CN113087753B - Pyrene-modified G-four-chain fluorescent probe and preparation method and application thereof - Google Patents

Pyrene-modified G-four-chain fluorescent probe and preparation method and application thereof Download PDF

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CN113087753B
CN113087753B CN202110295806.8A CN202110295806A CN113087753B CN 113087753 B CN113087753 B CN 113087753B CN 202110295806 A CN202110295806 A CN 202110295806A CN 113087753 B CN113087753 B CN 113087753B
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熊海
赵龙
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Abstract

The invention discloses a pyrene modified G-quadruplex fluorescent probe and a preparation method and application thereof. The pyrene-modified G-quadruplex DNA sequence is adsorbed on the surface of the reduced graphene oxide, so that the fluorescence of the pyrene-modified G-quadruplex DNA sequence is quenched, when the G-quadruplex DNA sequence is combined with thrombin, the adsorption capacity of the pyrene-modified G-quadruplex DNA sequence on the surface of the reduced graphene oxide is weakened, the fluorescence is enhanced, and the pyrene-modified G-quadruplex fluorescent probe well avoids the interference of a background signal. The method is simple, efficient, good in selectivity, wide in dynamic response range and convenient to use of related instruments, is a detection means with higher sensitivity in the existing chemiluminescence detection method, and has important potential application value.

Description

Pyrene-modified G-four-chain fluorescent probe and preparation method and application thereof
Technical Field
The invention relates to the field of molecular probes, in particular to a pyrene-modified G-four-chain fluorescent probe and a preparation method and application thereof.
Background
Cancer is a leading cause of death in humans, and idiopathic venous thrombosis is associated with occult cancer, and thrombin generated during thrombosis enhances the malignant phenotype of tumor cells.
Thrombin can activate the adhesion of tumor cells with platelets, endothelial cells and subendothelial matrix proteins, promote the growth of tumor cells, increase the seeding and spontaneous metastasis of tumor cells, and stimulate the angiogenesis of tumor cells. Therefore, detecting the thrombin content in tumor cells is of great importance for studying the proliferation process of cancer cells and diagnosing cancer.
Disclosure of Invention
In view of the defects of the prior art, the invention aims to provide a pyrene modified G-four-chain fluorescent probe, a preparation method and application thereof, and aims to detect thrombin.
The technical scheme of the invention is as follows:
the invention provides a pyrene modified G-quadruplex fluorescent probe, which comprises reduced graphene oxide and a pyrene modified G-quadruplex DNA sequence adsorbed on the surface of the reduced graphene oxide.
Optionally, the base sequence of the G-quadruplex DNA sequence is 5' -TTT TTU 1 GGG TTA GGG TTA GGG TTA GGG-3 'or 5' -TTT TU 1 U 1 GGG TTA GGG TTA GGG TTA GGG-3', wherein,
Figure GDA0003769578370000021
optionally, the base sequence of the pyrene modified G-quadruplex DNA sequence is 5' -TTT TTU 1 * GGG TTA GGG TTA GGG TTA GGG-3 'or 5' -TTT U 1 * U 1 * GGG TTA GGG TTA GGG TTA GGG-3', wherein,
Figure GDA0003769578370000022
the second aspect of the invention provides a preparation method of the pyrene-modified G-quadruplex fluorescent probe, which comprises the following steps:
providing a uracil phosphate derivative and reduced graphene oxide;
synthesizing the G-quadruplex DNA sequence by a DNA solid phase synthesizer by using the uracil phosphate derivative;
preparing the pyrene-modified G-quadruplex DNA sequence by using the G-quadruplex DNA sequence as a template through Click reaction;
and incubating the reduced graphene oxide and the pyrene-modified G-quadruplex DNA sequence to obtain the pyrene-modified G-quadruplex fluorescent probe.
Optionally, the step of preparing the uracil phosphate derivative comprises:
providing 5-tripropargylamine-2 '-deoxyuridine, 4,4' -bismethoxytrityl chloride, 2-cyanoethyl-N, N-diisopropylphosphorochloridite;
reacting the 5-tripropargylamine-2 ' -deoxyuridine with 4,4' -bismethoxytrityl chloride, and separating and purifying to obtain 5' -DMT-5-tripropargylamine-2 ' -deoxyuridine (wherein DMT is short for 4,4' -bismethoxytrityl);
and (3) reacting the 5 '-DMT-5-tripropargylamine-2' -deoxyuridine with the 2-cyanoethyl-N, N-diisopropyl chlorophosphite amide, and separating and purifying to obtain the uracil phosphate derivative.
Alternatively, the 5-tripropargylamine-2 '-deoxyuridine is prepared from 5-iodo-2' -deoxyuridine and tripropargylamine.
Optionally, the step of preparing the reduced graphene oxide comprises:
preparing graphene oxide by using graphite as a raw material by using a Hummer method;
and reducing the graphene oxide to obtain the reduced graphene oxide.
Optionally, the step of preparing the pyrene-modified G-quadruplex DNA sequence by a Click reaction with the G-quadruplex DNA sequence as a template comprises:
combining the G-quadruplex DNA sequence with CuSO 4 TBTA solution, 1- (azidomethyl) pyrene solution, naHCO 3 And mixing the aqueous solution and dimethyl sulfoxide, carrying out light-resistant reaction, and separating and purifying to obtain the pyrene-modified G-quadruplex DNA sequence.
Optionally, the step of incubating the reduced graphene oxide and the pyrene-modified G-quadruplex DNA sequence to obtain the pyrene-modified G-quadruplex fluorescent probe comprises:
adding the pyrene modified G-quadruplex DNA sequence into a buffer solution to perform first hatching at room temperature;
and then adding reduced graphene oxide, fully dispersing, and carrying out secondary incubation at room temperature to obtain the pyrene-modified G-four-chain fluorescent probe.
In a third aspect of the invention, the invention provides an application of the pyrene modified G-quadruplex fluorescent probe in thrombin detection.
Has the advantages that: the invention provides a pyrene modified G-quadruplex fluorescent probe and a preparation method and application thereof, wherein the pyrene modified G-quadruplex fluorescent probe comprises reduced graphene oxide and a pyrene modified G-quadruplex DNA sequence adsorbed on the surface of the reduced graphene oxide, and the pyrene modified G-quadruplex DNA sequence is adsorbed on the surface of the reduced graphene oxide, so that the fluorescence of the pyrene modified G-quadruplex DNA sequence is quenched, and when the G-quadruplex DNA sequence is combined with thrombin, the adsorption capacity of the pyrene modified G-quadruplex DNA sequence on the surface of the reduced graphene oxide is weakened, and the fluorescence is enhanced. The method is simple, efficient, good in selectivity, wide in dynamic response range and convenient to use of related instruments, is a detection means with higher sensitivity in the existing chemiluminescence detection method, and has important potential application value.
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FIG. 1 is an AFM image of pyrene-modified G-quadruplex fluorescent probe in an example of the present invention.
FIG. 2 is a graph showing the fluorescence quenching effect of pyrene-modified G-four-stranded fluorescent probes at different concentrations of reduced graphene oxide in example 1 of the present invention.
FIG. 3 is a graph showing the fluorescence quenching effect of pyrene-modified G-four-stranded fluorescent probes at different concentrations of reduced graphene oxide in example 1 of the present invention.
FIG. 4 is a graph showing the fluorescence quenching effect of the pyrene-modified G-four-stranded fluorescent probe with reduced graphene oxide concentration of 30. Mu.g/mL in example 2 of the present invention.
FIG. 5 is a graph showing the results of the fluorescence response of the G-quadruplex fluorescent probe modified with pyrene by the concentrations of different thrombins in example 2 of the present invention.
Detailed Description
The invention provides a pyrene modified G-four-chain fluorescent probe and a preparation method and application thereof, and the invention is further described in detail below in order to make the purpose, technical scheme and effect of the invention clearer and clearer. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
Graphene is a novel nano material, the internal structure of the novel nano material is very unique, the novel nano material has a hexagonal lattice structure, every two carbon atoms form a unit cell, each carbon atom and adjacent atoms can form 3 sigma bonds and one pi bond, the surface of graphene has super-strong adsorption capacity, and by utilizing the performance, graphene can adsorb dye molecules (or biomolecules marked by fluorescent groups) and perform efficient energy transfer to cause fluorescent signal quenching of the dye molecules. Due to the unique property of graphene, the graphene can be applied to the field of detection of biomolecules.
Based on the above, the embodiment of the invention provides a pyrene modified G-quadruplex fluorescent probe, which comprises reduced graphene oxide (rGO) and a pyrene modified G-quadruplex DNA sequence adsorbed on the surface of the reduced graphene oxide. Hereinafter, the G-quadruplex DNA sequence is designated as G DNA And the pyrene modified G-quadruplex DNA sequence is marked as G * DNA 。
An AFM image of the pyrene-modified G-four-chain fluorescent probe is shown in FIG. 1. White region represents reduced graphene oxide surface adsorbed G * DNA ，G * DNA Is less than 10nm. G * DNA The distribution on the surface of the reduced graphene oxide has no obvious selectivity.
In the pyrene-modified G-four-strand fluorescent probe, the G is used * DNA Adsorbing on the surface of the reduced graphene oxide so that G * DNA The fluorescence of (a) is quenched, and when the pyrene-modified G-four-chain fluorescent probe is applied to thrombin detection, G is detected * DNA And thrombin to bind said G * DNA The adsorption capacity on the surface of the reduced graphene oxide is weakened, and the fluorescence is enhanced. The pyrene-modified G-four-chain fluorescent probe is a double-optical-zone fluorescent probe (381nm, 395nm and 478nm), and eximer (excimer) fluorescence of the pyrene-modified G-four-chain fluorescent probe has better penetrability (the longer the wavelength is, the better the penetrating effect is).
In one embodiment, the reduced graphene oxide is reacted with G * DNA The mass concentration to molar concentration ratio of (1-50) μ g-mL: 1. Mu. Mol/L, in this ratio range, G * DNA The fluorescence of (a) can be quenched well.
In one embodiment, the G is DNA Has a base sequence of 5' -TTT TTU 1 GGG TTA GGG TTA GGG TTA GGG-3 'or 5' -TTT TU 1 U 1 GGG TTA GGG TTA GGG TTA GGG-3', wherein,
Figure GDA0003769578370000061
that is, G is the same as G in the present embodiment DNA There are two kinds of base sequences of (1): is 5' -TTT TTU 1 GGG TTA GGG TTA GGG TTA GGG-3', another is 5' -TTT TU 1 U 1 GGG TTA GGG TTA GGG TTA GGG-3', for ease of distinction, the base sequence is 5' -TTT TTU 1 GGG TTA GGG TTA GGG TTA GGG-3' DNA sequence G DNA-1 The base sequence is 5' -TTT TU 1 U 1 GGG TTA GGG TTA GGG TTA GGG-3' DNA sequence G DNA-2 。
In this example, the base sequence is a human telomerase DNA sequence, which can form a G quadruplex and recognize thrombin.
In one embodiment, the G is * DNA Has a base sequence of 5' -TTT TTU 1 * GGG TTA GGG TTA GGG TTA GGG-3 'or 5' -TTT U 1 * U 1 * GGG TTA GGG TTA GGG TTA GGG-3', wherein,
Figure GDA0003769578370000062
that is, G is the same as G in the present embodiment * DNA There are two kinds of base sequences of (1): is 5' -TTT TTU 1 * GGG TTA GGG TTA GGG TTA GGG-3', another is 5' -TTT T U 1 * U 1 * GGG TTA GGG TTA GGG TTA GGG-3', for ease of distinction, the base sequence is 5' -TTT TTU 1 * GGG TTA GGG TTA GGG TTA GGG-3' pyrene-modified DNA sequence was designated G * DNA-1 The base sequence is 5' -TTT T U 1 * U 1 * GGG TTA GGG TTA GGG TTA GGG-3' DNA sequence is denoted G * DNA-2 。
The embodiment of the invention also provides a preparation method of the pyrene modified G-four-chain fluorescent probe, which comprises the following steps:
s1, providing a uracil phosphate derivative and reduced graphene oxide;
s2, synthesizing the G by using the uracil phosphate derivative through a DNA solid phase synthesizer DNA ；
S3, with the G DNA As a template, the G is prepared by Click reaction * DNA ；
S4, reacting the reduced graphene oxide with the G * DNA And (4) incubating to obtain the pyrene modified G-four-strand fluorescent probe.
In step S1, in one embodiment, the step of preparing the uracil phosphate derivative comprises:
s11, providing 5-tripropargylamine-2 '-deoxyuridine, 4,4' -bismethoxytrityl chloride, 2-cyanoethyl-N, N-diisopropylphosphoroamidite chloride;
s12, reacting the 5-tripropargylamine-2 '-deoxyuridine with 4,4' -bis-methoxy trityl chloride, and separating and purifying to obtain 5 '-DMT-5-tripropargylamine-2' -deoxyuridine;
s13, reacting the 5 '-DMT-5-tripropargylamine-2' -deoxyuridine with the 2-cyanoethyl-N, N-diisopropyl chlorophosphorous amide, and separating and purifying to obtain the uracil phosphate derivative.
In step S11, in one embodiment, the 5-tripropargylamine-2 '-deoxyuridine is prepared from 5-iodo-2' -deoxyuridine and tripropargylamine.
The specific preparation process of the uracil phosphate derivative is as follows:
Figure GDA0003769578370000081
the following will describe in detail the preparation process of the uracil phosphate derivative in conjunction with the specific preparation process of the uracil phosphate derivative as described above:
the first step is to dissolve 5-iodo-2' -deoxyuridine and CuI in DMF, and then add Pd (PPh) in turn 3 ) 4 Anhydrous Et 3 N, propargylamine, reacting at room temperature, concentrating the reaction product, and performing column Chromatography (CH) 2 Cl 2 MeOH volume ratio of 20).
And secondly, dissolving 5-tripropargylamine-2 '-deoxyuridine in anhydrous pyridine, drying by spinning, dissolving the solution in the anhydrous pyridine, adding 4,4' -bismethoxytrityl chloride, reacting at room temperature, adding methanol, and continuing stirring. Concentrating the reaction product and then carrying out column Chromatography (CH) 2 Cl 2 MeOH volume ratio of 30).
In the third step, 5 '-DMT-5-tripropargylamine-2' -deoxyuridine was dissolved in anhydrous CH 2 Cl 2 In, add (iPr) 2 EtN and 2-cyanoethyl-N, N-diisopropylphosphoramidite chloride, at room temperature. Then using CH 2 Cl 2 Diluting, adding Na 2 CO 3 Extracting the solution, and extracting the organic layer with anhydrous Na 2 SO 4 Drying, concentrating, and performing column Chromatography (CH) 2 Cl 2 The volume ratio of MeOH is 40).
In step S1, in one embodiment, the step of preparing the reduced graphene oxide includes:
preparing graphene oxide by using graphite as a raw material by using a Hummer method;
and reducing the graphene oxide to obtain the reduced graphene oxide.
In an implementation methodIn the formula, naBH is adopted at 70 DEG C 4 And reducing the graphene oxide.
In step S2, in one embodiment, the G is synthesized by an H-8 type DNA solid phase synthesizer using the uracil phosphate derivative according to a previously designed base sequence DNA 。
In this step, the G is synthesized DNA In the raw material, the base U adopts the tripropargylamine modified uracil phosphate derivative, and other bases (A, G, T) adopt commercial products.
In the solid phase synthesis of DNA sequences, certain free groups on the nucleotide to be activated are first protected so that the reaction proceeds in the designed direction, and 5'-OH is protected with 4,4' -Dimethoxytriphenyl (DMT) and the protected groups do not participate in the subsequent reactions. Thus, dichloroacetic acid is used to remove DMT from DNA sequences after they have been synthesized.
Therefore, the step S2 further includes the steps of:
taking out CPG powder linked with DNA in the DNA synthetic column, placing in a glass bottle, adding ammonia water, sealing, and performing ammonolysis at 55 deg.C for 14h. And (3) taking the supernatant, concentrating, drying, adding distilled water for dissolving, and separating and purifying by using a high performance liquid chromatograph to obtain the 5' -DMT protected DNA sequence. Placing the DNA into a centrifuge tube, cooling to 0 ℃, adding dichloroacetic acid aqueous solution until the DNA is completely dissolved, adding triethylamine, concentrating and drying. Adding distilled water for dissolving, and desalting by using a High Performance Liquid Chromatography (HPLC) to obtain the DNA sequence with the 5' -DMT removed.
In step S3, in one embodiment, the G DNA As a template, the G is prepared by Click reaction * DNA Comprises the following steps:
subjecting the G to DNA Placing in a centrifuge tube, adding CuSO 4 TBTA solution, 1- (azidomethyl) pyrene solution, naHCO 3 Reacting the water solution and dimethyl sulfoxide in the dark, separating and purifying to obtain the G * DNA 。
In step S4, in one embodiment, the reduction is performedGraphene oxide and the G * DNA Incubating, wherein the step of preparing the pyrene-modified G-four-strand fluorescent probe comprises the following steps:
subjecting the G to * DNA Adding the mixture into a buffer solution to perform first incubation at room temperature;
and then adding reduced graphene oxide, fully dispersing, and carrying out secondary incubation at room temperature to obtain the pyrene-modified G-four-chain fluorescent probe.
Subjecting the G to * DNA Adding to buffer at room temperature for the first incubation in order to obtain G * DNA Completely dispersed in the buffer and formed a G quadruplex.
In one embodiment, the buffer is Phosphate Buffered Saline (PBS) having Na as a major component 2 HPO 4 、KH 2 PO 4 NaCl and KCl.
The embodiment of the invention also provides application of the pyrene modified G-four-chain fluorescent probe in thrombin detection.
The pyrene-modified G-quadruplex fluorescent probe comprises reduced graphene oxide and a pyrene-modified G-quadruplex DNA sequence adsorbed on the surface of the reduced graphene oxide, and the pyrene-modified G-quadruplex DNA sequence is adsorbed on the surface of the reduced graphene oxide, so that the fluorescence of the pyrene-modified G-quadruplex DNA sequence is quenched, and when the G-quadruplex DNA sequence is combined with thrombin, the adsorption capacity of the pyrene-modified G-quadruplex DNA sequence on the surface of the reduced graphene oxide is weakened, the fluorescence is enhanced, and the detection of the thrombin is realized.
The invention is further illustrated by the following specific examples.
Example 1
Pyrene-modified G-four-chain fluorescent probe (rGO/G) * DNA-1 ) Synthesis of (2)
1. Synthesis of uracil phosphate derivatives
5-iodo-2' -deoxyuridine (1.0g, 2.82mmol) and CuI (108mg, 0.56mmol) were dissolved in 15mL of DMF under nitrogen protectionSequentially adding Pd (PPh) 3 ) 4 (326mg, 0.28mmol), anhydrous Et 3 N (688mg, 6.8mmol) and tripropargylamine (3.6g, 27.4mmol) were reacted at room temperature for 6 hours. Concentrating the reaction product and then carrying out column Chromatography (CH) 2 Cl 2 MeOH volume ratio of 20).
5-Tripropargylamine-2 '-deoxyuridine (650mg, 1.78mmol) was dissolved in 20mL of anhydrous pyridine, dried, and further dissolved in 20mL of anhydrous pyridine under nitrogen protection, and 4,4' -bismethoxytrityl chloride (812mg, 2.38mmol) was added to the solution to react at room temperature for 6 hours. 5mL of methanol was added and stirring was continued for 0.5h. Concentrating the reaction product and then performing column Chromatography (CH) 2 Cl 2 MeOH, 30 vol) to give 5 '-DMT-5-tripropargylamine-2' -deoxyuridine as a pale yellow solid foam after isolation and purification (600mg, 51%).
The resulting pale yellow solid foam, 5 '-DMT-5-tripropargylamine-2' -deoxyuridine (600mg, 0.91mmol), was dissolved in 10mL anhydrous CH under nitrogen protection 2 Cl 2 In, add (iPr) 2 EtN (300. Mu.L, 1.8 mmol) and 2-cyanoethyl-N, N-diisopropylphosphoramidite chloride (378. Mu.L, 1.62 mmol) were reacted at room temperature for 1h. Then the reaction system uses CH 2 Cl 2 Diluted (50 mL), and 50mL of Na was added 2 CO 3 Extracting the solution (5 wt%), and extracting the organic layer with anhydrous Na 2 SO 4 Drying, concentrating, and performing column Chromatography (CH) 2 Cl 2 MeOH, 40 vol) to give a white solid, foamy uracil phosphate derivative (650mg, 85%) after isolation and purification. 31 P NMR(CDCl 3 ):150.25，149.6。
2. Synthesis of reduced graphene oxide (rGO)
Preparing graphene oxide by using graphite as a raw material by using a Hummer method;
50mL of 0.5mg/mL graphene oxide solution and 50mL of 150mM/L NaBH 4 And mixing the solutions, and reducing for 1h at 70 ℃ to obtain rGO.
3. G-quadruplex DNA sequence (G) DNA-1 ) Synthesis of (2)
A G-quadruplex DNA sequence was synthesized in a solid phase using uracil phosphate derivatives and other commercially available basic phosphates (DMT-dA (Bz) -CE phosphate, DMT-dG (dmf) -CE phosphate, DMT-dC (Ac) -CE phosphate, DMT-dT-CE phosphate, all available from Hippon Dinaraceae Biotech Co., ltd.) according to a previously designed base sequence using an H-8 type DNA synthesizer. After the synthesis, the CPG powder linked with DNA in the DNA synthesis column was taken out, placed in a glass bottle, added with ammonia (1 mL), sealed, and aminolyzed at 55 ℃ for 14h. And (3) taking the supernatant, concentrating, drying, adding 0.5mL of distilled water for dissolving, and separating and purifying by using a high performance liquid chromatograph (HPLC, acetonitrile/acetic acid-triethylamine buffer) to obtain the DMT protected DNA sequence of the 5' -position.
The mixture was placed in a centrifuge tube, cooled to 0 deg.C, 0.3mL of 2.5% aqueous dichloroacetic acid was added until the DNA was completely dissolved (less than 5 minutes), 0.25mL of triethylamine was added, and the mixture was concentrated and dried. Dissolving in 0.5mL of distilled water, desalting with High Performance Liquid Chromatography (HPLC) to obtain DNA sequence with 5' -DMT removed, i.e. G DNA-1 ：TTT TTU 1 GGG TTA GGG TTA GGG TTA GGG-3'。
4. Pyrene-modified G-quadruplex DNA sequence (G) * DNA-1 ) Synthesis of (2)
Form G DNA-1 (5260 units) in a centrifuge tube, 50. Mu.L of freshly prepared CuSO was added sequentially 4 TBTA (mass ratio 1:1) solution (solute 20mM, solvent volume ratio H) 2 DMSO: t-BuOH =4, 1), 60. Mu.L of a 1- (azidomethyl) pyrene solution (solute 20mM, solvent in volume ratio H 2 O dioxane DMSO =1 in 1), 50 μ L of freshly prepared aqueous tris (2-carboxyethyl) phosphine (solute 20 mM), 50 μ L of NaHCO 3 Aqueous solution (solute: 100 mM), 50. Mu.L DMSO, and reaction for 12h in the dark. Concentrating, drying, adding 0.5mL of distilled water for dissolving and filtering, and performing separation and purification by using a High Performance Liquid Chromatograph (HPLC) to obtain a benzopyrene modified DNA sequence, namely G * DNA-1 ：5'-TTT TTU 1 * GGG TTA GGG TTA GGG TTA GGG-3'。
5. Pyrene-modified G-four-chain fluorescent probe (rGO/G) * DNA-1 ) Synthesis of (2)
G is to be * DNA-1 (1. Mu. Mol) was added to 20mM PBS buffer (1 mL) and incubated at room temperature for 0.5h; then adding rGO (referring to the concentration of the rGO in the added system) with the concentration of 1 mu G/ml, 2 mu G/ml, 10 mu G/ml, 20 mu G/ml, 30 mu G/ml, 40 mu G/ml and 50 mu G/ml respectively to fully disperse the rGO, and incubating for 0.5h at room temperature to form a stable pyrene modified G-four-chain fluorescent probe which is marked as rGO/G * DNA-1 。
Test 1
G-four-chain fluorescent probe (rGO/G) modified by pyrene with different rGO concentrations * DNA-1 ) Fluorescence intensity measurements were performed.
Example 2
Pyrene-modified G-four-strand fluorescent probe (G) * DNA-2 ) Synthesis of (2)
The synthesis of uracil phosphate derivatives and the synthesis of reduced graphene oxide were the same as in example 1.
1. G-quadruplex DNA sequence (G) DNA-2 ) Synthesis of (2)
A G-quadruplex DNA sequence was synthesized in a solid phase using uracil phosphate derivatives and other commercially available basic phosphates (DMT-dA (Bz) -CE phosphate, DMT-dG (dmf) -CE phosphate, DMT-dC (Ac) -CE phosphate, DMT-dT-CE phosphate, all available from Hippon Dinaraceae Biotech Co., ltd.) according to a previously designed base sequence using an H-8 type DNA synthesizer. After the synthesis, the CPG powder linked with DNA in the DNA synthesis column was taken out, placed in a glass bottle, added with ammonia (1 mL), sealed, and aminolyzed at 55 ℃ for 14h. And (3) taking the supernatant, concentrating, drying, adding 0.5mL of distilled water for dissolving, and separating and purifying by using a high performance liquid chromatograph (HPLC, acetonitrile/acetic acid-triethylamine buffer) to obtain the DMT protected DNA sequence of the 5' -position.
The DNA fragment was placed in a centrifuge tube, cooled to 0 ℃ and added with 0.3mL of an aqueous solution of dichloroacetic acid (mass fraction: 2.5%) until the DNA was completely dissolved (less than 5 minutes), added with 0.25mL of triethylamine, concentrated and dried. Dissolving in 0.5mL of distilled water, desalting with High Performance Liquid Chromatography (HPLC) to obtain DNA sequence with 5' -DMT removed, i.e. G DNA-2 ：5'-TTT TU 1 U 1 GGG TTA GGG TTA GGG TTA GGG-3'。
2. Pyrene-modified G-quadruplex DNA sequence (G) * DNA-2 ) Synthesis of (2)
Form G DNA-2 (5 260units) were placed in centrifuge tubes, and 50. Mu.L of freshly prepared CuSO was added to each tube 4 -TBTA (Mass ratio 1:1) solution (20 mM, solvent in volume ratio H 2 O DMSO: t-BuOH =4, mixed solution of 1) 2 O dioxane: DMSO =1 mixed solution) 50 μ L of freshly prepared aqueous solution of tris (2-carboxyethyl) phosphine (solute 20 mM), 50 μ L of NaHCO 3 Aqueous solution (solute: 100 mM), 50. Mu.L DMSO, and reaction for 12h in the dark. Concentrating, drying, adding 0.5mL of distilled water for dissolving and filtering, and separating and purifying by using a High Performance Liquid Chromatography (HPLC) to obtain a benzopyrene modified DNA sequence G * DNA-2 ：5'-TTT T U 1 * U 1 * GGG TTA GGG TTA GGG TTA GGG-3'。
3. Synthesis of pyrene-modified G-four-chain fluorescent probe
G is to be * DNA-2 (1. Mu. Mol) was added to 20mM PBS buffer (1 mL) and incubated at room temperature for 0.5h; then adding rGO (referring to the concentration of the rGO in the added system) with the concentration of 1 mu G/mL, 2 mu G/mL, 10 mu G/mL, 20 mu G/mL, 30 mu G/mL, 40 mu G/mL and 50 mu G/mL respectively to fully disperse the rGO, and incubating for 0.5h at room temperature to form a stable pyrene modified G-four-chain fluorescent probe which is marked as rGO/G * DNA-2 。
Test 2
Pyrene modified G-four-chain fluorescent probe (rGO/G) with rGO concentration of 30 mu G/mL * DNA-2 ) Fluorescence intensity measurements were performed.
Test 3
rGO/G * DNA-2 Detection of Bovine Serum Albumin (BSA)
In the presence of rGO/G * DNA -2 to 1mL of 20mM PBS buffer solution, bovine serum albumin solutions (0 units/mL, 50units/mL, 75units/mL, 100units/mL, 125units/mL, 250units/mL, 375units/mL, 500 units/mL) at different concentrations were addedThe albumin contains thrombin and is incubated for 0.5h at room temperature. Finally, pyrene-modified G-four-stranded fluorescent probes (rGO/G) were measured separately * DNA-2 ) Fluorescence intensity at different concentrations of bovine serum albumin.
The test results were as follows:
as shown in fig. 2, 3 and 4, it can be seen that reduced graphene oxide can quench the fluorescence of pyrene-modified DNA sequences. In FIGS. 2 and 3, the fluorescence quenching effect on pyrene-modified DNA sequences was enhanced with increasing rGO content.
Differential Thrombin concentration vs rGO/G * DNA-2 The response of the fluorescence signal is shown in FIG. 5. In FIG. 5, the fluorescence curve from bottom to top corresponds to a thrombin concentration of 0units/mL to 500units/mL, and it can be seen from the graph that when the G-quadruplex DNA sequence binds to thrombin in the presence of thrombin, the adsorption capacity of the pyrene-modified G-quadruplex DNA sequence on the surface of reduced graphene oxide is reduced, and the fluorescence is enhanced.
In summary, the pyrene-modified G-quadruplex fluorescent probe and the preparation method and application thereof provided by the invention comprise reduced graphene oxide and a pyrene-modified G-quadruplex DNA sequence adsorbed on the surface of the reduced graphene oxide, and the pyrene-modified G-quadruplex DNA sequence is adsorbed on the surface of the reduced graphene oxide, so that the fluorescence of the pyrene-modified G-quadruplex DNA sequence is quenched, and when the G-quadruplex DNA sequence is combined with thrombin, the adsorption capacity of the pyrene-modified G-quadruplex DNA sequence on the surface of the reduced graphene oxide is weakened, and the fluorescence is enhanced. The method is simple, efficient, good in selectivity, wide in dynamic response range and convenient to use of related instruments, is a detection means with higher sensitivity in the existing chemiluminescence detection method, and has important potential application value.
It is to be understood that the invention is not limited to the examples described above, but that modifications and variations may be effected thereto by those of ordinary skill in the art in light of the foregoing description, and that all such modifications and variations are intended to be within the scope of the invention as defined by the appended claims.

Claims (8)

1. A pyrene-modified G-quadruplex fluorescent probe is characterized by comprising reduced graphene oxide and a pyrene-modified G-quadruplex DNA sequence adsorbed on the surface of the reduced graphene oxide;
the base sequence of the pyrene modified G-quadruplex DNA sequence is 5' -TTT TTU 1 * GGG TTA GGG TTA GGG TTA GGG-3 'or 5' -TTT U 1 * U 1 * GGG TTA GGG TTA GGG TTA GGG-3'，
Wherein the content of the first and second substances,
Figure FDA0003769578360000011
2. the method for preparing the pyrene-modified G-quadruplex fluorescent probe according to claim 1, comprising the steps of:
providing a uracil phosphate derivative and reduced graphene oxide;
synthesizing the G-quadruplex DNA sequence by a DNA solid phase synthesizer by using the uracil phosphate derivative;
preparing the pyrene-modified G-quadruplex DNA sequence by using the G-quadruplex DNA sequence as a template through a click reaction;
and incubating the reduced graphene oxide and the pyrene-modified G-quadruplex DNA sequence to obtain the pyrene-modified G-quadruplex fluorescent probe.
3. The method for preparing a pyrene-modified G-quadruplex fluorescent probe according to claim 2, wherein the step of preparing the uracil phosphate derivative comprises:
providing 5-tripropargylamine-2 '-deoxyuridine, 4,4' -bismethoxytrityl chloride, 2-cyanoethyl-N, N-diisopropylphosphorochloridite;
reacting the 5-tripropargylamine-2 '-deoxyuridine with the 4,4' -bis-methoxy trityl chloride, and separating and purifying to obtain 5 '-DMT-5-tripropargylamine-2' -deoxyuridine;
and (3) reacting the 5 '-DMT-5-tripropargylamine-2' -deoxyuridine with the 2-cyanoethyl-N, N-diisopropyl chlorophosphite amide, and separating and purifying to obtain the uracil phosphate derivative.
4. The method for preparing a pyrene-modified G-quadruplex fluorescent probe according to claim 3, wherein the 5-tripropargylamine-2 '-deoxyuridine is prepared from 5-iodo-2' -deoxyuridine and tripropargylamine.
5. The method for preparing a pyrene-modified G-quadruplex fluorescent probe according to claim 2, wherein the step of preparing reduced graphene oxide comprises:
preparing graphene oxide by using graphite as a raw material by using a Hummer method;
and reducing the graphene oxide to obtain the reduced graphene oxide.
6. The method for preparing a pyrene-modified G-quadruplex fluorescent probe according to claim 2, wherein the step of preparing the pyrene-modified G-quadruplex DNA sequence by click reaction using the G-quadruplex DNA sequence as a template comprises:
combining the G-quadruplex DNA sequence with CuSO 4 TBTA solution, 1- (azidomethyl) pyrene solution, naHCO 3 And mixing the aqueous solution and dimethyl sulfoxide, carrying out a light-resistant reaction, and separating and purifying to obtain the pyrene-modified G-quadruplex DNA sequence.
7. The method for preparing a pyrene-modified G-quadruplex fluorescent probe according to claim 2, wherein the step of incubating the reduced graphene oxide with the pyrene-modified G-quadruplex DNA sequence to obtain the pyrene-modified G-quadruplex fluorescent probe comprises:
adding the pyrene modified G-quadruplex DNA sequence into a buffer solution to perform first hatching at room temperature;
and then adding reduced graphene oxide, fully dispersing, and carrying out secondary incubation at room temperature to obtain the pyrene-modified G-four-chain fluorescent probe.
8. Use of the pyrene-modified G-quadruplex fluorescent probe of claim 1 in the preparation of a thrombin detection fluorescent probe.
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