CN104194379B - A kind of thiazole orange class Molecule of Cyanine Dyes and application thereof - Google Patents

Abstract

The invention discloses a kind of thiazole orange class Molecule of Cyanine Dyes and application thereof.The structure of this dye molecule includes: conjugated group, enzyme mark group, crosslinked group and connecting portion.This molecule is for cell fluorescence dyeing and the survival condition judging antibacterial based on bacterial lactone enzymatic activity, and the amplification in PCR reacts of the Selective depression dead DNA of bacteria, thus reaching the purpose to antibacterial Quantitative detection of living.Molecular structure of dye of the present invention is simple, and preparation cost is low, and esterase degradation rate is high.

Description

A kind of thiazole orange cyanine dye molecule and its application

technical field

The invention relates to a molecular structure design for live bacteria detection and its application in bacteria detection technology. The thiazole orange cyanine dye molecule (abbreviated as BOMA) involved in the present invention can be applied to the technical fields of cell staining and bacterial detection, and utilizes the presence or absence of intracellular esterase activity as the basis for judging the living state of bacteria, and inhibits the death of bacteria in the fluorescent quantitative PCR reaction. The DNA amplification of bacteria can achieve the purpose of quantitatively detecting the content of live bacteria in the sample.

Background technique

Foodborne pathogenic bacteria are one of the main factors causing foodborne diseases, and have attracted much attention in the field of food safety in the world. In the natural environment, microorganisms exist in a variety of physiological states: viable bacteria that can be cultivated, viable bacteria that are in a non-culturable state (Viable but Nonculturable, VBNC), dead bacteria with complete structures but no biological activity (“Ghosts”) and Membrane damage kills bacteria. Only "live bacteria" retain the virulence and pathogenicity of the original bacteria, posing a potential threat to food safety, and bacteria that have been inactivated by high temperature, ultraviolet rays, etc. have lost their pathogenicity, so food The key to the inspection of source pathogenic bacteria is the existence and accurate quantification of "live" pathogenic bacteria in food.

The current criteria used to distinguish live bacteria from dead bacteria are: whether they can be cultivated, whether they have metabolic activity, and whether the cell membrane is intact. The isolation and culture method will miss the detection of live non-culturable bacteria. The existing live bacteria PCR technology (EMA/PMA-qPCR live bacteria detection technology) actually distinguishes between "dead" and "live" cells through the integrity of the cell membrane. The live bacteria detection technology uses a new nucleic acid dye EMA (ethidium azide bromide) or PMA (propidium azide bromide) to infiltrate membrane-damaged cells, and then covalently binds to DNA, thereby inhibiting the DNA of membrane-damaged cells from Amplification in a PCR reaction. This method utilizes the characteristics that ethidium bromide and propidium bromide in EMA/PMA molecules cannot penetrate the cell membrane, selectively binds the DNA of membrane damaged cells, and then under the action of visible light, the cross-linking group and DNA Covalent cross-linking occurs to achieve the effect of inhibiting the amplification of the cross-linked DNA in the PCR reaction. However, cell membrane damage may be an extreme form of cell death, and the integrity of the cell membrane as a detection standard for distinguishing dead and living bacteria is flawed. First of all, in theory, EMA/PMA cannot enter living cells, but for some cells with reversible partial membrane damage (such as bacteria present in environmental samples) and live bacteria with partially intact membranes, the dye will still penetrate the cell membrane, Cause different degrees of loss of live bacteria DNA, resulting in false negative results. Secondly, some sterilization methods do not directly act on the cell membrane, such as ultraviolet sterilization, freezing sterilization, etc. In this case, EMA- or PMA-based live PCR methods cannot be used to evaluate the effect of the sterilization method, otherwise the number of viable cells will be seriously overestimated. Therefore, using the integrity of the cell membrane as the detection standard for distinguishing live bacteria from dead bacteria is still flawed, and the analysis of live bacteria based on biological metabolic activity will overcome the insufficiency of the PMA-qPCR method in principle, and can directly pass the intracellular metabolic activity. Whether to distinguish between dead and live bacteria. Although trace amounts of metabolic activity remain in a small number of dead cells, in the vast majority of cases this activity is lost faster than, or roughly equal to, the damage to the membrane. Therefore, a set of live bacteria analysis method based on biological metabolic activity is established, which can not only effectively detect live bacteria (including VBNC bacteria), but also overcome the insufficiency of the original live bacteria detection technology. Dead cells with membrane damage are not detected, which also meets the basic requirements for detection of pathogenic bacteria.

Contents of the invention

The object of the present invention is to provide a thiazole orange cyanine dye molecule for detecting living bacteria based on bacterial esterase activity.

Another object of the present invention is to provide applications of such molecules.

The invention relates to a molecular structure of a thiazole orange cyanine dye used in the detection technology of live bacteria and its application in the detection of live bacteria. The molecular structure extends thiazole orange dyes from the design and application of molecular probes to the molecular design and application of living bacteria detection. Molecules designed using this molecular structure can be used in the technical fields of cell staining and bacterial detection. By using the characteristics of the molecule to freely penetrate the cell membrane, the living state of the bacteria can be judged by the activity of the intracellular esterase, and the DNA of the dead bacteria can be detected in the fluorescent quantitative PCR reaction. The inhibitory effect of amplification can achieve the purpose of quantitatively detecting the content of live bacteria.

A kind of thiazole orange cyanine dye molecule, molecular structure is as follows:

R is a C2-C5 alkyl group.

The specific molecular structure is:

The molecular structure includes four parts: a binding group, an enzyme labeling group, a cross-linking group, and a linking part. Taking an alkyl group whose linking site R is C2 as an example, the positions of the above groups are shown in FIG. 1 .

The above-mentioned thiazole orange cyanine dye molecule is used for the detection of the viable bacteria content and the rapid fluorescent staining of the cells.

The present invention is implemented through the following steps:

According to the chemical reaction formula

Compound I' is obtained by reacting compound I with ethanol azide; the reaction is carried out in the following solvent: N,N-dicyclohexylcarbodiimide, N,N-dimethylformamide or a mixed solvent of the above solvents; the reaction A base needs to be added as a catalyst such as 4-dimethylaminopyridine; usually the reaction is from 0°C to 30°C; the reaction time takes about 1 to 24 hours; after the reaction is completed, it is generally extracted and collected with ether, and compound I' is obtained after drying; The product is proved by methods such as NMR.

Compound I" is obtained by reacting chemical compound I' with 3-methyl-2-methylmercaptobenzothiazole: the reaction is carried out under triethanolamine: usually the reaction temperature is from 0°C to 30°C, generally at room temperature, and the reaction time is about It takes 1 to 36 hours; after the reaction is completed, it is generally extracted and collected with diethyl ether, washed with a mixed solvent of diethyl ether and ethanol, and then dried to obtain compound I"; the obtained product is proved by methods such as NMR.

The structure of the thiazole orange cyanine dye molecule involved in the present invention connects the crosslinking group, the enzyme labeling group and the binding group through the linking part to form a kind of dye that can freely penetrate cell membranes, is sensitive to esterase activity, and has DNA crosslinking properties. linked molecules. The function of the binding group is to allow the molecule to freely penetrate the cell membrane, so the molecule can enter all cells. One end of the ester bond in the enzyme labeling group is connected to the crosslinking group, and the other end is connected to the DNA binding group through the linking site. Since the ester bond in the enzyme-labeled group is sensitive to the activity of biological esterase, the ester bond is broken under the action of esterase hydrolysis, and the binding group and the crosslinking group can be separated from each other. This is also a key part of the molecular structure involved in the present invention. The cross-linking group can inhibit the amplification of nucleic acid in PCR reaction after covalently binding to nucleic acid under light. In active cells, when the ester bond in the molecule is broken by enzymolysis, the cross-linking group will fall off from the molecule; on the contrary, in inactive cells, the cross-linking group will not fall off, and it will bind to DNA under the action of visible light. Covalent crosslinks form covalent compounds that inhibit subsequent DNA amplification. For the excess molecules in the free state, the crosslinking groups are inactivated by reacting with water under the action of visible light to form hydroxylamine. Therefore, the molecules designed with the structure provided by the present invention are combined with quantitative PCR technology to establish a novel live bacteria detection technology to distinguish live and dead bacteria, which will depend on the esterase activity in the cell rather than the integrity of the cell membrane, which will be comparable The previous live bacteria detection technology more accurately detects the live bacteria content.

The present invention has following advantage and beneficial effect:

(1) The present invention designs a BOMA molecule for cell staining and live bacteria detection. Tests have shown that BOMA molecules can effectively penetrate the bacterial wall membrane and have a good binding ability with nucleic acids; BOMA is hydrolyzed with lipase, and the hydrolysis rate is as high as 95%, indicating that the ester bond of the molecule can be broken under the action of related enzymes; Combined with real-time fluorescent quantitative PCR, the molecule has obvious inhibitory effect on bacterial DNA amplification. The test results show that the cell membrane penetration, esterase sensitivity and inhibition of DNA PCR amplification of BOMA molecules can be realized, so this molecule can be used for cell fluorescence staining and detection of live bacteria. Whether the enzyme activity exists can determine the living state of the bacteria, and the purpose of quantitatively detecting the content of live bacteria in the sample can be achieved by inhibiting the nucleic acid amplification of the dead bacteria in the fluorescent quantitative PCR reaction.

(2) Compared with the prior art, on the premise of not affecting the function of the molecular binding group, the present invention removes a benzene ring structure in the binding group, which makes the preparation method of BOMA more convenient and reduces the synthesis cost. It can be seen that the improved BOMA molecule not only retains the key functional characteristics of the original molecule, but also has lower preparation cost, simpler preparation method, and high esterase degradation rate; the linking site is the C ₅ alkyl thiazole orange cyanine dye molecule ( The lipase degradation rate of BOMA-5) is 92%, especially the lipase degradation rate of the thiazole orange cyanine dye molecule (BOMA- ₂ ) whose linking position is C2 alkyl is increased to 95%.

(3) BOMA with improved molecular structure is analyzed from the synthesis technology, and the length of the (connecting part) alkyl group is shortened from C ₅ to C ₂ structure, which can greatly reduce the probability of molecular isomers during the synthesis process, thereby improving Yield, reduce the loss caused by isomers in the preparation process.

(4) Compared with the prior art, BOMA molecules can not only carry out rapid fluorescent staining to dead and living cells, but also use the BOMA-qPCR method to carry out rapid quantitative detection of the number of viable bacteria through the presence or absence of bacterial lactonase activity, so it can Avoid the long time-consuming traditional culture method, the missed detection of VBNC bacteria, and the false positive and false negative results of the PMA-qPCR method due to the quantitative detection of live bacteria through the integrity of the cell membrane.

Description of drawings

Fig. 1 is the chemical structural formula of the thiazole orange molecule (abbreviated as BOMA- ₂ molecule) whose connection site is C2 alkyl and marks the position of each group.

Fig. 2 is a fluorescence microscope image of 10 μg/mL concentration of BOMA-2 molecules mixed with Escherichia coli O157: H7 and incubated for 10 minutes.

Figure 3 is the variation of the degradation rate of BOMA-2 molecules with the amount of lipase.

Figure 4 shows the inhibitory effects of different concentrations of BOMA-2 on the amplification of Staphylococcus aureus, Escherichia coli O157 and Listeria monocytogenes DNA in PCR reactions. DNA from Escherichia coli O157 and Listeria monocytogenes.

Fig. 5 is the PCR reaction result of live Escherichia coli DNA treated with BOMA-2 and without BOMA-2.

Fig. 6 is the result of PCR reaction of dead Escherichia coli DNA treated with BOMA-2 and not treated with BOMA-2.

Fig. 7 is the PCR reaction result of mixed E. coli DNA treated with BOMA-2 and not treated with BOMA-2.

Figure 8 shows the variation of the degradation rate of BOMA-5 molecules with the amount of lipase.

Figure 9 shows the inhibitory effects of different concentrations of BOMA-5 on the amplification of Staphylococcus aureus, Escherichia coli O157, and Listeria monocytogenes DNA in PCR reactions. DNA from Escherichia coli O157 and Listeria monocytogenes.

detailed description

The following examples illustrate the application of the molecular structure design covered by the present invention, the preparation of BOMA molecules and further description of its application, but this is only an example and does not limit the present invention. For the experimental methods without specific conditions indicated in the following examples, conventional conditions are generally followed, such as filtration, rotary evaporation, and purification with purification columns.

Example 1

A kind of thiazole orange cyanine dye molecule (BOMA-2), its preparation method is as follows:

The overall chemical reaction formula is

Concrete preparation steps are as follows:

Preparation of compound 2:

1.50 g of compound 1 was mixed with 2.00 g of bromoacetic acid, and heated at 120° C. for 5 hours. After the reaction temperature was lowered to room temperature, the obtained brown solid was dissolved in methanol and concentrated in vacuo. The concentrated material was dissolved in 20 mL of dichloromethane and cooled to 0°C. 40 mL of acetone was slowly added dropwise, and the solid was collected by filtration. After washing three times with 15 mL of acetone, the obtained crude material was resuspended in 20 mL of dichloromethane and stirred for 30 minutes. The solid was collected by filtration and washed three times with 15 mL of dichloromethane to obtain 1.32 g of a light gray solid with a yield of 39%.

Preparation of Compound 3:

1. After stirring 30 g of compound 2 with dichloromethane and 4-dimethylaminopyridine evenly, add an appropriate amount of N,N-dicyclohexylcarbotriimide in an ice bath and stir evenly. Add 80 mL of azide ethanol, react at room temperature for 24 hours, and collect the product by filtration. After the product was rotary evaporated to a paste, ethyl acetate was added, and it was placed in the refrigerator for 48 hours to remove insoluble matter. After column purification, 1.13 g of a light gray solid was obtained with a yield of 56%.

Preparation of Compound 4 (BOMA-2):

1.13 g of compound 3 and 1.00 g of 3-methyl-2-methylmercaptobenzothiazole were dissolved in 10 mL of dichloromethane, and 948 μL of triethylamine was added to obtain a yellow mixture after reaction, which was stirred at room temperature for 24 hours. 50 mL of diethyl ether was slowly added dropwise to the mixture, and the solid was collected by filtration. Each was washed three times with 15 mL of diethyl ether to obtain crude material. Resuspend with 75 mL of 1:2 (v:v) ethanol:ether mixture and stir for 1 hour. The solid was collected by filtration and washed three times with 15 mL of ether. Then re-dissolve the solid with 75mL of 1:2 (v:v) ethanol:ether mixed solvent, stir for 1 hour, collect the solid by filtration and wash with 15mL of ether three times to obtain 0.78g of yellow solid with a yield of 49%.

The molecular structure of BOMA-2 is shown in Figure 1, and its relative molecular mass is 382. BOMA is a yellow solid at room temperature, slightly soluble in water, and easily soluble in organic solvents such as dimethyl sulfoxide. Its hydrogen spectrum chemical shift of nuclear magnetic resonance is as follows: 1H-NMR (600MHz, d6-DMSO): δ2.21-2.28 (t, 2H, CH ₂ ), 3.19-3.26 (t, 2H, CH ₂ ), 3.82-3.87 (s, 3H, CH ₃ ), 3.98-4.03 (t, 2H, CH ₂ ), 4.38-4.43 (t, 2H, CH ₂ ), 6.75-6.79 (s, 1H, CH=), 7.19-7.23 (d ,1H,Ar-H),7.46-7.52(t,1H,Ar-H),7.57-7.60(d,1H,Ar-H),7.69-7.75(t,1H,Ar-H),7.79-7.83 (d, 1H, Ar-H), 7.87-7.90 (d, 1H, Ar-H), 8.36-8.39 (d, 1H, Ar-H), 8.55-8.61 (d, 1H, Ar-H).

Binding experiment of BOMA-2 molecules with Escherichia coli, Staphylococcus aureus, Listeria monocytogenes and other bacteria:

(1) Penetration of BOMA-2 to bacterial cell membrane

50 mg of BOMA powder was dissolved in 1 mL of 20% (v/v) dimethyl sulfoxide (DMSO) to obtain a stock solution with a concentration of 50 mg/mL. Add 10 μL BOMA-2 stock solution to 90 μL sterile ultrapure water and mix well to obtain 5 mg/mL BOMA-2 working solution.

Take 500 μL of freshly cultivated E. coli O157:H7 bacteria solution in a 1.5 mL centrifuge tube, centrifuge at 6000 rpm for 3 minutes, and resuspend with an equal volume of sterile water. Add 1 μL of BOMA working solution to the bacterial solution to make the final concentration of BOMA-2 in the bacterial solution 10 μg/mL, mix well, and incubate at room temperature in the dark for 10 minutes. Place the centrifuge tube horizontally on ice, and light it at 20cm below the 650W tungsten halogen lamp for 5 minutes, and shake the ice box gently to ensure that the solution is fully irradiated.

The cells treated with BOMA-2 were fixed with 10% (v/v) buffered formalin. Aspirate 5 μL of the fixed bacterial solution, drop it on the center of the glass slide without autofluorescence, add glycerol mounting medium, cover with a cover glass and seal with transparent nail polish. After the sample is made, observe it with a laser confocal microscope (excitation wavelength 455nm, emission wavelength 490nm, 100×oil lens).

The bacteria treated with BOMA were all stained and emitted strong fluorescence under the excitation light, confirming that BOMA-2 can penetrate the cell membrane and enter the bacteria body, and can perform rapid fluorescent staining on the cells. The results are shown in Figure 2.

(2) Sensitivity of BOMA-2 to lipase

Enzyme treatment conditions: 5 mL (2.5 μmoL) of 0.212 mg/mL BOMA solution, preheated in a water bath at 40 ° C for 2 minutes, lipase (immobilized lipase Novozym435, Novozym, Denmark) dosage 0.5 ~ 3.5 U, fully mixed at 40 ° C , 150r/min under the dark treatment for 20 minutes. Membrane filtration, and the filtrate was used for high performance liquid chromatography (HPLC) analysis to detect hydrolyzed products (protected from light). HPLC conditions: the chromatographic column adopts ZorbaxSB-C18 column (4.6mm×250mm, 5μm); the mobile phase is water: acetonitrile=60:40 (v/v) phosphate buffer (0.02mol/L, pH value 6.99～7.01 ); column temperature 30°C; flow rate 1mL/min; injection volume 10μL; PDA detector, excitation wavelength 455nm, emission wavelength 490nm. In the control group, an equal concentration of BOMA solution without enzymatic hydrolysis was used instead of enzymatic hydrolysis solution. The result is expressed in degradation rate, and the calculation formula is as follows:

Degradation rate = (content of BOMA-2 before enzymolysis (mg/mL) - content of BOMA-2 after enzymolysis (mg/mL))/content of BOMA in the control group (mg/mL) × 100%

Under the catalysis of lipase, the hydrolysis rate of BOMA-2 is as high as 95%, indicating that BOMA-2 molecules are sensitive to lipase activity and can be rapidly hydrolyzed under the action of corresponding enzymes. The results are shown in Figure 3.

(3) Inhibitory effect of BOMA-2 on DNA amplification

Take 500 μL of Staphylococcus aureus, Escherichia coli O157:H7, and Listeria monocytogenes respectively in 1.5 mL centrifuge tubes, centrifuge at 6000 rpm for 3 minutes, collect the bacteria, and resuspend in an equal volume of sterile water. Genomic DNA was extracted according to the instructions of the Bacterial Genomic DNA Rapid Extraction Kit. Add BOMA-2 solution to the extracted 3 kinds of bacterial DNA respectively to make the final concentration reach 0, 0.5, 1.0, 3.0, 5.0, 10.0, 15.0, 20.0 μg/mL, mix thoroughly and let stand in the dark for 5 minutes, then Put it sideways on crushed ice, and light it at about 20cm below the tungsten halogen lamp for 5 minutes to cross-link BOMA and DNA, and at the same time passivate the free BOMA-2 molecules in the solution. The light-treated DNA solution was used as a real-time fluorescent quantitative PCR reaction template to investigate the inhibitory effect of BOMA-2 on DNA amplification.

The total volume of the fluorescent quantitative PCR reaction system is 25 μL, which contains 2.5 μL of 10×Buffer, 3.5 μL of 25mmol/LMg2+ solution, 1 μL of 25mmol/LdNTPs, 1 μL of primers before and after 15 μmol/L, 1 μL of 10 μmol/L probe, 2 μL of template solution, and TaqDNA polymerization Enzyme 2.5U, DEPC water 12.5μL. Reaction conditions: pre-denaturation at 95°C for 2 minutes; 95°C for 5 seconds, 60°C for 40 seconds and collecting fluorescence signals for 40 cycles (cycle here refers to collecting fluorescence from 95°C for 5 seconds to 60°C for 40 seconds Signal). After the reaction, keep warm at 40°C for 2 minutes. For each fluorescent quantitative PCR reaction, three parallel experiments were performed, and the average Ct value (the number of cycles experienced by the fluorescent signal reaching the set threshold value) and the sample standard deviation SD value were calculated.

BOMA-2 at a concentration of 10 μg/mL had obvious inhibitory effects on the PCR reaction amplification of the DNA of the three bacteria, and the results are shown in FIG. 4 .

(4) BOMA-2 detects the live E. coli content in the sample

Take two equal concentrations of 1.5 mL freshly cultured Escherichia coli liquid in 2 ml centrifuge tubes, wash the bacteria twice with sterile water and resuspend with equal volume of sterile water. A portion of the bacteria solution was placed in a boiling water bath at 100° C. for 5 minutes for heat inactivation to obtain dead Escherichia coli bacteria solution. Take two parts of 500 μL live bacteria solution, dead bacteria solution, and mixed bacteria solution with a ratio of 1:1 of dead and live bacteria, and add 5 mg/mL BOMA-2 to one part of each bacteria solution to work. The final concentration of BOMA-2 in the bacterial solution was 10 μg/mL (in another part, sterile water was used instead of BOMA working solution). After mixing well, incubate at room temperature in the dark for 10 minutes. Place the centrifuge tube horizontally on ice, and light it at 20cm below the 650W tungsten halogen lamp for 5 minutes, and shake the ice box gently to ensure that the solution is fully irradiated. Centrifuge the BOMA-2-treated and non-BOMA-2-treated bacterial solutions at 10,000 rpm for 5 minutes, and remove the supernatant. DNA extraction was performed using a bacterial genomic DNA extraction kit. The extracted DNA was used as template for fluorescent quantitative PCR reaction.

The total volume of the fluorescent quantitative PCR reaction system is 25 μL, which contains 2.5 μL of 10×Buffer, 3.5 μL of 25mmol/LMg2+ solution, 1 μL of 25mmol/LdNTPs, 1 μL of primers before and after 15 μmol/L, 1 μL of 10 μmol/L probe, 2 μL of template solution, and TaqDNA polymerization Enzyme 2.5U, DEPC water 12.5μL. Reaction conditions: pre-denaturation at 95°C for 2 minutes; 40 cycles at 95°C for 5 seconds, 60°C for 40 seconds and collecting fluorescence signals. After the reaction, keep warm at 40°C for 2 minutes.

After extracting DNA from live Escherichia coli treated with 10 μg/mL BOMA-2 and without BOMA treatment, the purified DNA was used as a template for qPCR reaction, and the results are shown in Figure 5.

The results show that the Ct values (both 23) of the qPCR reaction results of the live E. coli DNA treated with BOMA-2 and those without BOMA-2 treatment are quite close, indicating that BOMA-2 does not affect the PCR of live bacterial DNA. Reaction amplification has basically no effect on the qPCR quantitative detection results of live bacteria.

After extracting DNA from dead Escherichia coli treated with 10 μg/mL BOMA-2 and without BOMA-2, the purified DNA was used as a template for qPCR reaction, and the results are shown in Figure 6.

The results indicated that DNA of dead bacteria without BOMA-2 treatment could participate in nucleic acid amplification in qPCR reaction (Ct value was about 23); DNA cannot participate in the amplification in the qPCR reaction (no Ct value), indicating that BOMA can inhibit the amplification of dead bacterial DNA in the PCR reaction.

After extracting DNA from dead Escherichia coli treated with 10 μg/mL BOMA-2 and without BOMA-2, the purified DNA was used as a template for qPCR reaction, and the results are shown in Figure 7.

The results show that the Ct value of the mixed bacterial sample without BOMA-2 treatment is 23, which reflects the total DNA content of dead and viable bacteria in the sample; while the mixed bacterial sample treated with BOMA-2 , the Ct value of the test result is 26 (compared with the Ct value of the sample without BOMA treatment, ΔCt=3), indicating that after BOMA-2 treatment, the live bacteria in the sample can be quantitatively detected under the background of dead bacteria, To achieve the purpose of differential detection of live bacteria.

Example 2

A kind of thiazole orange cyanine dye molecule (BOMA-5), its preparation method is as follows:

Preparation of compound 2:

1.50 g of compound 1 was mixed with 2.26 g of 6-bromohexanoic acid, and heated at 120° C. for 5 hours. After the reaction temperature was lowered to room temperature, the obtained brown solid was dissolved in methanol and concentrated in vacuo. The concentrated material was dissolved in 20 mL of dichloromethane and cooled to 0°C. 40 mL of acetone was slowly added dropwise, and the solid was collected by filtration. After washing three times with 15 mL of acetone, the obtained crude material was resuspended in 20 mL of dichloromethane and stirred for 30 minutes. The solid was collected by filtration and washed three times with 15 mL of dichloromethane to obtain 1.44 g of a light gray solid with a yield of 41%.

Preparation of Compound 3:

After 1.4 g of compound 2 was stirred evenly with dichloromethane and 4-dimethylaminopyridine, an appropriate amount of N,N-dicyclohexylcarbotriimide was added under ice cooling and stirred evenly. Add 80 mL of azide ethanol, react at room temperature for 24 hours, and collect the product by filtration. After the product was rotary evaporated to a paste, ethyl acetate was added, and it was placed in the refrigerator for 48 hours to remove insoluble matter. After column purification, 0.94 g of light gray solid was obtained with a yield of 52%.

Preparation of Compound 4 (BOMA-5):

0.94 g of compound 3 and 1.00 g of 3-methyl-2-methylmercaptobenzothiazole were dissolved in 10 mL of dichloromethane, and 948 μL of triethylamine was added to obtain a yellow mixture after reaction, which was stirred at room temperature for 24 hours. 50 mL of diethyl ether was slowly added dropwise to the mixture, and the solid was collected by filtration. Each was washed three times with 15 mL of diethyl ether to obtain crude material. Resuspend with 75 mL of 1:2 (v:v) ethanol:ether mixture and stir for 1 hour. The solid was collected by filtration and washed three times with 15 mL of ether. The solid was redissolved with 75 mL of 1:2 (v:v) ethanol:ether mixed solvent, stirred for 1 hour, collected by filtration and washed three times with 15 mL of ether to obtain 0.65 g of a yellow solid with a yield of 47%.

The relative molecular mass of BOMA-5 is 424. It is a yellow solid at room temperature, slightly soluble in water, and easily soluble in organic solvents such as dimethyl sulfoxide. Its hydrogen spectrum chemical shift of nuclear magnetic resonance is as follows: 1H-NMR (600MHz, d6-DMSO): δ1.23-1.35 (m, 2H, CH ₂ ), 1.42-1.51 (m, 2H, CH ₂ ), 1.73-1.77 (m,2H,CH ₂ ),2.16-2.25(t,2H,CH ₂ ),3.25-3.30(t,2H,CH ₂ ),3.85-3.91(s,3H,CH ₃ ),4.03-4.07(t ,2H,CH ₂ ),4.43-4.49(t,2H,CH ₂ ),6.80-6.84(s,1H,CH=),7.23-7.27(d,1H,Ar-H),7.51-7.57(t, 1H, Ar-H), 7.60-7.63(d, 1H, Ar-H), 7.74-7.80(t, 1H, Ar-H), 7.83-7.87(d, 1H, Ar-H), 7.91-7.94( d, 1H, Ar-H), 8.42-8.45 (d, 1H, Ar-H), 8.62-8.66 (d, 1H, Ar-H).

Binding experiment of BOMA-5 molecules with Escherichia coli, Staphylococcus aureus, Listeria monocytogenes and other bacteria:

(1) Penetration of BOMA-5 to bacterial cell membrane

50 mg of BOMA-5 powder was dissolved in 1 mL of 20% (v/v) dimethyl sulfoxide (DMSO) to obtain a stock solution with a concentration of 50 mg/mL. Add 10 μL BOMA-5 stock solution to 90 μL sterile ultrapure water and mix well to obtain 5 mg/mL BOMA-5 working solution.

Take 500 μL of freshly cultured E. coli O157:H7 bacteria solution in a 1.5 mL centrifuge tube, centrifuge at 6000 r/min for 3 minutes, and resuspend with an equal volume of sterile water. Add 1 μL of BOMA-5 working solution to the bacterial solution to make the final concentration of BOMA-5 in the bacterial solution 10 μg/mL, mix well, and incubate at room temperature in the dark for 10 minutes. Place the centrifuge tube horizontally on ice, and light it at 20cm below the 650W tungsten halogen lamp for 5 minutes, and shake the ice box gently to ensure that the solution is fully irradiated.

The cells treated with BOMA-5 were fixed with 10% (v/v) buffered formalin. Aspirate 5 μL of the fixed bacterial solution, drop it on the center of the glass slide without autofluorescence, add glycerol mounting medium, cover with a cover glass and seal with transparent nail polish. After the samples were prepared, the results were observed with a laser confocal microscope (excitation wavelength 455nm, emission wavelength 490nm, 100×oil lens).

(2) Sensitivity of BOMA-5 to lipase

Enzyme treatment conditions: 5 mL (2.5 μmoL) of 0.212 mg/mL BOMA-5 solution, preheated in a water bath at 40 ° C for 2 minutes, lipase (immobilized lipase Novozym435, Novozym, Denmark) dosage 0.5 ~ 3.5 U, fully mixed Treat at 40°C, 150r/min in the dark for 20 minutes. Membrane filtration, and the filtrate was used for high performance liquid chromatography (HPLC) analysis to detect hydrolyzed products (protected from light). HPLC conditions: the chromatographic column adopts ZorbaxSB-C18 column (4.6mm×250mm, 5μm); the mobile phase is water: acetonitrile=60:40 (v/v) phosphate buffer (0.02mol/L, pH value 6.99～7.01 ); column temperature 30°C; flow rate 1mL/min; injection volume 10μL; PDA detector, excitation wavelength 455nm, emission wavelength 490nm. In the control group, an equal concentration of BOMA-5 solution without enzymatic hydrolysis was used instead of enzymatic hydrolysis solution. The result is expressed in degradation rate, and the calculation formula is as follows:

Degradation rate = (content of BOMA-5 before enzymolysis (mg/mL) - content of BOMA-5 after enzymolysis (mg/mL))/content of BOMA-5 in the control group (mg/mL) × 100%

Under the catalysis of lipase, the hydrolysis rate of BOMA-5 is about 92%, indicating that BOMA-5 molecules are sensitive to lipase activity and can be rapidly hydrolyzed under the action of corresponding enzymes. The results are shown in Figure 8.

(3) Inhibitory effect of BOMA-5 on DNA amplification

Take 500 μL of Staphylococcus aureus, Escherichia coli O157:H7, and Listeria monocytogenes respectively in 1.5mL centrifuge tubes, centrifuge at 6000r/min for 3 minutes, collect the bacteria, and resuspend with an equal volume of sterile water. Genomic DNA was extracted according to the instructions of the Bacterial Genomic DNA Rapid Extraction Kit. Add BOMA-5 solution to the extracted three kinds of bacterial DNA respectively to make the final concentration reach 0, 0.5, 1.0, 3.0, 5.0, 10.0, 15.0, 20.0 μg/mL, mix well and let stand in the dark for 5 minutes, then Put it sideways on crushed ice, and light it at about 20cm below the tungsten halogen lamp for 5 minutes to cross-link BOMA-5 with DNA and passivate free BOMA-5 molecules in the solution. The light-treated DNA solution was used as a real-time fluorescent quantitative PCR reaction template to investigate the inhibitory effect of BOMA-5 on DNA amplification.

The total volume of the fluorescent quantitative PCR reaction system is 25 μL, which contains 2.5 μL of 10×Buffer, 3.5 μL of 25mmol/LMg2+ solution, 1 μL of 25mmol/LdNTPs, 1 μL of primers before and after 15 μmol/L, 1 μL of 10 μmol/L probe, 2 μL of template solution, and TaqDNA polymerization Enzyme 2.5U, DEPC water 12.5μL. Reaction conditions: pre-denaturation at 95°C for 2 minutes; 95°C for 5 seconds, 60°C for 40 seconds and collecting fluorescence signals for 40 cycles (cycle here refers to collecting fluorescence from 95°C for 5 seconds to 60°C for 40 seconds Signal). After the reaction, keep warm at 40°C for 2 minutes. For each fluorescent quantitative PCR reaction, three parallel experiments were performed, and the average Ct value (the number of cycles experienced by the fluorescent signal reaching the set threshold value) and the sample standard deviation SD value were calculated.

When the concentration of BOMA-5 reached 10 μg/mL, the DNA amplification of the three bacteria began to be significantly inhibited, and the results are shown in Figure 9.

Claims (4)

1. a thiazole orange class Molecule of Cyanine Dyes, it is characterised in that molecular structure is as follows:

R is the alkyl of C2～C5.

2. thiazole orange class Molecule of Cyanine Dyes according to claim 1, it is characterised in that molecular structure is:

3. the application of thiazole orange class Molecule of Cyanine Dyes described in claim 1 or 2, it is characterised in that this molecule is for the detection of viable bacteria content.

4. the application of thiazole orange class Molecule of Cyanine Dyes described in claim 1 or 2, it is characterised in that this molecule is for carrying out rapid fluorescence dyeing to cell.