KR101336867B1 - Mesoporous Silica Nanoparticle Immobilized Colorimetric Chemosensor for Nerve Agent - Google Patents

Abstract

The present invention relates to a chemical sensor for nerve agent detection, the chemical sensor using an azo-pyridine-based compound immobilized on the mesoporous silica can effectively detect the nerve agent through UV / vis spectrum and color change, regeneration in solution It is very practical because it is possible.

Description

Chemistry sensor for detecting nerve agent including azopyridine-based compound immobilized on mesoporous silica nanoparticle {Mesoporous Silica Nanoparticle Immobilized Colorimetric Chemosensor for Nerve Agent}

The present invention relates to a chemical sensor for nerve agent detection, and more particularly, to fix the azo-pyridine-based compound having a selectivity to the nerve agent in the mesoporous silica nanoparticles to effectively detect the nerve agent in accordance with absorbance or color change. It is about a chemical sensor.

A chemical warfare agent is a substance intended for military operations because of its physiological effects in order to kill or injure people or cause serious harm to humans. Modern chemical warfare agents have been divided into five categories: nerve agents, blistering agents, choking agents, blood agents and incapacitating agents:

During World War II, chemical warfare agents focused on the so-called "neurologic agents," which are fast-acting poisons that attack the nervous system. Among chemical warfare agents, nerve agents are extremely dangerous, and due to their high toxicity and ease of manufacture, the necessity of detecting these deadly chemicals is important through a fast and reliable process.

Neuronal agents, a family of phosphorus-containing organic chemicals that disrupt the mechanisms of message delivery to nerves through organs, have been extensively studied because of their rapid and harsh effects on human and animal health systems. To date, detection methods for many neurological agents have been developed based on fluorescence and colorimetric methods. Nevertheless, these methods have some limitations such as slow response, lack of specificity, limited selectivity, low sensitivity, difficulty in real time measurement or acceptability.

In particular, the neuroagent-like substance is easily hydrolyzed in water within minutes, so in most cases a non-aqueous solution should be used to detect the neuroagent in relation to absorption and emission spectrophotometric spectral changes. Greatly limit the analytical possibilities. Therefore, nowadays, a simple and efficient method for detecting neuronal agents in aqueous media is of great interest.

On the other hand, silica-based nanoparticles are of great interest for biomedical and environmental research such as bio-separation, drug targeting, cell separation, enzyme fixation and protein purification due to their biocompatibility and stability. Receptor-fixed nanoparticles have several important and distinct advantages as solid chemistry sensors.

Firstly, these nanoparticles are easily synthesized by sol-gel condensation, a universal technique that enables the presence of chemical functionality. Second, the receptor immobilized on the inorganic carrier is capable of removing organic guest molecules (toxic metal ions or anions) from the contaminating solution. Third, nanoparticles are easy to separate from contaminants by filtration and can be repeatedly utilized by appropriate treatment. In this respect, the homogeneous porosity and large surface area of the mesoporous silicas (SBA-15 and MCM-41) are very promising as inorganic carriers. If hybrid nanomaterials can be provided as portable systems for chemical sensors, silica-based hybrid nanomaterials can be used as semi-permanent sensors because they can easily detect specific guest molecules in the biological and environmental fields.

The problem to be solved by the present invention is to provide a chemical sensor that can be detected through the UV / vis spectrum and color change, excellent selectivity, easy synthesis, renewable neuronal agent detection.

The present invention provides an azo-pyridine-based compound represented by formula (1) having neuroagent selectivity.

(1)

(One)

In another aspect, the present invention provides a chemical sensor for nerve agent detection characterized in that it comprises an azo-pyridine-based compound fixed to the mesoporous silica nanoparticles represented by the following structural formula (A).

According to an embodiment of the present invention, the chemical sensor may be manufactured in the form of a disk pellet including azo-pyridine-based compound fixed to the mesoporous silica nanoparticles, thereby implementing a portable chemical sensor.

According to one embodiment of the invention, the chemical sensor is characterized in that it is reusable after the detection of neurons, more specifically after the detection of the neurons can be reused by treatment with NaOH to separate the neurons.

In another aspect, the present invention provides a chemical sensor for detecting a neurochemical agent fixed to the mesoporous silica nanoparticles by dissolving the azo-pyridine-based compound of formula (4) in toluene, adding mesoporous silica nanoparticles and stirring under reflux conditions It can manufacture.

In this case, the compound of Formula (4) may be prepared according to the following <Reaction Scheme 1>.

<Reaction Scheme 1>

     (1) (4)

In another aspect, the present invention provides a method for detecting a neurological agent by UV / vis spectrum or color change using a chemical sensor for neurological agent detection characterized in that it comprises an azo-pyridine-based compound immobilized on the mesoporous silica nanoparticles do.

In the present invention, azo pyridine-based compound having DCP selectivity was synthesized and fixed to mesoporous silica, thereby developing a chemical sensor capable of detecting nerve agent through UV / vis spectrum and color change. The chemical sensor according to the present invention is not only very good in selectivity but also has high utility in that it is easy to synthesize and reproducible in aqueous solution.

1 is a schematic diagram showing the change that occurs when the chemical sensor according to the present invention reacts with a neuroagent.
2 is a view showing the chemical structure of the formula 1-4 and MSIAP.
Of Figure 3 (a) is CH ₃ CN, and in the DCP, PMP, DMMP, TEP and TBP UV / Vis spectrum of formula 1 (20.0 μM) in accordance with the addition (10 equiv.), (B ) the CH ₃ CN in DCP UV / Vis titration spectrum of the formula (1) (20.0 μM) according to the addition amount change.
4 is a view showing a mechanism for predicting the change of ICT of Formula 1 when detecting DCP.
Fig. 5 is a graph of absorbance of MSIAP when alternately immersed in 1.0 μM DCP aqueous solution and 10.0 μM NaOH. The cycle number represents the number of repetitions of the immersion / wash cycle, and the vertical axis represents the absorbance of MSIAP at 405 nm.
6 is a photograph of (a) MSIAP and (b) TEM.
7 is a UV / Vis spectrum of MSIAP (20.0 μM) with addition of DCP, PMP, DMMP, TEP and TBP (10 equiv.) In H ₂ O.
8 is a photograph of (a) MSIAP (10 mg) in the presence of (b) DCP, (c) DMP, (d) DMMP, (e) TEP, and (f) TBP (10 equiv).
9 is a UV / Vis spectrum of 1 (20.0 μM), 1 + HClO4, and 1 + DCP (10 equiv.) In CH ₃ CN.
10 is a UV / Vis spectrum of 1-3 (20.0 μM) in CH ₃ CN with addition of DCP, PMP, DMMP, TEP and TBP (10 equiv.).
11 is a FAB-Mass spectrum of Formula 1 + DCP.
Figure 12 shows (A) The nitrogen adsorption-desorption isotherms of (a) mesoporous silica and (b) nitrogen absorption / separation isotherms of MSIAP, and (B) shows (a) mesoporous silica and (b) Barrett- of MSIAP. Joyner-Halenda (BJH) pore diameter.
Figure 13 is an FT-IR spectrum of (a) mesoporous silica and (b) MSIAP.
14 is the TOF-SIMS spectrum of Formula 4. FIG.
Figure 15 is an XPS spectrum of (a) mesoporous silica and (b) MSIAP.
FIG. 16 is a graph showing the time change for the absorbance intensity of MSIAP (20.0 μM) upon addition of DCP (10 equiv) in H 2 O (405 nm).
Figure 17 (a) is the absorbance spectrum of MSIAP (20 μM) upon addition of DCP (100 μM) at different pH values, (b) is a graph showing the change in pH value for the absorbance of MSIAP.
FIG. 18 is a ¹ H NMR (CDCl ₃ , 400 MHz) spectrum of Formula 4.
FIG. 19 is a ¹³ C (CDCl ₃ , 100 MHz) spectrum of Formula 4.
20 is a FAB-Mass spectrum of Formula 4. FIG.

Hereinafter, the present invention will be described in detail.

In the present invention, a novel and easy to prepare colorimetric azo-pyridine-based compound (Formula 1 and Formula 4) for neuronal agent detection and reproducible mesoporous silica-fixed nanoparticles were synthesized using the same. (Fig. 2)

Neuronal agent selectivity according to the present invention, azo-pyridine-based compound is represented by the following formula (1).

(1)

(One)

In another aspect, the present invention provides a chemical sensor for nerve agent detection characterized in that it comprises an azo-pyridine-based compound fixed to the mesoporous silica nanoparticles represented by the following structural formula (A).

The binding site comprising an azo-pyridine group can selectively detect neuroagent DCP (diethylchlorophosphate), which is one of the analogs of chemical warfare agents, compared to other series of phosphate compounds. The compound of Formula 1 exhibits a red-shifted proportional change of about 60 nm in absorption spectroscopy when DCP is added by intramolecular charge transfer (ICT) change. The color change of the receptor (Formula 1) from yellow to red in the concentration range of ˜1.0 × 10 ⁻⁶ M is of considerable value for the selective detection of DCP neuroagent-like substances by the naked eye.

In addition, in the present invention, in terms of a solid phase application, mesoporous silica nanoparticles (MSIAP) using the compound of Formula 1 were prepared by a sol-gel grafting reaction. MSIAP changed color from red to yellow when MSIAP was immersed in DCP aqueous solution, but treated with NaOH solution turned red again, releasing non-toxic diethylphosphate. Changes in MSIAP uptake by DCP are consistent in the pH 3-9 range.

Hereinafter, the present invention will be described in more detail with reference to preferred embodiments. However, these examples are presented by way of example to aid the understanding of the present invention, and the scope of the present invention is not limited thereto.

Synthetic example  One: AZO Synthesis of Pyridine Compound

UV-vis absorption spectra were recorded using a Sinco S-3100 spectrophotometer. NMR and mass spectra were recorded using a Varian instrument (400 MHz) and JMS-700 MStation mass spectrometer, respectively. Transmission electron microscopy (TEM) images were taken with a JEOL JEM.2100 F microscope. Nitrogen adsorption isotherms were measured at 78 K using a Micromeritics ASAP 2010 analyzer. Time-of-flight secondary ion mass spectrometry (TOF-SIMS) was analyzed using a model PHI 7200 with Cs and Ga ion guns for mass and negative ion mass detection. XPS spectra were recorded using a Thermo VG Scientific spectrometer. Reactants 2-hydrazinopyridine, 1,4-benzoquinone, phenol, sodium nitrite, aniline, and diethylchlorophosphate (DCP), pinacrylylphosphonate (PMP), dimethylmethylphosphonate (DMMP), Organophosphorus compounds of triethylphosphate (TEP) and tributylphosphate (TBP) were purchased from Aldrich and used as received. All solvents were analytical reagent grade chemicals purchased from Deoksan Pharmaceutical Co., Ltd. MeCN for absorbance spectra was HPLC reagent grade.

First, an azo-compound of Formula 1 was prepared by reacting 2-hydrazinopyridine with 1,4-benzoquinone in an acidic medium. Under nitrogen, a solution of the compound of formula 1 (0.1 g, 0.5 mmol) and 3-triethoxysilylpropylisocyanate (0.125 g, 0.5 mmol) synthesized in the above synthesis example was suspended in dry CHCl ₃ (10 ml). Et ₃ N (10 mL) was then added and the resulting mixture was refluxed under N _{2 for} 48 h. The mixture was evaporated with a rotary evaporator and the resulting crude was purified by column chromatography (silica, ethyl acetate / hexane 1: 4) to give 0.2 g of the compound of formula 4 (yield 89%).

¹ H NMR (400 MHz, CDCl ₃ , δ: 8.70 (d, J = 4.8 Hz, 1H), 8.04 (d, J = 8.9 Hz, 2H), 7.86 (t, J = 8.0 Hz, 1H), 7.78 ( d, J = 8.0 Hz, 1H), 7.37 (t, J = 4.8 Hz, 1H), 7.28 (d, J = 8.8 Hz, 2H), 5.65 (t, J = 5.8 Hz, 1H), 3.76 (q, 6H), 3.23 (q, 2H ), 1.67 (q, 2H), 1.22 (t, J = 8.2 Hz, 9H), 0.68 (t, J = 8.2 Hz, 2H). 13 C NMR (100 MHz, CDCl 3 , δ: 162.9, 154.4, 154.1, 149.6, 138.6, 125.1, 125.0, 122.3, 115.7, 58.7, 43.8, 31.8, 23.2, 18.5, 14.3, 7.9.MS (FAB): [M ⁺ H] ⁺ C ₂₁ H ₃₁ Calc. For N ₄ O ₅ Si, 447.58. Found 447.3.

Synthetic example  2: Medium porosity  Synthesis of silica

To synthesize the mesoporous silica, a hydrochloric acid solution of P-123 [(poly (ethylene oxide) -poly (propyleneoxide) -poly (ethyleneoxide) terpolymer)] was prepared. Tetraethyl orthosilicate (TEOS) was then added and the resulting mixture was stirred at 40 ° C. for 20 hours. The molar composition of TEOS: HCl: H ₂ O: P-123 was 1: 5.9: 193: 0.017. The solid obtained was aged at 65 ° C. for one day, then filtered and washed and dried at 90 ° C.

To separate the mold to obtain mesopores, 1.0 g of the as-synthesized SBA-15 was mixed with 100 mL of 60% by weight H ₂ SO ₄ solution and refluxed at 95 ° C. for one day. The obtained product was washed with water and recovered, and then dried at 90 ° C. To form the mesopores, the acid treated sample was heated to 200 ° C. in air. In order to remove the cationic surfactant from the resulting dried fibrous aggregates and particles, the samples were fired at a temperature of 1 ° C./min for 5 hours in a box furnace at 20 ° C. in air.

Synthetic example  3: Medium porosity  Fixation of Receptor (Formula 4) on Silica

Compound of formula 4 (100 mg) was dissolved in toluene (10 mL). Mesoporous silica nanoparticles (100 mg) were added as a solid. The resulting silica suspension was stirred for 24 h in toluene under reflux conditions. The collected solid was then washed several times with toluene (50 mL) to wash off excess compound 4 and then dried in vacuo.

Comparative Example Synthesis of Compounds of Chemical Formulas 2 and 3

As controls, compounds of formulas (2) and (3) have been previously reported (H. Xu, X. Zeng, Bioorg . Med. Chem . Lett . 2010, 20 , 4193 and ND Paul, T. Kramer, JE McGrady, S. Goswami , Chem . Commun . 2010, 46 , 7124.).

Experimental Example : DCP  Selectivity testing

First, the reaction of Formula 1 with diethylchlorophosphate (DCP), pinacrylylphosphonate (PMP), dimethylmethylphosphonate (DMMP), triethylphosphate (TEP) and tributylphosphate (TBP) in acetonitrile Tested. These organic phosphate compounds have been used extensively as sarin analogues because they exhibit similar reactivity with neurological agents such as salvia, sarin and soman, but lack toxicity.

The absorption spectrum of the stable trans isomer of the azo-pyridine chemical sensor (Formula 1) shows a strong peak (ε = 2.3 × 10 ⁴ M ⁻¹ cm ⁻¹ ) at 345 nm, corresponding to the π-π * transition. The addition of 10 equivalents of DCP to the solution of 1 causes the absorption band to red shift to 405 nm (ε = 2.5 × 10 ⁴ M ⁻¹ cm ⁻¹ ) to change from yellow to red in the organic medium (FIG. 4A). This spectroscopic change is due to increased intramolecular electron transfer (ICT). Promoted by the strong push-pull effect between the electron-donor oxygen (OH) and the electron acceptor pyridinium group, the enhanced ICT has the effect of shifting the maximum wavelength toward the 60 nm long wavelength in the UV / vis spectrum. Figure 3b shows the detailed change in absorption upon gradual titration of DCP (0-20 equivalents) (Figure 5.

As shown in FIG. 9, a strong absorption band of 1, centered at 405 nm in acidic solution, can support that pyridinium ion formation of 1 would enhance ICT when reacted with DCP. The new band at 405 nm is also similar to the band of 2-pyridine nitrogen methylated azo compounds previously reported by Velasco.

In order to elucidate the role of pyridine nitrogen atom while reflecting the progress of ICT in the nucleophilic interaction in Formula 1, compounds of 2 and 3, which are structural analogs, were synthesized. However, for the addition of DCP, the compound of formula 2 showed no spectral change, suggesting that the DCP binding to formula 1 depends on the pyridine nitrogen atom, not on phenolic OH. In the absence of an electron donor such as OH, Compound 3 had a smaller spectral change (20 nm) for DCP compared to Compound 1 (FIG. 10), which allows the push-pull mediated ICT of pyridine N reactor from OH to detect DCP. It can prove to be very important for.

This 1: 1 binding is evidenced by the peak at m / z 337.2 (calculated 337.12) corresponding to [Formula 1+ DCP + H] ²⁺ in the ESI-MS spectrum of the mixture of Formula 1 and 20 μM DCP (FIG. 11). ).

It is well known that strong bases such as NaOH can react with phosphates (DCP or DFP) to produce less toxic hydrolysis products (phosphates). In addition, the present inventors confirmed that the treatment of NaOH from the change in absorbance and the color regeneration from red to yellow can easily dissociate DCP from the 1-DCP complex and restore the original compound 1 (FIG. 5).

In consideration of expanding the usefulness of the compound of Formula 1, MSIAP was prepared by reacting the compound of Formula 4 with mesoporous silica. MSIAP was obtained by immobilizing the receptor (Formula 4) at reflux conditions for 24 hours. In this process, the triethoxysilyl group of compound 4 is hydrolyzed and covalently attached to the surface of the mesoporous silica. After cooling to room temperature, the red solid product obtained was filtered, washed with THF and dried overnight. The characteristics of MSIAP were analyzed by transmission electron microscopy (TEM), FT-IR, BET isotherm, TOF-SIMS and X-ray photoelectron spectroscopy (XPS).

In FIG. 6, the TEM image of MSIAP clearly shows that the mesoporous channels before and after attachment of Compound 4 are well aligned in a hexagonal arrangement. In order to investigate the porosity change of the mesoporous silica induced by the introduction of compound 4, the inventors measured the surface area, pore volume and pore diameter of mesoporous silica and MSIAP using nitrogen adsorption-desorption isotherm (FIG. 12). It was. Mesoporous silica has a surface area of Brunauer-Emmett-Teller (BET) of 1050.8 m ² / g and a pore volume of 0.47 cm ³ / g.

On the other hand, the inventors observed that MSIAP has a BET surface area of 850.35 m ² / g and a pore volume of 0.32 cm ³ / g. Mesoporous silica and MSIAP have BJH pore diameters of 8.53 and 7.20 nm, respectively. The small surface area and pore diameter of MSIAP may be due to the attachment of compound 4 to the mesoporous silica (FIG. 12).

Furthermore, IR spectroscopy was used for mesoporous silica and MSIAP to obtain more structural evidence of MSIAP. IR peaks of the mesoporous silica were found at 3450, 1658 and 1084 cm ^-1 , whereas in MSIAP at 3010, 2976, 2933, 1646, 1633, 1540, 1471, 1446, 1428 and 1382 cm ^-1 (FIG. 13) The new peaks derived from azo-pyridine 4 provide convincing evidence that the compound of formula 4 is attached to the surface of the mesoporous silica.

A fragment of Compound 4 ( m / z = 473.25) appears in the TOF-SIMS spectrum of MSIAP, providing evidence that Compound 4 was anchored to the surface of the silica particles (FIG. 14). Mesoporous silica nanoparticles were further identified by XPS before and after functionalization of compound 4 (FIG. 15). While the XPS spectrum of the fixed former methoxy early porous silica nanoparticles of the compound 4 are shown a Si _2P O _1S and the binding energy (Fig. 15a), the MSIAP the C and N _{1S 1S} for carbon and nitrogen appear (Fig. 15b).

Instability of suitable chlorides containing electrophiles hinders the development of neuroagent sensors in aqueous conditions (DCP hydrolyzes in minutes in water buffered to pH 7). One of the important advantages of MSIAP according to the present invention is that it is more practical in that it provides a neuroagent detector that is 100% water soluble and renewable. In the absence of neuroagonist analogs, the aqueous suspension of MSIAP was initially red. However, in the presence of DCP, the suspension of MSIAP changed color from red to yellow. On the other hand, no significant change in absorption was observed in parallel experiments using PMP, DMMP, TEP and TBP (Fig. 7).

From these spectroscopic changes, the inventors noted that MSIAP has high selectivity for DCP and similar spectroscopic reactions with the compound of formula 1 mentioned above. These results indicate that MSIAP is a novel organic-inorganic hybrid sensor for detecting neuronal DCP, which is highly applicable to the environmental field. The time-path estimate of the change in absorption of MSIAP at 405 nm (FIG. 16) shows that the absorption strength of MSIAP begins to increase immediately after the addition of DCP and nearly saturates after 30 seconds. In other words, this fast response time can provide a quick and convenient method for the quantification of DCP in aqueous solution.

To extend the above performance to the portable chemical sensor kit, disc-shaped pellets were prepared from MSIAP (FIG. 8). The color of the disc-shaped pellets prepared from MSIAP changed from red to yellow when immersed in a DCP (10 μM) aqueous solution. On the other hand, no significant changes in absorption were observed in parallel experiments such as PMP, DMMP, TEP and TBP solutions (10 μM). In addition, the solid absorption spectrum of the disk-shaped pellets of MSIAP including DCP was the same as that obtained from MSIAP dispersed in aqueous solution. Fluorescence changes after washing with disk-shaped pellets prepared from MSIAP and NaOH (10 μM) solution were found to be completely reversible. These results show that disk-shaped pellets prepared from MSIAP can be applied as portable chemical sensors for the detection of DCP in the biological and environmental fields.

The inventors also investigated the effect of pH on the spectral spectral behavior of MSIAP in the absence and presence of DCP because sensing must be practical over a wide pH range in order to ideally apply MSIAP to biological and environmental applications. Over the pH 3-9 range, MSIAP showed no absorption in the absence of DCP, while DCP addition showed almost the same absorption intensity at all pH values investigated within this pH range. From these results, it is clearly confirmed that MSIAP is suitable for use in a physiological environment ranging from pH 3-9 (FIG. 17).

We have synthesized chromogenic azo-pyridine (Formula 1 and Formula 4) and azo-pyridine (MSIAP) immobilized on recyclable mesoporous silica for detecting neuronal agent DCP. MSIAP has higher sensitivity and selectivity to DCP than other organophosphorus compounds in absorption change in 100% aqueous solution, and the color change can be easily visually recognized. Furthermore, MSIAP can not only detect toxins but also simultaneously dissolve the toxins into less toxic molecules by NaOH treatment and then regenerate them back to the original MSIAP. Therefore, it is very interesting to use azo-pyridine basic structure as an ICT perspective for DCP detection, and further, based on the present invention, it is possible to construct a more advanced system for other neurological agents through design modification.

Claims (8)

delete The azo-pyridine-based compound represented by the following formula (4) capable of selectively binding to a neuron agent and detecting the neuron agent through color change or NV / vis spectrum includes mesoporous silica nanoparticles immobilized on the surface. Chemical sensors for detecting nerve agents using:

Formula (4). 3. The method of claim 2,
The mesoporous silica nanoparticles in which the azo-pyridine-based compound represented by the formula (4) is fixed to the surface is in the form of a disc-shaped pellet, wherein the chemical sensor for detecting nerve agent. 3. The method of claim 2,
Wherein said chemical sensor is reusable after detecting the nerve agent. 5. The method of claim 4,
The chemical sensor is a nerve agent detection chemical sensor, characterized in that reusable by treatment with NaOH after separation of the nerve agent detection. A method for preparing a chemical sensor for detecting neurochemicals immobilized on mesoporous silica nanoparticles by dissolving azo-pyridine compound of formula (4) in toluene, adding mesoporous silica nanoparticles, and stirring under reflux conditions:

Formula (4). The method according to claim 6,
Method of producing a chemical sensor for detecting a neurochemical agent, characterized in that the compound of Formula (4) is prepared according to the following <Scheme 1>:
<Scheme 1>

Formula (1) Formula (4) The azo-pyridine-based compound represented by the following formula (4) comprises a mesoporous silica nanoparticle fixed on the surface of the nerve agent by using a color or UV / vis spectrum change of the chemical sensor for nerve agent detection How to detect:

Formula (4).

Images