CN103242306A - Bacteria nitroreductase detection kit and special-purpose fluorescence probe thereof - Google Patents

Abstract

(式I)The invention discloses a bacterial nitroreductase detection kit and a special fluorescent probe thereof. The structural formula of the fluorescent probe is shown in Formula I. The bacterial nitroreductase detection kit includes a reagent stock solution 1 and a reagent stock solution 2; wherein, the reagent stock solution 1 is a buffer containing coenzyme, and the reagent stock solution 2 is a solution of the fluorescent probe. Experiments have found that the fluorescence intensity of the probe itself is very weak. After adding nitroreductase, the probe undergoes a 1,6-rearrangement elimination reaction and releases the resorufin fluorescent parent. The fluorescence of the solution is significantly enhanced and the color changes from almost no color into purple, indicating that the method can be used for the detection of nitroreductase. The invention has the advantages of simple operation, low cost, high speed, high efficiency and sensitivity, etc., is easy to popularize and apply, and has great application prospects in the fields of food and medicine.

(Formula I)

Description

A bacterial nitroreductase detection kit and its special fluorescent probe

technical field

The invention relates to a bacterial nitroreductase detection kit and a special fluorescent probe thereof.

Background technique

Bacterial nitroreductase (nitroreductase, NTR) belongs to flavin mononucleotide (flavinmononucleotide, FMN) dependent nitroreductase superfamily, and usually exists in dimer form. They can use reduced nicotinamide adenine dinucleotide (phosphate) (reduced nicotinamide adenine dinucleotide (phosphate), NAD(P)H) as an electron donor to catalyze a variety of exogenous nitroaromatic, nitroheterocyclic , the reduction reaction of quinones and flavin compounds, the nitro group is a common functional group in biomedicine, antibacterial agents and pesticides, and its reduction is a key step to achieve the degradation of nitroaryl pollutants. It also plays an important role in activation and detoxification mechanisms. Currently, there are few reports on methods for detecting bacterial nitroreductase.

Contents of the invention

The object of the present invention is to provide a fluorescent probe for detecting bacterial nitroreductase and a preparation method thereof.

The fluorescent probe for detecting bacterial nitroreductase provided by the present invention is 7-[(5-nitrothiophen-2-yl)methoxy]-3H-phenoxazin-3-one, and its structural formula is as formula I Shown:

The preparation method of the compound shown in the above-mentioned formula I comprises the following steps: in the presence of a catalyst, 7-hydroxyphenoxazinone sodium salt shown in formula II and 2-bromomethyl-5 shown in formula III -Nitrothiophene is mixed and reacted to obtain the compound shown in formula I.

In the above method, the catalyst is selected from at least one of organic bases and inorganic bases; the organic base is selected from at least one of triethylamine and pyridine; the inorganic base is selected from potassium carbonate, sodium hydroxide, At least one of sodium carbonate and sodium bicarbonate.

The molar ratio of the 7-hydroxyphenoxazinone sodium salt, 2-bromomethyl-5-nitrothiophene and catalyst is 1:0.5～5:0.5～5, preferably 1:1-4:1-4 , more preferably 1:1:1 or 1:4:4.

The reaction temperature of the reaction is 0° C. to 60° C., preferably room temperature (about 20° C.), and the reaction time is 1 to 24 hours. The reaction is carried out in an organic solvent; the organic solvent is selected from at least one of N,N-methylformamide (DMF), triethylamine, tetrahydrofuran and acetonitrile. The amount of the organic solvent used is based on completely dissolving the reactants, and there is no need to specifically limit the amount used.

The compound represented by the above formula I can be used to prepare a bacterial nitroreductase detection kit.

Another object of the present invention is to provide a bacterial nitroreductase detection kit.

The bacterial nitroreductase detection kit includes a reagent stock solution 1 and a reagent stock solution 2; the reagent stock solution 1 is a buffer containing coenzymes, and the reagent stock solution 2 is 7-[(5-nitro A solution of thiophen-2-yl)methoxy]-3H-phenoxazin-3-one.

In the reagent stock solution 1, the buffer is a phosphate buffer with a pH value of 6-9, and the phosphate is selected from at least one of Na ₂ HPO ₄ , NaH ₂ PO ₄ and KH ₂ PO ₄ The concentration of the phosphate is 0.01-0.5M, specifically 10mM; the coenzyme is nicotinamide adenine dinucleotide or nicotinamide adenine dinucleotide phosphate, and its concentration is 0.01-0.1mM. In the reagent stock solution 2, the concentration of the 7-[(5-nitrothiophen-2-yl)methoxy]-3H-phenoxazin-3-one is 1mM; the solvent is ethanol or dimethyl Sulfoxide, specifically dimethylsulfoxide.

The above bacterial nitroreductase detection kit may also only consist of reagent stock solution 1 and reagent stock solution 2 .

The application of the bacterial nitroreductase detection kit provided by the present invention in measuring the content of nitroreductase, especially the application in the content of nitroreductase produced in the growth process of detecting bacteria (such as Escherichia coli), all belong to the present invention scope of protection.

The method for measuring nitroreductase content comprises the steps:

1) Make a standard curve:

a) with 550nm as the excitation wavelength, measure the fluorescence intensity of a series of different concentrations of the nitroreductase standard substance at the emission wavelength of 585nm, denoted as F; and measure the fluorescence intensity of the reagent blank at the emission wavelength of 585nm, Denote it as F ₀ , take the concentration C of nitroreductase as the abscissa, and the fluorescence intensity change value ΔF as the ordinate, and draw a standard curve;

Wherein, ΔF=FF ₀ ; the solution of a series of nitroreductase standard products with different concentrations is composed of reagent stock solution 1, reagent stock solution 2 and the standard of nitroreductase in the nitroreductase detection kit Obtained by mixing the stock solution;

2) Detect the content of nitroreductase in the sample to be tested:

Replace the nitroreductase standard substance in the step 1) with the sample to be tested, and detect the fluorescence intensity of the sample to be tested at an emission wavelength of 585 nm according to the method described in the step 1), denoted as F', Substitute the F' into the standard curve obtained in step 1), and then obtain the content of nitroreductase in the sample to be tested.

In the above detection method, in the series of solutions of nitroreductase standard products with different concentrations, the concentrations of the nitroreductase are 0, 0.005, 0.01, 0.025, 0.05, 0.1, 0.15, 0.2, 0.3, 0.6, 0.8 and 1 μg/mL.

The volumes of the solutions of the series of nitroreductase standard products with different concentrations are all 2 mL.

In the standard stock solution of nitroreductase, the solvent is water; the concentration of nitroreductase in the standard stock solution of nitroreductase is 500 μg/mL.

The volume ratio of the reagent stock solution 1 and the reagent stock solution 2 in the nitroreductase detection kit is 2 mL:10 μL.

The bacterial nitroreductase detection kit provided by the invention has the following characteristics:

1) Reagent stock solution 2 is slightly orange-yellow and has no fluorescence; after reacting with nitroreductase, it can appear purple-red and emit strong fluorescence.

2) The reaction speed is fast, and the color can be developed within 20 minutes.

3) High sensitivity, when the concentration of nitroreductase is ≥25ng/mL, the obvious purple-red can be observed with naked eyes.

4) The color reaction occurs only in the presence of nitroreductase, and other common inorganic salts, sugars, amino acids, vitamins, and biological thiol species do not interfere.

5) It has a long fluorescence emission wavelength (585nm), can be detected by fluorescence spectroscopy, and the determination limit can reach 0.1ng/mL, and can be used to detect the content of nitroreductase produced in the process of bacterial growth.

Therefore, the kit is an excellent and easy-to-use bacterial nitroreductase detection device, and is expected to provide a powerful research tool in modern biology, physiology, medicine and other related fields.

The present invention uses resorufin (resorufin) fluorescent dye as a parent and 5-nitrothiophene as a specific response group to design and synthesize a fluorescent probe (compound shown in formula I) for detecting bacterial nitroreductase. It was found that the fluorescence intensity of the probe itself was very weak. After adding nitroreductase, the probe undergoes a 1,6-rearrangement elimination reaction, releasing the fluorescent parent resorufin. The fluorescence of the solution is significantly enhanced and the color changes from almost colorless to purple. Red, indicating that the method can be used for the detection of nitroreductase. In order to verify the practicability of this method and apply it to the quantitative determination of Escherichia coli metabolic nitroreductase, we prepared a bacterial nitroreductase detection kit using this principle. The invention has the advantages of simple and convenient operation, low cost, quickness, high efficiency and sensitivity, etc., and is easy to popularize and apply.

Description of drawings

Fig. 1 is the chemical reaction equation of the present invention to prepare the compound shown in formula I.

Figure 2 is the fluorescence spectrum of the bacterial nitroreductase detection kit reacting with different concentrations of nitroreductase.

Fig. 3 is the fluorescence emission spectrum of the bacterial nitroreductase detection kit used in the reaction of various interfering species.

Figure 4 shows the change in absorbance of the nitroreductase produced by the bacterial nitroreductase detection kit during the detection of Escherichia coli growth.

Figure 5 shows the changes in fluorescence intensity of nitroreductase produced by the bacterial nitroreductase detection kit during the detection of Escherichia coli growth.

Detailed ways

The present invention will be described below through specific examples, but the present invention is not limited thereto.

The experimental methods described in the following examples, unless otherwise specified, are conventional methods; the reagents and biological materials, unless otherwise specified, can be obtained from commercial sources.

Example 1, preparation of 7-[(5-nitrothiophen-2-yl)methoxy]-3H-phenoxazin-3-one shown in formula I

Prepare according to the chemical reaction flow chart shown in Figure 1.

The operation steps are as follows: Dissolve 7-hydroxyphenoxazinone sodium salt (235mg, 1mmol) in DMF (10mL), add 2-bromomethyl-5-nitrothiophene (223mg, 1mmol), catalyst potassium carbonate (210mg , 1 mmol) in the reaction solution, and reacted at 20°C for 6 hours. After completion of the reaction, after cooling, the solvent was concentrated under reduced pressure to obtain a crude product, which was purified by column chromatography using n-hexane/ethyl acetate (2:1, v/v) as eluent to obtain 180 mg of the product.

The structural characterization data results of the product are as follows:

¹ H NMR (600MHz, 298K, CF ₃ COOD): δ8.40(d, J=9.4Hz, 1H), 8.36(d, J=9.4Hz, 1H), 7.92(d, J=4.2Hz, 1H) , 7.69(t, J=2.8Hz, 1H), 7.67(t, J=2.8Hz, 1H), 7.56(d, J=2.5Hz, 1H), 7.48(d, J=2.3Hz, 1H), 7.20 (d, J=4.2Hz, 1H), 5.62(s, 2H).

¹³ C NMR (150MHz, 298K, CF ₃ COOD): δ175.2, 170.5, 151.3, 150.6, 149.3, 144.4, 138.6, 136.2, 136.1, 134.7, 129.0, 126.4, 125.2, 123.2, 101.7, 99.2, 66.5.

Elemental Analysis 1 (C ₁₇ H ₁₀ N ₂ O ₅ S): Calculated C57.62, H2.84, N7.91, S9.05%; Measured: C57.18, H2.93, N8.00, S8 .98%.

It can be seen from the above that the structure of the product is correct, which is 7-[(5-nitrothiophen-2-yl)methoxy]-3H-phenoxazin-3-one shown in formula I.

Embodiment 2: Spectral properties of reagent 1 (i.e. compound shown in formula I) reacting with different concentrations of nitroreductase

Weigh 3.38 mg of reagent 1 and make 10 ml of dimethyl sulfoxide solution as mother solution (1 mM).

Add 50 μl of the above mother solution dropwise to a certain amount of 10 mM phosphate buffer solution containing 50 μM coenzyme (nicotinamide adenine dinucleotide), then add different concentrations of nitroreductase solutions, and then use 10 mM phosphate buffer solution The solution was adjusted to 10ml. After reacting at 37° C. for 20 min, measure its UV-Vis absorption spectrum and fluorescence emission spectrum. When measuring the fluorescence emission spectrum, de-excite at 550nm; the slit width for excitation and emission is 10nm; the voltage is 400V.

Fig. 2 is the fluorescence spectrum of the reaction between reagent 1 and 0-1 μg/ml nitroreductase.

Fig. 2 result shows, reagent 1 among the present invention has the following characteristics:

1) The probe is colorless and non-fluorescent in solution, but with the addition of nitroreductase, the probe absorbs at about 570nm and produces purple-red fluorescence at 585nm;

2) The intensity of ultraviolet-visible absorption and fluorescence intensity increase with the increase of the concentration of nitroreductase;

3) When 5 μM reagent 1 is used, the fluorescence enhancement is linearly related to the concentration of nitroreductase in the range of 5-300 ng/ml.

Embodiment 3: Reagent 1 (compound shown in formula I) reacts with other species (selectivity research)

Various substances were added to the 5μM reagent 1 solution: potassium chloride (150mM), calcium chloride (2.5mM), magnesium chloride (2.5mM), glucose (10mM), vitamin C (1mM), vitamin B ₆ (1mM ), human serum albumin (100μM), hydrogen peroxide (10μM), hydroxyl radical (10μM), glutamic acid (1mM), arginine (1mM), serine (1mM), glutathione (5mM), semi Cystine (1 mM), homocysteine (1 mM), dithiothreitol (1 mM) and nitroreductase (0.5 μg/mL). After reacting at 37°C for 20 min, the fluorescence emission spectrum was measured. When measuring the fluorescence emission spectrum, de-excite at 550nm; the slit width for excitation and emission is 10nm; the voltage is 400V.

50 μl of reagent 1 stock solution (1 mM) was added to 10 ml of the solution mixed with the above-mentioned substances, and nitroreductase was added to make the final concentration 0.5 μg/mL.

Fig. 3 is a fluorescence emission spectrum obtained when reagent 1 (5 μM) is mixed with various other substances.

The experimental results showed that only nitroreductase could elicit an obvious light signal response from reagent 1, proving that the reagent was highly selective for nitroreductase, while the presence of other species did not interfere with the determination of nitroreductase.

Example 4: Quantitative determination of the concentration of nitroreductase produced during the growth of Escherichia coli with bacterial nitroreductase kit

1) Preparation of Escherichia coli test sample solution:

First prepare Luria-Bertani (LB) medium, its composition is as follows: tryptone 10g/L, yeast extract 5g/L, NaCl 10g/L. After preparation, it is sterilized in a sterilizing pot, and E. coli grows obvious colonies after being cultivated at 37°C for 48 to 72 hours. The culture medium containing E. coli was diluted to a fixed concentration according to a certain ratio, divided into 15 portions and fixed to 2 mL, and cultured in a shaker at 37°C for 0, 1, 2, 3, and 4 hours respectively.

2) Making of working curve

Take 0.5 mg of nitroreductase and dissolve it in 1 mL of secondary water to prepare a standard stock solution of 500 μg/mL nitroreductase; in addition, add 2 mL of LB medium and 5 μL of reagent stock solution 2 to the sample bottle in sequence, and Make the final concentration of pernitroreductase 0, 0.005, 0.01, 0.025, 0.05, 0.1, 0.15, 0.2, 0.3, 0.6, 0.8 and 1 μg/mL in sequence, shake well, and react in a shaking table at 37°C for 20min, take the reaction solution in a 1 cm quartz cuvette, using 550nm as the excitation wavelength, measure the fluorescence intensity at 585nm to obtain the fluorescence intensity F of a series of standard solutions, and measure the fluorescence intensity F ₀ of the corresponding reagent blank solution. Taking the concentration C (μg/mL) of nitroreductase as the abscissa, and the fluorescence intensity change value ΔF (ΔF=FF ₀ ) as the ordinate, draw the working curve, and obtain the corresponding linear regression equation as ΔF=4.0×10 ³ C (μg/mL) + 22.2 (R = 0.995).

Reagent stock solution 2 is a dimethyl sulfoxide solution of reagent 1 (the compound shown in formula I), and the final concentration of the reagent is 5 μM;

3) Determination of the content of nitroreductase produced during the growth of Escherichia coli

Add reagent stock solution 2 to the medium of Escherichia coli cultured for different times, measure the fluorescence intensity at 585nm, record it as F', and substitute F' into the above step 2) to obtain the linear regression equation ΔF=4.0×10 ³ C (μg /mL)+22.2 (R=0.995), the content of nitroreductase produced during the growth of Escherichia coli was obtained. The experiment was repeated three times, and the experimental results are shown in Table 1 below.

Table 1. Detection results of nitroreductase content produced during the growth of Escherichia coli

Figure 4 is the change in absorbance of the nitroreductase produced by the kit during the detection of Escherichia coli growth; it can be seen from Figure 4 that the number of Escherichia coli is also increasing with the increase of time. Figure 5 shows the change in fluorescence intensity of the nitroreductase produced by the kit during the growth of Escherichia coli; it can be seen from Figure 5 that the content of nitroreductase produced by Escherichia coli increases with time, and more resorufin is released Fluorophore, fluorescence is enhanced.

Finally, it should be noted that: the above-mentioned embodiment only enumerates that reagent 1 is a fluorescent reagent, and the concentrations of 1 and phosphate buffer in the reaction system are respectively 5 μM and 10 mM, and the coenzyme (nicotinamide adenine dinucleotide) is The case of 50μM reaction for 20min. The results of other fluorescent reagent concentrations and reaction times are not listed one by one, but they are not used to limit the present invention. Any person skilled in the art should be able to make various modifications and changes without departing from the spirit and scope of the present invention.

Claims (10)

1. the compound shown in the formula I:

2. the method for preparing the compound shown in the described formula I of claim 1, comprise the steps: under the condition that catalyzer exists, 2-brooethyl-5-nitrothiophene shown in 7-hydroxyl Phenazoxine ketone sodium salt shown in the formula II and the formula III is reacted, obtain compound shown in the described formula I;

3. method according to claim 2 is characterized in that: described catalyzer is selected from least a in organic bases and the mineral alkali; Described organic bases is selected from least a in triethylamine and the pyridine; Described mineral alkali is selected from least a in salt of wormwood, sodium hydroxide, yellow soda ash and the sodium bicarbonate.

4. according to claim 2 or 3 described methods, it is characterized in that: the molar ratio of described 7-hydroxyl Phenazoxine ketone sodium salt, 2-brooethyl-5-nitrothiophene and catalyzer is 1: 0.5～5: 0.5～5, preferred 1: 1-4: 1-4, more preferably 1: 1: 1 or 1: 4: 4;

The temperature of reaction of described reaction is 0 ℃～60 ℃, is preferably room temperature, and the reaction times is 1～24 hour; Described being reflected in the organic solvent carried out, and described organic solvent is selected from N, at least a in dinethylformamide, triethylamine, tetrahydrofuran (THF) and the acetonitrile.

5. bacterium nitroreduction enzyme detection kit, it comprises reagent storing solution 1 and reagent storing solution 2; Described reagent storing solution 1 is for containing the damping fluid of coenzyme, and described reagent storing solution 2 is the solution of the compound shown in the claim 1 Chinese style I.

6. test kit according to claim 5, it is characterized in that: in the described reagent storing solution 1, described damping fluid is that the pH value is 6～9 phosphate buffered saline buffer, and described phosphoric acid salt is selected from Na ₂HPO ₄, NaH ₂PO ₄And KH ₂PO ₄In at least a; Described phosphatic concentration is 0.01～0.5M; Described coenzyme is Reduced nicotinamide-adenine dinucleotide or Triphosphopyridine nucleotide, reduced, and its concentration is 0.01～0.1mM;

In the described reagent storing solution 2, the compound concentrations shown in the described formula I is 0.5～2mM; Solvent is ethanol or dimethyl sulfoxide (DMSO).

7. according to claim 5 or 6 described test kits, it is characterized in that: described bacterium nitroreduction enzyme detection kit is made up of described reagent storing solution 1 and reagent storing solution 2.

8. the application of each described test kit in measuring nitroreductase content among the claim 5-7.

9. application according to claim 8 is characterized in that: measure the method for nitroreductase content, comprise the steps:

1) production standard curve:

A) with 550nm as excitation wavelength, the solution of measuring the nitroreductase standard substance of a series of different concns is the fluorescence intensity at 585nm place at emission wavelength, is designated as F; And to measure reagent blank be the fluorescence intensity at 585nm place at emission wavelength, is designated as F ₀, be X-coordinate with the concentration C of nitroreductase, fluorescence intensity changing value Δ F is ordinate zou, the drawing standard curve;

Wherein, Δ F=F-F ₀The solution of the nitroreductase standard substance of described a series of different concns is got by the standard inventory solution mixing of reagent storing solution 1, reagent storing solution 2 and nitroreductase in each described nitroreduction enzyme detection kit among the claim 5-7;

2) content of nitroreductase in the detection testing sample:

The described nitroreductase standard substance of described step 1) are replaced with testing sample, detecting described testing sample according to the described method of described step 1) is the fluorescence intensity at 585nm place at emission wavelength, be designated as F ', with the described step 1) gained of described F ' substitution typical curve, and then obtain the content of nitroreductase in the described testing sample.

10. application according to claim 9 is characterized in that: in the solution of the nitroreductase standard substance of described a series of different concns, the concentration of described nitroreductase is followed successively by 0,0.005, and 0.01,0.025,0.05,0.1,0.15,0.2,0.3,0.6,0.8 and 1 μ g/mL;

The volume of the solution of the nitroreductase standard substance of described a series of different concns is 2mL;

In the standard inventory solution of described nitroreductase, solvent is water; The concentration of nitroreductase is 500 μ g/mL in the standard inventory solution of described nitroreductase;

Reagent storing solution 1 in the described nitroreduction enzyme detection kit and the volume ratio of described reagent storing solution 2 are 2mL: 10 μ L.

Images