CN110156734B - Fluorescent probe and fluorescent test paper for detecting nerve gas simulant and preparation method thereof - Google Patents

Abstract

The invention discloses a fluorescent probe for detecting a nerve gas simulant, fluorescent test paper and a preparation method thereof, wherein the structural formula of the fluorescent probe for detecting the nerve gas simulant is shown as

Wherein R is positioned at any substituted position on the benzene ring, and R is H, OCH₃Or Cl. The fluorescent probe has fluorescence specificity for halophosphate nerve gas, and can be subjected to fluorescence lightening type fluorescence sensing, so that the halophosphate nerve gas can be detected, the detection sensitivity is high, and the detection limit is low; the prepared fluorescent test paper can be used for detecting halophosphate nervotoxic gas in the gas atmosphere, and has the advantages of simple detection method, low preparation cost and convenient carrying.

Description

A fluorescent probe, fluorescent test paper and preparation thereof for the detection of nerve gas simulants backup method

technical field

The invention relates to the field of small molecule fluorescent probes, in particular to a fluorescent probe used for the detection of nerve gas simulants, a fluorescent test paper and a preparation method thereof.

Background technique

Sarin (GB) and soman (soman, GD) are two representative nerve gases, both of which are halophosphates; these compounds can irreversibly react with acetylcholinesterase in the human central nervous system. This disrupts nerve impulses, causing suffocation, cardiac arrest, and death within minutes; and these compounds have been used in large quantities in chemical weapons and pesticides since World War I. Therefore, it is of great significance to develop a sensing method for the rapid and accurate detection of halophosphate nerve gases.

As a detection method with short response time, high sensitivity, high temporal and spatial resolution, and visualization, fluorescence sensing has been widely used in the quantitative and qualitative detection of various environmental parameters and biochemical substrates. It has been reported that using diethyl chlorophosphate (DCP) as a nerve gas mimic, the fluorescent materials used in the detection of halophosphate nerve gas include conjugated polymers, small molecule fluorescent materials, bioprinting materials, aggregates. Compared with other materials, such as induced luminescence materials and nanomaterials, small molecule materials have the advantages of low cost and simple synthesis method; however, there are still few small molecule fluorescent materials used in the detection of halophosphate nerve gas, and they cannot be used in Low-cost preparation of fluorescent test strips.

Therefore, the existing technology still needs to be improved and developed.

SUMMARY OF THE INVENTION

In view of the above-mentioned deficiencies in the prior art, the purpose of the present invention is to provide a fluorescent probe, fluorescent test paper and preparation method thereof for the detection of nerve gas simulants, aiming to solve the existing problems in the detection of halophosphonate nerve gas. There are still few small molecule fluorescent materials, and there is a problem that fluorescent test strips cannot be prepared at low cost.

The technical scheme of the present invention is as follows:

其中，R位于所在苯环上的任意取代位置，R为H、OCH₃或Cl。A fluorescent probe for the detection of nerve gas simulants, the structural formula of the fluorescent probe for the detection of nerve gas simulants is:

Wherein, R is located at any substituted position on the benzene ring, and R is H, OCH ₃ or Cl.

The fluorescent probe for the detection of nerve gas simulants, wherein, R=H.

A method for preparing a fluorescent probe for the detection of nerve gas simulants as described above, comprising the steps of:

A, R-substituted 2-hydroxyacetophenone, 4-(diethoxymethyl)benzaldehyde and inorganic base were placed in a polar solvent, heated to reflux for 12-36h; after dropping to room temperature, hydrogen peroxide was added, and stirred at room temperature for 12 hours ~36h, then add excess hydrochloric acid to stir, after concentration and column chromatography, the fluorescent probe intermediate is obtained;

B. The fluorescent probe intermediate and hydroxylamine hydrochloride are refluxed in CH ₂ Cl ₂ for 4 to 36 hours, and concentrated and recrystallized to obtain the fluorescent probe.

In the method for preparing a fluorescent probe for the detection of nerve gas simulants, in step A, the inorganic base is KOH or NaOH; the polar solvent is methanol, ethanol or ethylene glycol.

The preparation method of the fluorescent probe for the detection of nerve gas simulants, wherein, in step A, the R-substituted 2-hydroxyacetophenone, 4-(diethoxymethyl)benzaldehyde, inorganic The molar ratio of alkali and hydrogen peroxide is 1:3～5:5～40:10～50.

In the method for preparing a fluorescent probe for nerve gas simulant detection, in step B, the molar ratio of the fluorescent probe intermediate to hydroxylamine hydrochloride is 1:2-10.

A method for preparing a fluorescent test paper for nerve gas simulant detection, comprising the steps of:

C, placing the fluorescent probe for nerve gas simulant detection as claimed in claim 1 or 2 and the host material in an organic solvent, and mixing uniformly;

D, then spread the above mixed solution on the substrate, dry it to form a film, and cut it out.

The preparation method of the fluorescent test paper for the detection of nerve gas simulants, wherein, in step C, the weight ratio of the fluorescent probe for the detection of nerve gas simulants to the matrix material is 1:10-1000; The matrix material is PEG, PP, or PB; the boiling point of the organic solvent is lower than 100°C.

A fluorescent test paper for the detection of nerve gas simulants, which is prepared by the above-mentioned preparation method of the fluorescent test paper for the detection of nerve gas simulants.

Beneficial effects: the fluorescent probe of the present invention has a novel structure, uses a flavonoid dye with high fluorescence quantum yield, good photostability, clear light-emitting mechanism, simple synthesis and low toxicity as the fluorescent probe parent, and uses a highly reactive oxime group as the precursor of the fluorescent probe. It is the recognition group of the halophosphate type nerve gas, and realizes the fluorescent lighting type fluorescence sensing, so as to realize the detection of the halophosphate type nerve gas; the fluorescent probe of the present invention has the advantages of Fluorescence specificity, can be directly used for qualitative and quantitative detection of halophosphate nerve gas in solution, with high sensitivity and low detection limit; the fluorescence test paper prepared with it can be used to detect halophosphate nerve gas in gas atmosphere , the detection method is simple, the preparation cost of the fluorescent test paper is low, and it is convenient to carry.

Description of drawings

FIG. 1 is a graph showing the change over time of the fluorescence response of HOFO to the solution to be tested containing different concentrations of DCP in Example 1 of the present invention.

FIG. 2 is a graph of the fluorescence response of HOFO to solutions to be tested containing different concentrations of DCP in Example 1 of the present invention.

FIG. 3 is a comparison diagram of the fluorescence response of HOFO to the solution to be tested containing different compounds (the same concentration) in Example 1 of the present invention.

4 is a graph showing the fluorescence response of solid HOFO in the fluorescent test paper in Example 1 of the present invention to gas atmospheres with different DCP vapor concentrations.

5 is a comparison diagram of the fluorescence response of solid HOFO in the fluorescent test paper in Example 1 of the present invention to different compound vapors (same vapor concentrations).

Detailed ways

The present invention provides a fluorescent probe for the detection of nerve gas simulants, a fluorescent test paper and a preparation method thereof. It should be understood that the specific embodiments described herein are only used to explain the present invention, but not to limit the present invention.

其中，R位于所在苯环上的任意取代位置，R为H、OCH₃或Cl；进一步，R＝H。The present invention provides a fluorescent probe for the detection of nerve gas simulants, wherein the structural formula of the fluorescent probe for the detection of nerve gas simulants is:

Wherein, R is located at any substitution position on the benzene ring, and R is H, OCH ₃ or Cl; further, R=H.

The fluorescent probe of the invention has a novel structure, uses a flavonoid dye with high fluorescence quantum yield, good photostability, clear luminescence mechanism, simple synthesis and low toxicity as the fluorescent probe parent, and uses a highly reactive oxime group as a halophosphoric acid The recognition group of the ester nerve gas; that is, the oxime hydroxyl group of the fluorescent probe of the present invention can act as a nucleophile to attack the phosphorus (V) center of the halophosphoric acid ester nerve gas, so that the photoinduced interaction between the oxime substituent and the flavonoid skeleton can be achieved. Electron transfer (photo induced electron transfer, PET) process is inhibited to cause fluorescence enhancement, so as to realize the fluorescence sensing of halophosphate nerve gas, so as to realize its detection. Compared with the fluorescence quenching sensor, the fluorescent probe of the present invention is more convenient for naked eye observation as a fluorescent light-up sensor, and more conforms to the requirement of accurate detection of halophosphate type nerve gas.

The present invention also provides a method for preparing a fluorescent probe for the detection of nerve gas simulants as described above, comprising the steps of:

A, R-substituted 2-hydroxyacetophenone, 4-(diethoxymethyl)benzaldehyde and inorganic base were placed in a polar solvent, heated to reflux for 12-36h; after dropping to room temperature, hydrogen peroxide was added, and stirred at room temperature for 12 hours ~36h, then add excess hydrochloric acid to stir, after concentration and column chromatography, the fluorescent probe intermediate is obtained;

B. The fluorescent probe intermediate and hydroxylamine hydrochloride are refluxed in CH ₂ Cl ₂ for 4 to 36 hours, and concentrated and recrystallized to obtain the fluorescent probe.

The preparation route of the fluorescent probe for the detection of nerve gas simulants of the present invention is as follows:

In the invention, fluorescent probe intermediates (flavonoid dyes) are prepared at low cost through a "one-pot two-step method" through Claisen-Schmidt condensation reaction and Algar-Flynn-Oyamada oxidative cyclization reaction in turn; and then through Schiff base condensation reaction to prepare Obtain fluorescent probes. Further, in step A, the inorganic base is KOH or NaOH; the polar solvent is methanol, ethanol or ethylene glycol; the R-substituted 2-hydroxyacetophenone, 4-(diethoxymethyl) ) The molar ratio of benzaldehyde, inorganic base and hydrogen peroxide is 1:3～5:5～40:10～50; the eluent used for the column chromatography is n-hexane and acetic acid whose volume ratio is 10～4:1 mixed solvent of ethyl ester. In step B, the molar ratio of the fluorescent probe intermediate to hydroxylamine hydrochloride is 1:2-10; the solvent used for the recrystallization is tetrahydrofuran (THF).

The preparation method of the fluorescent probe of the present invention is simple and low in cost, and is suitable for expanded production in practice.

The present invention also provides a method for preparing a fluorescent test paper for nerve gas simulant detection, comprising the steps of:

C, placing the above-mentioned fluorescent probe for nerve gas simulant detection and the host material in an organic solvent, and mixing uniformly;

D, then spread the above mixed solution on the substrate, dry it to form a film, and cut it out.

Further, in step C, the matrix material can be selected from but not limited to one of polyethylene glycol (Polyethyleneglycol, PEG), polypropylene (Polypropylene, PP), polybutadiene (polybutadiene, PB); The weight ratio of the fluorescent probe used for the detection of the nerve gas simulant to the matrix material is 1:10-1000; the boiling point of the organic solvent is lower than 100° C., preferably ethyl acetate. In step D, the substrate may be selected from, but not limited to, one of a glass sheet and a quartz sheet; the drying temperature is 60-90°C.

The present invention also provides a fluorescent test paper for the detection of nerve gas simulants, which is prepared by using the above-mentioned preparation method of the fluorescent test paper for the detection of nerve gas simulants.

The invention adopts the preparation method of first swelling and then drying to prepare the fluorescent test paper, which is simple, convenient, easy to realize, and suitable for expanding scale production in practice; the invention uses a cheap polymer material as a matrix material, which greatly reduces the usage amount of the fluorescent probe, The production cost is further reduced. The fluorescent test paper prepared by the invention maintains high detection sensitivity to halophosphate ester nerve gas and is convenient to carry. The method for detecting the halophosphate ester nerve poison gas in the gas of the present invention is simple and convenient.

The fluorescent probe of the fluorescent test paper for the detection of nerve gas simulants of the present invention, the preparation method of the fluorescent test paper and the performance thereof will be described in detail below by way of examples.

Example 1

¹H-NMR(600MHz,DMSO-d6),δ:10.09(br s,2H),8.44(d,J＝8.2Hz,2H),8.13(d,J＝7.9Hz,1H),8.09(d,J＝8.2Hz,2H),7.84(t,J＝7.7Hz,1H),7.80(d,J＝8.3Hz,1H),7.49(t,J＝7.4Hz,1H).¹³C-NMR(150MHz,DMSO-d6),δ:192.77,173.27,154.71,143.54,140.41,136.25,134.16,129.54,128.10,124.78,121.29,118.57。MS(HR ESI-TOF):m/z calcd for C₁₆H₁₀O₄:266.0652，found[M+H]⁺:267.0647。(1) 2-Hydroxyacetophenone (40mmol) and terephthalaldehyde diacetal (160mmol) were dissolved in ethanol, then KOH (400mmol) was added, heated to reflux for 24h, cooled to room temperature; after adding hydrogen peroxide (20mL) Continue the reaction for 24h, add hydrochloric acid to pH=1, stir for 5min, remove the solvent under reduced pressure, and obtain the fluorescent probe intermediate by column chromatography (the eluent is a mixed solvent of n-hexane and ethyl acetate with a volume ratio of 7:1). 4-(3-hydroxy-4-oxo-4H-chromen-2-yl)benzaldehyde (abbreviated as HOFA), the yield is 45%, and the structural formula of the fluorescent probe intermediate is:

¹ H-NMR (600MHz, DMSO-d6), δ: 10.09 (br s, 2H), 8.44 (d, J=8.2Hz, 2H), 8.13 (d, J=7.9Hz, 1H), 8.09 (d, J=8.2Hz, 2H), 7.84 (t, J=7.7Hz, 1H), 7.80 (d, J=8.3Hz, 1H), 7.49 (t, J=7.4Hz, 1H). ¹³ C-NMR (150MHz) , DMSO-d6), δ: 192.77, 173.27, 154.71, 143.54, 140.41, 136.25, 134.16, 129.54, 128.10, 124.78, 121.29, 118.57. MS (HR ESI-TOF): m/z calcd for C ₁₆ H ₁₀ O ₄ : 266.0652, found[M+H] ⁺ : 267.0647.

¹H-NMR(600MHz,DMSO-d6),δ:9.62(s,1H),8.18(d,J＝8.3Hz,2H),8.09(d,J＝8.0Hz,1H),7.79–7.73(m,2H),7.71(s,1H),7.61(d,J＝8.4Hz,2H),7.44(t,J＝7.4Hz,1H),7.05(br s,1H).¹³C-NMR(150MHz,DMSO-d6),δ:172.86,154.57,145.16,139.05,137.98,136.76,133.75,129.81,127.87,125.00,124.82,124.60,121.36,118.44.MS(HR ESI-TOF):m/z calcd forC₁₆H₁₁O₄N:281.0761,found[M+H]⁺:282.0754。(2) HOFA (4 mmol) and hydroxylamine hydrochloride (20 mmol) were refluxed in dichloromethane for 24 h, cooled and then removed under reduced pressure to remove the solvent, and recrystallized in THF to obtain the fluorescent probe 4-(3-hydroxy-4-oxo- 4H-chromen-2-yl)benzaldehyde oxime (HOFO for short), the yield is 89%, the structural formula of the fluorescent probe is

¹ H-NMR (600MHz, DMSO-d6), δ: 9.62(s, 1H), 8.18(d, J=8.3Hz, 2H), 8.09(d, J=8.0Hz, 1H), 7.79-7.73(m , 2H), 7.71(s, 1H), 7.61(d, J=8.4Hz, 2H), 7.44(t, J=7.4Hz, 1H), 7.05(br s, 1H). ¹³ C-NMR(150MHz, DMSO-d6),δ:172.86,154.57,145.16,139.05,137.98,136.76,133.75,129.81,127.87,125.00,124.82,124.60,121.36,118.44.MS(HR ESI-TOF):m/z 6 H dcalc _{forC 11} O ₄ N: 281.0761, found[M+H] ⁺ : 282.0754.

(3) Using diethyl chlorophosphate (DCP) as the simulant of halophosphate nerve gas, the fluorescence response of HOFO in solution to halophosphate nerve gas was tested.

Dissolve HOFO in methanol to prepare 4 HOFO solutions of the same concentration (0.01 mM); add different amounts of DCP to the 4 HOFO solutions to obtain 4 solutions containing different concentrations of DCP to be tested. The concentrations of DCP are 0.05 mM and 0.2 mM, respectively. mM, 0.4 mM, 0.8 mM; then the above-mentioned solutions to be tested were tested by fluorescence spectrum respectively. The fluorescence response of HOFO to solutions containing different concentrations of DCP with time was shown in Figure 1. It can be seen that after 60s, the effect of HOFO on DCP The response has tended to be complete, indicating that HOFO has a very fast response rate to DCP. The HOFO solution (0.01 mM) was excited by the light of 365 nm. When the concentration of DCP in the HOFO solution increased from 0 to 0.8 mM, the fluorescence response of HOFO to different concentrations of DCP is shown in Figure 2. The fluorescence emission peak of HOFO at 560 nm varies with the concentration of DCP. The detection limit of HOFO for DCP can reach 1.3ppb (ie 7.8nmol/L) with increasing concentration. Diethyl chlorophosphate (DCP) and cyanophosphate were added to 5 parts of HOFO solution (0.01mM) respectively. Diethyl cyanophosphonate (DCNP), dimethyl methylphosphonate (DMMP), triethyl phosphate (TEP), acetic acid (Acetic acidglacial, AcOH) to obtain 5 different test solutions (The concentrations of the above five compounds are all 0.8 mM), the solution to be tested is excited with 365 nm light, and the fluorescence responses of HOFO to the above five compounds are shown in Figure 3, which shows that the fluorescent probe is specific for DCP in the solution. sexual response.

(4) Preparation of fluorescent test paper.

With PEG (Mw=10000) as the matrix material, 1 g of PEG was dissolved in 300 mL of ethyl acetate, and 50 mg of HOFO was added, heated to make it evenly mixed, and then poured onto a glass sheet, dried at 60 °C to form a film, and cut into pieces according to experimental needs. HOFO-loaded fluorescent test strips of suitable shape (eg, rectangle).

(5) The detection performance test of HOFO-loaded fluorescent test paper for DCP vapor.

The fluorescent test paper loaded with HOFO is placed in a gas atmosphere with different DCP vapor concentrations for 10 to 600 s (for example, 60 s). The spectral response of solid HOFO in the fluorescent test paper to DCP vapor is shown in Figure 4. It can be seen that the fluorescence intensity of solid HOFO varies with DCP. The change of vapor concentration is still increasing. The inserted picture is the fluorescence comparison result of the fluorescent test paper before the test and the fluorescent test paper after the test under the portable UV lamp. The detection limit of solid HOFO in the fluorescent test paper to DCP vapor can reach 2.6ppb; The comparison results of the fluorescence responses of solid HOFO in the test paper to different compound vapors (compound vapor concentrations are all 120 ppm) are shown in Figure 5. It can be seen that the solid HOFO in the fluorescent test paper still has a specific response to DCP.

In summary, the present invention provides a fluorescent probe for the detection of nerve gas simulants, a fluorescent test paper and a preparation method thereof. The fluorescent probe of the present invention has a novel structure, high fluorescence quantum yield and good photostability. , Flavonoid dyes with clear luminescence mechanism, simple synthesis and low toxicity are used as fluorescent probe precursors, and the highly reactive oxime group is used as the recognition group of halophosphonate nerve gas to realize fluorescent light-on fluorescence sensing. , so as to realize the detection of halophosphate nerve gas; the fluorescent probe of the present invention has fluorescence specificity to halophosphate nerve gas, and can be directly used for qualitative and quantitative detection of halophosphate nerve gas in solution , high sensitivity and low detection limit; the fluorescent test paper prepared with it can be used to detect halophosphate nerve gas in gas atmosphere, the detection method is simple, the preparation cost of fluorescent test paper is low, and it is convenient to carry.

It should be understood that the application of the present invention is not limited to the above examples. For those of ordinary skill in the art, improvements or transformations can be made according to the above descriptions, and all these improvements and transformations should belong to the protection scope of the appended claims of the present invention.

Claims (9)

1. a fluorescent probe for the detection of nerve gas simulants, is characterized in that, the structural formula of described fluorescent probe is

2. a preparation method of the fluorescent probe that is used for the detection of nerve gas simulants as claimed in claim 1, is characterized in that, comprises the steps: A. 2-Hydroxyacetophenone, 4-(diethoxymethyl)benzaldehyde and inorganic base were placed in a polar solvent, heated to reflux for 12-36h; after cooling to room temperature, hydrogen peroxide was added, and stirred at room temperature for 12-36h, Then add excess hydrochloric acid and stir, through concentration and column chromatography, the fluorescent probe intermediate is obtained; B. The fluorescent probe intermediate and hydroxylamine hydrochloride are refluxed in CH ₂ Cl ₂ for 4 to 36 hours, and concentrated and recrystallized to obtain the fluorescent probe. 3. The method for preparing a fluorescent probe for nerve gas simulant detection according to claim 2, wherein in step A, the inorganic base is KOH or NaOH; the polar solvent is methanol, ethanol or ethylene glycol. 4. The method for preparing a fluorescent probe for nerve gas simulant detection according to claim 2, wherein in step A, the 2-hydroxyacetophenone, 4-(diethoxymethyl) ) The molar ratio of benzaldehyde, inorganic base and hydrogen peroxide is 1:3～5:5～40:10～50. 5. The method for preparing a fluorescent probe for nerve gas simulant detection according to claim 2, wherein in step B, the molar ratio of the fluorescent probe intermediate to hydroxylamine hydrochloride is 1:2～ 10. 6. a preparation method of the fluorescent test paper for the detection of nerve gas simulants, is characterized in that, comprises the steps: C, placing the fluorescent probe for nerve gas simulant detection as claimed in claim 1 and the host material in an organic solvent, and mixing uniformly; D, then spread the above mixed solution on the substrate, dry it to form a film, and cut it out. 7. the preparation method of the fluorescent test paper that is used for the detection of nerve gas simulants according to claim 6, is characterized in that, in step C, the weight ratio of the described fluorescent probe for the detection of nerve gas simulants and host material is 1:10-1000; the matrix material is PEG, PP, or PB; the boiling point of the organic solvent is lower than 100°C. 8 . The method for preparing a fluorescent test paper for nerve gas simulant detection according to claim 7 , wherein in step D, the substrate is a glass sheet or a quartz sheet. 9 . 9 . A fluorescent test paper for the detection of nerve gas simulants, characterized in that, it is prepared by the method for preparing a fluorescent test paper for the detection of nerve gas simulants according to any one of claims 6 to 8 .

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