC state, and the intestinal environment can drive the resuscitation of VBNC bacteria, restoring their metabolic activity, thereby increasing the risk of environmental infection. This study systematically reveals the situation of Vibrio parahaemolyticus entering the VBNC state under acidic stress and the potential risk of its resuscitation, providing theoretical support for the prevention and control of Vibrio parahaemolyticus environmental pollution and human health, and verifying the feasibility of the method for evaluating Vibrio parahaemolyticus VBNC state in the digestive tract in vitro.

Claims

1. A method for evaluating the VBNC state of Vibrio parahaemolyticus in the digestive tract in vitro, comprising the following steps: Preparation of Vibrio parahaemolyticus sample dilution and simulated digestive juice respectively; Simulated gastric digestion: adding simulated gastric juice with pH of 2.5, 3.5 and 4.5 into centrifuge tubes containing Vibrio parahaemolyticus bacterial solution, respectively, which are recorded as test group 1, test group 2 and test group 3; adding simulated gastric juice with pH of 2.5, 3.5 and 4.5 into centrifuge tubes containing Vibrio parahaemolyticus bacterial solution and glucose, respectively, which are recorded as test group 4, test group 5 and test group 6; placing all samples in a 37℃ constant temperature water bath shaker for 30min, 60min and 120min to obtain gastric juice; Simulated intestinal digestion: adding simulated intestinal juice into the gastric juice obtained after 120min of culture in the six groups of samples, controlling the pH of the mixed solution to be 7, and culturing in a 37℃ constant temperature water bath shaker for 120min to obtain intestinal juice; Systematic evaluation: Extracting the simulated digestive juice from all samples at 30min, 60min, 120min and 240min, respectively, and performing plate counting, PMAxx-PCR, flow cytometry, ATP detection, SEM observation and cell membrane permeability detection on the simulated digestive juice, respectively, to evaluate the growth and metabolism of Vibrio parahaemolyticus in the digestive tract and the situation of entering the VBNC state, thereby realizing the rapid and systematic evaluation of Vibrio parahaemolyticus in the digestive tract.

2. The method for evaluating the VBNC state of V. parahaemolyticus in the digestive tract in vitro according to claim 1, characterized in that, The preparation steps of the Vibrio parahaemolyticus sample dilution are as follows: adding 20mL of laboratory preserved Vibrio parahaemolyticus into 200mL of LB broth medium and placing it in a constant temperature water bath shaker at 37℃ for 12h.

3. The method for assessing the VBNC state of V. parahaemolyticus in the digestive tract in vitro according to claim 1, characterized in that, The preparation steps of the simulated gastric juice are as follows: weighing 2.075g of protease-peptone, 0.5125g of NaCl, 0.0925g of KCl, 0.15g of KH2PO4 and 0.0275g of CaCl2, adding 250mL of sterile water into a 250mL conical flask, configuring two 250mL solutions according to the proportion, sterilizing them in a 121℃ high pressure sterilization pot for 15min, and cooling them for standby use; dividing the 250mL solution into three 80mL portions and adjusting the pH to 2.5, 3.5 and 4.5 by using HCl, and adding 4g of glucose into each of the three portions; finally, adding 0.08g of bovine bile, 0.16g of lysozyme and 0.02128g of pepsin into each solution to obtain six groups of simulated gastric juice; The preparation steps of the simulated intestinal juice are as follows: weighing 0.334g of NaCl, 0.072g of KCl, 0.106g of KH2PO4 and 0.088g of CaCl2, respectively, adding 200mL of sterile water into a 250mL conical flask, sterilizing it in a 121℃ high pressure sterilization pot for 15min, and cooling it for standby use; weighing 0.02g of trypsin into 50mL of sterile water; weighing 0.2g of choline salt into 50mL of sterile water for standby use; mixing 12mL of simulated intestinal juice, adjusting the pH to 7 by using NaOH solution, adding 4mL of trypsin solution and 2.4mL of choline salt solution.

4. The method for evaluating the VBNC state of V. parahaemolyticus in the digestive tract in vitro according to claim 1, characterized in that, The volume ratio of the simulated gastric juice to the Vibrio parahaemolyticus bacterial solution is 49mL:1mL.

5. The method for evaluating the VBNC state of V. parahaemolyticus in the digestive tract in vitro according to claim 1, wherein ​ 6. The method for evaluating the VBNC state of V. parahaemolyticus in the digestive tract in vitro according to claim 1, wherein The volume ratio of simulated gastric juice to simulated intestinal fluid was 20 mL: 12 mL.

7. The method for evaluating the VBNC state of V. parahaemolyticus in the digestive tract in vitro according to claim 1, wherein the medium in the plate counting process is trypticase soy agar (TSA) medium.

8. The method for assessing the presence of VBNC state of V. parahaemolyticus in vivo in the digestive tract according to claim 1, wherein, During the PMAxx treatment of the bacterial solution, the PMAxx concentration was 20 μM / L, 10 μL of PMAxx was added to 1 mL of the sample, the incubation time in the dark was 10 min, and the photo-crosslinking time was 15 min.

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