CN101648908A - New water-soluble rare-earth organic chelate fluorescent probe and preparation method thereof - Google Patents

Abstract

The invention relates to a novel water-soluble rare earth organic chelate fluorescent probe and a preparation method thereof. The rare earth fluorescent probe consists of a bifunctional organic ligand 2,4,6-pyridine tricarboxylic acid (PTA) and SmCl ₃ and DyCl respectively. ₃ is prepared by coordination reaction in aqueous solution at a molar ratio of 3:1. The labeled probe has the advantages of strong fluorescence intensity, good water solubility and stability, easy biomolecular labeling, and little impact on the labeled biomolecules, and can be widely used in labeled immunoassays, nucleic acid assays, and disease diagnosis. , Bacteria, virus, microorganism detection, etc. In addition, because of its simple preparation process, low cost of raw materials and low investment in equipment, it also has practical development value in expanding its application range.

Description

Novel water-soluble rare earth organic chelate fluorescent probe and preparation method thereof

technical field

The invention belongs to the field of fluorescence immunoassay and biological detection, and specifically relates to a bifunctional organic ligand 2,4,6-pyridine tricarboxylic acid, its water-soluble rare earth fluorescent probes PTA:Sm ³⁺ and PTA:Dy ³⁺ and its Preparation.

technical background

Time-resolved immunoassay technique is a non-radioactive immunoassay technique established by Soini and Kojola in the early 1980s. Due to its high sensitivity, simple operation, stable tracer, no radioactive pollution, and the linear range of the standard curve is significantly better than Other technologies have attracted the most attention. As metal ions, rare earth elements are difficult to directly bind to organisms. Therefore, a chelating agent with dual functional groups is required for labeling. One end is connected to rare earth metal ions, and the other end is connected to free amino or carboxyl groups of organisms.

Rare earth fluorescent probes are mainly complexes of some rare earth elements Sm ³⁺ , Eu ³⁺ , Tb ³⁺ , and Dy ³⁺ , which can emit strong fluorescence under the irradiation of ultraviolet light. Rare earth complexes have the advantages of being used as probe labels for immunoassay: (1) The excitation spectrum band is wide, which is beneficial to increase the excitation energy and the specific activity of the marker; (2) The emission spectrum band is very narrow, which is conducive to reducing The background improves the resolution; (3) Stokes shift is large, which is beneficial to eliminate the interference of non-specific fluorescence; (4) the fluorescence lifetime is long, generally 100-1000μs, while the background fluorescence decay time is only 1-10ns, with a difference of 5 ~6 orders of magnitude, so time-resolved detection technology can be used to delay the measurement time and measure after the background fluorescence is completely attenuated. The measured fluorescence is the fluorescence of rare earth organic complexes, thereby eliminating the interference of protein background fluorescence; (5) rare earth The markers are relatively stable and can be stored for 1 to 2 years, overcoming the shortcomings of markers such as isotopes and enzymes.

Contents of the invention

One of the objectives of the present invention is to provide a bifunctional organic ligand 2,4,6-pyridinetricarboxylic acid PTA.

The second object of the present invention is to provide a water-soluble rare earth fluorescent probe PTA: Sm ³⁺ and PTA: Dy ³⁺ .

The third object of the present invention is to provide the preparation method of the above two kinds of water-soluble rare earth fluorescent probes.

To achieve the above object, the present invention adopts the following technical solutions:

A bifunctional organic ligand 2,4,6-pyridinetricarboxylic acid, characterized in that the structural formula of the organic ligand is:

A water-soluble rare earth fluorescent probe is characterized in that the rare earth fluorescent probe has the following two structures:

a. Sodium tris(2,4,6-pyridinetricarboxylic acid) samarium(III) acid binary complex, namely Na ₆ [Sm(PTA) ₃ ]·18H ₂ O, the specific structure is:

b. Sodium tris(2,4,6-pyridinetricarboxylic acid) dysdysprosium(III) binary complex, namely Na ₆ [Dy(PTA) ₃ ]·18H ₂ O, the specific structure is:

A method for preparing the above-mentioned water-soluble rare earth fluorescent probe is characterized in that the specific steps of the method are:

a. Mix 2,4,6-collidine and deionized water in a certain volume ratio, slowly add excess oxidant KMnO ₄ under stirring at room temperature, stir at room temperature for 10-15 hours, and stir at 40-50°C for 10-15 hours hours, filter while hot, wash with hot water, adjust the pH of the filtrate=1~2, white solid precipitates, filter, wash with cold water, slowly add concentrated HCl dropwise at 95°C until most of the white solid dissolves, filter while hot, 0~5 After standing at ℃, a white solid precipitated, and the bifunctional organic ligand 2,4,6-pyridinetricarboxylic acid was obtained;

b. The preparation method of water-soluble rare earth fluorescent probe: the PTA obtained in step a is dissolved in deionized water, and slowly added dropwise to SmCl ₃ aqueous solution or to DyCl ₃ aqueous solution; wherein the molar ratio of SmCl ₃ or DyCl ₃ to PTA is 1: 3 ; adjust the pH of the solution to 8-9, stir and react at 60°C for 8-12 hours, filter, leave at room temperature until it precipitates, wash, and dry in vacuum to obtain colorless crystals, which are water-soluble rare earth fluorescent probes Na ₆ [ Sm(PTA) ₃ ]·18H ₂ O or Na ₆ [Dy(PTA) ₃ ]·18H ₂ O.

The characteristics of the rare earth fluorescent probes PTA: Sm ³⁺ and PTA: Dy ³⁺ prepared by the present invention are:

a. It has good water solubility and stability;

b. It has strong fluorescence intensity and large fluorescence quantum yield;

c. It is easy to label biomolecules, has low toxicity, and has little impact on the labeled biomolecules;

d. The preparation process is simple, the cost is low, no organic solvent is used, and the environment will not be polluted.

Because the labeled probe has the advantages of strong fluorescence intensity, good water solubility and stability, easy biomolecular labeling, and little impact on the labeled biomolecules, it can be widely used in labeled immunoassays, nucleic acid assays, disease Diagnosis, detection of bacteria, viruses, microorganisms, etc. In addition, because of its simple preparation process, low cost of raw materials and low investment in equipment, it has practical significance in expanding its application range.

Description of drawings

Figure 1 shows the excitation and emission spectra of Na ₆ [Sm(PTA) ₃ ]·18H ₂ O and Na ₆ [Dy(PTA) ₃ ]·18H ₂ O in the solid state, where (a) is Na ₆ [Dy (PTA) ₃ ]·18H ₂ O excitation and emission spectra, (b) is the excitation and emission spectra of Na ₆ [Sm(PTA) ₃ ]·18H ₂ O (5300PC, high sensitivity, Na ₆ [Dy( The excitation and emission slits of PTA) ₃ ] are both 1.5 nm, and the excitation and emission slits of Na ₆ [Sm(PTA) ₃ ] are 3 nm and 1.5 nm, respectively).

Figure 2 shows the excitation and emission spectra of Na ₆ [Sm(PTA) ₃ ] and Na ₆ [Dy(PTA) ₃ ] in aqueous solution (concentration of 1.0×10 ^-4 mol/L), where (a) is The excitation and emission spectra of Na ₆ [Dy(PTA) ₃ ]·18H ₂ O, (b) is the excitation and emission spectra of Na ₆ [Sm(PTA) ₃ ]·18H ₂ O (5300PC, high sensitivity, Na The excitation and emission slits of the ₆ [Sm(PTA) ₃ ] solution are 10 nm and 5 nm, respectively, and the excitation and emission slits of the Na ₆ [Dy(PTA) ₃ ] solution are both 3 nm).

Detailed ways

The present invention will be further described below with examples, but not limited thereto.

Implementation example one: the specific preparation method of bifunctional organic ligand 2,4,6-pyridinetricarboxylic acid (PTA) is as follows:

Using 2,4,6-collidine (purity 99.99%) and KMnO ₄ (AR) as raw materials, mix 2,4,6-collidine and deionized water at a volume ratio of 1:20, room temperature Slowly add an appropriate amount of KMnO ₄ under stirring, then stir at room temperature for 10-15 hours, stir at 40-50°C for 10-15 hours, filter while hot, wash with hot water three times, adjust the filtrate to pH = 1 with concentrated HCl, a white solid precipitates, filter, Wash with cold water three times, slowly add concentrated HCl dropwise at 95°C until most of the white solid dissolves, filter while hot, and white solid (PTA) precipitates after standing at 0-5°C to obtain the target dihydrate product. Yield 54.5%, mp232.8 ℃ (decomposition), the structure of this compound is:

Elemental analysis: C ₈ H ₅ NO ₆ , experimental value (theoretical value) C: 38.62 (38.88), N: 5.64 (5.67), H: 3.65 (3.67), C/N: 6.85 (6.86).IR (KBr) /cm ^-1 : 3501.8 (v _COO-H ), 3157.7 (v _CH ), 1729.6 (v _C=O ), 1701.7 (v _C=O ), 1614.9 (v _{C=N, in Py} ), 1568.6 (v _{C =C,, in Py} ). ¹ H-NMR (DMSO, δ/ppm): 8.52 (s, 2H, py).

Example 2: The specific preparation method of Na ₆ [Sm(PTA) ₃ ]·18H ₂ O is as follows:

Dissolve 1.2mmol of PTA in a small amount of deionized water. After all the solids are dissolved, add deionized water to make the total volume of the system reach 50mL. Then 2.0 mL (0.4 mmol) of SmCl ₃ aqueous solution was slowly added to the above PTA solution, and the pH was adjusted to about 8 with 1.0 mol L ^-1 NaOH aqueous solution, stirred in a water bath at 60°C for 8 hours, and left at room temperature for several days to precipitate out Color crystal Na ₆ [Sm(PTA) ₃ ]·18H ₂ O, yield 60%.

Elemental analysis: SmNa ₆ C ₂₄ H ₄₂ N ₃ O ₃₆ (Na ₆ [Sm(PTA) ₃ ]·18H ₂ O), experimental value (theoretical value) C: 23.2 (23.31), N: 3.26 (3.40), H : 3.34 (3.42), C/N 7.12 (6.86).IR(KBr)/cm ^-1 : 3452.0 (v _COO-H ), 1633.3 (v _C=O ), 1558.9 (v _{C=N, in Py} ), 1437.4 (v _{C=C, in Py} ), 1374.6 (v _C=O ), 463.3 (v _Sm-O ).

Example 3: The specific preparation method of Na ₆ [Dy(PTA) ₃ ]·18H ₂ O is as follows:

This preparation method is basically the same as the preparation method of Na ₆ [Sm(PTA) ₃ ]·18H ₂ O in Example 2, except that the SmCl ₃ aqueous solution in Example 2 is replaced by DyCl ₃ aqueous solution.

Elemental analysis: DyNa ₆ C ₂₄ H ₄₂ N ₃ O ₃₆ (Na ₆ [Dy(PTA) ₃ ]·18H ₂ O), experimental value (theoretical value) C 22.43 (23.08), H 3.09 (3.39), N 3.35 ( 3.36), C/N 6.70(6.86).IR(KBr)/cm ^-1 : 3441.8(v _COO-H ), 1628.7(v _{as, COO-} ), 1367.4(v _{s, COO-} ), 1558.8(v _{C = N, in Py} ), 1437.5 (v _{C = C, in Py} ), 539.7 (v _Dy-O )

Figure 1 shows the excitation and emission spectra of Na ₆ [Sm(PTA) ₃ ]·18H ₂ O and Na ₆ [Dy(PTA) ₃ ]·18H ₂ O in solid state (5300PC, high sensitivity, Na ₆ [Dy The excitation and emission slits of (PTA) ₃ ] are both 1.5 nm, and the excitation and emission slits of Na ₆ [Sm(PTA) ₃ ] are 3 nm and 1.5 nm, respectively). Figure 2 shows the excitation and emission spectra of Na ₆ [Sm(PTA) ₃ ] and Na ₆ [Dy(PTA) ₃ ] in aqueous solution (concentration: 1.0×10 ^-4 mol/L) (5300PC, high sensitivity, The excitation and emission slits of the Na ₆ [Sm(PTA) ₃ ] solution are 10 nm and 5 nm, respectively, and the excitation and emission slits of the Na ₆ [Dy(PTA) ₃ ] solution are both 3 nm). It can be seen from the figure that the probe has a wide excitation spectrum band, which is beneficial to increase the excitation energy and the specific activity of the marker; the emission spectrum band is very narrow, which is beneficial to reduce the background and improve the resolution; It has strong fluorescence intensity, is easily soluble in water, and can exist stably in aqueous solution. It is expected to be widely used in the fields of marker immunoassay, nucleic acid determination, disease diagnosis, detection of bacteria, viruses, and microorganisms.

Claims (3)

1. a kind of bifunctional organic ligand 2,4,6-pyridinetricarboxylic acid, it is characterized in that the structural formula of this organic ligand is:

2. A water-soluble rare earth fluorescent probe adopts the bifunctional organic ligand 2,4,6-pyridine tricarboxylic acid according to claim 1 as a ligand, and is characterized in that the rare earth fluorescent probe has the following two structures : a. Sodium tris(2,4,6-pyridinetricarboxylic acid) samarium(III) acid binary complex, namely Na ₆ [Sm(PTA) ₃ ]·18H ₂ O, the specific structure is:

b. Sodium tris(2,4,6-pyridinetricarboxylic acid) dysdysprosium(III) binary complex, namely Na ₆ [Dy(PTA) ₃ ]·18H ₂ O, the specific structure is:

3. a method for preparing the water-soluble rare earth fluorescent probe according to claim 2, is characterized in that the concrete steps of the method are: a. Mix 2,4,6-collidine and deionized water in a certain volume ratio, slowly add excess oxidant KMnO ₄ under stirring at room temperature, stir at room temperature for 10-15 hours, and stir at 40-50°C for 10-15 hours hours, filter while hot, wash with hot water, adjust the pH of the filtrate=1~2, white solid precipitates, filter, wash with cold water, slowly add concentrated HCl dropwise at 95°C until most of the white solid dissolves, filter while hot, 0~5 After standing at ℃, a white solid precipitated, and the bifunctional organic ligand 2,4,6-pyridinetricarboxylic acid was obtained; b. The preparation method of water-soluble rare earth fluorescent probe: the PTA obtained in step a is dissolved in deionized water, and slowly added dropwise to SmCl ₃ aqueous solution or to DyCl ₃ aqueous solution; wherein the molar ratio of SmCl ₃ or DyCl ₃ to PTA is 1: 3 ; adjust the pH of the solution to 8-9, stir and react at 60°C for 8-12 hours, filter, leave at room temperature until it precipitates, wash, and dry in vacuum to obtain colorless crystals, which are water-soluble rare earth fluorescent probes Na ₆ [ Sm(PTA) ₃ ]·18H ₂ O or Na ₆ [Dy(PTA) ₃ ]·18H ₂ O.

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