CN113087753A - Benzopyrene-modified G-four-chain fluorescent probe and preparation method and application thereof - Google Patents
Benzopyrene-modified G-four-chain fluorescent probe and preparation method and application thereof Download PDFInfo
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- CN113087753A CN113087753A CN202110295806.8A CN202110295806A CN113087753A CN 113087753 A CN113087753 A CN 113087753A CN 202110295806 A CN202110295806 A CN 202110295806A CN 113087753 A CN113087753 A CN 113087753A
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- FMMWHPNWAFZXNH-UHFFFAOYSA-N Benz[a]pyrene Chemical compound C1=C2C3=CC=CC=C3C=C(C=C3)C2=C2C3=CC=CC2=C1 FMMWHPNWAFZXNH-UHFFFAOYSA-N 0.000 claims abstract description 60
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- C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
- C07H21/04—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids with deoxyribosyl as saccharide radical
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- G01N21/64—Fluorescence; Phosphorescence
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Abstract
The invention discloses a benzopyrene-modified G-quadruplex fluorescent probe and a preparation method and application thereof. The G-quadruplex DNA sequence modified by benzopyrene is adsorbed on the surface of the reduced graphene oxide, so that the fluorescence of the G-quadruplex DNA sequence modified by benzopyrene is quenched, and when the G-quadruplex DNA sequence is combined with thrombin, the adsorption capacity of the G-quadruplex DNA sequence modified by benzopyrene 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.
Description
Technical Field
The invention relates to the field of molecular probes, in particular to a benzopyrene 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 benzopyrene 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 benzopyrene modified G-quadruplex fluorescent probe, which comprises reduced graphene oxide and a benzopyrene 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 TTU1GGG TTA GGG TTA GGG TTA GGG-3 'or 5' -TTT TU1U1GGG TTA GGG TTA GGG TTA GGG-3', wherein,
optionally, the base sequence of the benzopyrene modified G-quadruplex DNA sequence is 5' -TTT TTU1 *GGG TTA GGG TTA GGG TTA GGG-3 'or 5' -TTT U1 *U1 *GGG TTA GGG TTA GGG TTA GGG-3', wherein,
the second aspect of the invention provides a preparation method of the benzopyrene modified G-four-chain 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 benzopyrene modified G-quadruplex DNA sequence by taking the G-quadruplex DNA sequence as a template through Click reaction;
and incubating the reduced graphene oxide and the G-quadruplex DNA sequence modified by the benzopyrene to obtain the G-quadruplex fluorescent probe modified by the benzopyrene.
Alternatively, the step of preparing the uracil phosphate derivative comprises:
providing 5-tripropargylamine-2 '-deoxyuridine, 4' -bismethoxytrityl chloride, 2-cyanoethyl-N, N-diisopropylphosphoroamidite;
reacting the 5-tripropargylamine-2 ' -deoxyuridine with the 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 benzopyrene-modified G-quadruplex DNA sequence by taking the G-quadruplex DNA sequence as a template through a Click reaction comprises:
combining the G-quadruplex DNA sequence with CuSO4TBTA solution, 1- (azidomethyl) pyrene solution, NaHCO3And mixing the aqueous solution and dimethyl sulfoxide, carrying out a light-resistant reaction, and separating and purifying to obtain the benzopyrene modified G-quadruplex DNA sequence.
Optionally, the step of incubating the reduced graphene oxide and the benzopyrene-modified G-quadruplex DNA sequence to obtain the benzopyrene-modified G-quadruplex fluorescent probe comprises:
adding the benzopyrene modified G-quadruplex DNA sequence 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 benzopyrene modified G-four-chain fluorescent probe.
In a third aspect of the invention, the invention provides an application of the benzopyrene modified G-four-chain fluorescent probe in thrombin detection.
Has the advantages that: the invention provides a benzopyrene modified G-four-chain fluorescent probe and a preparation method and application thereof, the benzopyrene-modified G-quadruplex fluorescent probe comprises reduced graphene oxide and a benzopyrene-modified G-quadruplex DNA sequence adsorbed on the surface of the reduced graphene oxide, since the G-quadruplex DNA sequence modified by benzopyrene is adsorbed on the surface of the reduced graphene oxide, so that the fluorescence of the G-quadruplex DNA sequence modified by benzopyrene is quenched, when the G-quadruplex DNA sequence is combined with thrombin, the adsorption capacity of the G-quadruplex DNA sequence modified by benzopyrene on the surface of the reduced graphene oxide is weakened, the fluorescence is enhanced, and the interference of a background signal is well avoided by the design of the G-quadruplex fluorescent probe modified by benzopyrene. 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.
Drawings
FIG. 1 is an AFM image of a benzopyrene-modified G-quadruplex fluorescent probe in an example of the present invention.
FIG. 2 is a graph showing the fluorescence quenching effect of a benzopyrene-modified G-four-stranded fluorescent probe with different concentrations of reduced graphene oxide in example 1 of the present invention.
FIG. 3 is a graph showing the fluorescence quenching effect of benzopyrene-modified G-four-stranded fluorescent probes with different concentrations of reduced graphene oxide in example 1 of the present invention.
FIG. 4 is a graph showing the fluorescence quenching effect of a benzopyrene-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 fluorescence response of different thrombin concentrations to a benzopyrene-modified G-quadruplex fluorescent probe in example 2 of the present invention.
Detailed Description
The invention provides a benzopyrene 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 more clear. 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 benzopyrene-modified G-four-chain fluorescent probe, which comprises reduced graphene oxide (rGO) and benzopyrene-modified G-four-chain fluorescent probe adsorbed on the surface of the reduced graphene oxideA strand DNA sequence. Hereinafter, the G-quadruplex DNA sequence is designated as GDNAAnd the G-quadruplex DNA sequence modified by benzopyrene is marked as G* DNA。
An AFM image of the benzopyrene-modified G-four-chain fluorescent probe is shown in FIG. 1. White region represents reduced graphene oxide surface adsorbed G* DNA，G* DNAIs less than 10 nm. G* DNAThe distribution on the surface of the reduced graphene oxide has no obvious selectivity.
In the benzopyrene modified G-four-chain fluorescent probe, the G is used* DNAAdsorbing on the surface of the reduced graphene oxide so that G* DNAThe fluorescence of the fluorescent probe is quenched, and when the G-four-chain fluorescent probe modified by benzopyrene is applied to thrombin detection, G is obtained* DNAAnd thrombin to bind said G* DNAThe adsorption capacity on the surface of the reduced graphene oxide is weakened, and the fluorescence is enhanced. The benzopyrene-modified G-four-chain fluorescent probe is a double-light-zone fluorescent probe (381nm,395nm and 478nm), and eximer (excimer) fluorescence 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* DNAThe ratio of the mass concentration to the molar concentration of (1-50) μ g/mL: 1. mu. mol/L, in this ratio range, G* DNAThe fluorescence of (a) can be quenched well.
In one embodiment, the G isDNAHas a base sequence of 5' -TTT TTU1GGG TTA GGG TTA GGG TTA GGG-3 'or 5' -TTT TU1U1GGG TTA GGG TTA GGG TTA GGG-3', wherein,
that is, G is the same as G in the present embodimentDNAThere are two kinds of base sequences of (1): is 5' -TTT TTU1GGG TTA GGG TTA GGG TTA GGG-3', another is 5' -TTT TU1U1GGG TTA GGG TTA GGG TTA GGG-3', for easy distinction, the base sequence is 5' -TTT TTU1GGG TTA GGG TTA GGG TTA GGG-3' DNA sequence GDNA-1The base sequence is 5' -TTT TU1U1GGG TTA GGG TTA GGG TTA GGG-3' DNA sequence GDNA-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* DNAHas a base sequence of 5' -TTT TTU1 *GGG TTA GGG TTA GGG TTA GGG-3 'or 5' -TTT U1 *U1 *GGG TTA GGG TTA GGG TTA GGG-3', wherein,
that is, G is the same as G in the present embodiment* DNAThere are two kinds of base sequences of (1): is 5' -TTT TTU1 *GGG TTA GGG TTA GGG TTA GGG-3', the other is 5' -TTT TU1 *U1 *GGG TTA GGG TTA GGG TTA GGG-3', for easy distinction, the base sequence is 5' -TTT TTU1 *GGG TTA GGG TTA GGG TTA GGG-3' benzopyrene modified DNA sequence is marked as G* DNA-1The base sequence is 5' -TTT T U1 *U1 *GGG TTA GGG TTA GGG TTA GGG-3' DNA sequence G* DNA-2。
The embodiment of the invention also provides a preparation method of the benzopyrene modified G-four-chain fluorescent probe, which comprises the following steps:
s1, providing uracil phosphate derivatives and reduced graphene oxide;
s2 Synthesis of G from the uracil phosphate derivative by DNA solid phase SynthesisDNA；
S3, andg isDNAAs a template, the G is prepared by Click reaction* DNA；
S4, reacting the reduced graphene oxide with the G* DNAAnd incubating to obtain the benzopyrene modified G-four-chain fluorescent probe.
In step S1, in one embodiment, the step of preparing the uracil phosphate derivative comprises:
s11, providing 5-tripropargylamine-2 '-deoxyuridine, 4' -bismethoxytrityl chloride, 2-cyanoethyl-N, N-diisopropylphosphorochloridite;
s12, reacting the 5-tripropargylamine-2 '-deoxyuridine with the 4,4' -bismethoxytrityl 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 chlorophosphite 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:
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 turn3)4Anhydrous Et3N, propargylamine, reacting at room temperature, concentrating the reaction product, and performing column Chromatography (CH)2Cl2MeOH in a volume ratio of 20:1) to obtain 5-tripropargylamine-2' -deoxyuridine as pale yellow solid foam after separation and purification.
And secondly, dissolving 5-tripropargylamine-2 '-deoxyuridine in anhydrous pyridine, drying by spinning, dissolving 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)2Cl2MeOH in a volume ratio of 30:1) to obtain a pale yellow solid foam of 5 '-DMT-5-tripropargylamine-2' -deoxyuridine after separation and purification.
In the third step, 5 '-DMT-5-tripropargylamine-2' -deoxyuridine was dissolved in anhydrous CH2Cl2In, add (iPr)2EtN and 2-cyanoethyl-N, N-diisopropylphosphoramidite chloride, at room temperature. Then using CH2Cl2Diluting, adding Na2CO3Extracting the solution, and extracting the organic layer with anhydrous Na2SO4Drying, concentrating, and performing column Chromatography (CH)2Cl2The volume ratio of MeOH is 40:1), and white solid foamed tripropargylamine-modified uracil phosphate derivatives are obtained after separation and purification.
In step S1, in one embodiment, the step of preparing 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 one embodiment, NaBH is employed at 70 deg.C4And reducing the graphene oxide.
In step S2, in one embodiment, the G is synthesized by H-8 type DNA solid phase synthesizer using the uracil phosphate derivative according to a previously designed base sequenceDNA。
In this step, the G is synthesizedDNAThe base U is the tripropargylamine-modified uracil phosphate derivative, and other bases (A, G, T) are all 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, the 5'-OH group is protected with 4,4' -dimethoxytriphenyl-phenyl (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 14 h. 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, G isDNAAs a template, the G is prepared by Click reaction* DNAComprises the following steps:
subjecting the G toDNAPlacing in a centrifuge tube, adding CuSO4TBTA solution, 1- (azidomethyl) pyrene solution, NaHCO3Reacting the water solution and dimethyl sulfoxide in the dark, separating and purifying to obtain the G* DNA。
In step S4, in one embodiment, the reduced graphene oxide is mixed with the G* DNAIncubating, wherein the step of preparing the benzopyrene modified G-four-chain fluorescent probe comprises the following steps:
subjecting the G to* DNAAdding 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 benzopyrene modified G-four-chain fluorescent probe.
Subjecting the G to* DNAAdding to buffer at room temperature for the first incubation in order to obtain G* DNACompletely dispersed in buffer and formed G quadruplexes.
In one embodiment, the buffer is Phosphate Buffered Saline (PBS) having Na as a major component2HPO4、KH2PO4NaCl and KCl.
The embodiment of the invention also provides application of the benzopyrene modified G-four-chain fluorescent probe in thrombin detection.
The G-quadruplex fluorescent probe modified by benzopyrene comprises reduced graphene oxide and a G-quadruplex DNA sequence modified by benzopyrene adsorbed on the surface of the reduced graphene oxide, and the G-quadruplex DNA sequence modified by benzopyrene is adsorbed on the surface of the reduced graphene oxide, so that the fluorescence of the G-quadruplex DNA sequence modified by benzopyrene is quenched, and when the G-quadruplex DNA sequence is combined with thrombin, the adsorption capacity of the G-quadruplex DNA sequence modified by benzopyrene 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
Benzopyrene-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 protection, and Pd (PPh) was added sequentially3)4(326mg, 0.28mmol), Anhydrous Et3N (688mg, 6.8mmol) and tripropargylamine (3.6g, 27.4mmol) were reacted at room temperature for 6 h. Concentrating the reaction product and then carrying out column Chromatography (CH)2Cl2MeOH in a 20:1 volume ratio) to give, after isolation and purification, 5-tripropargylamine-2' -deoxyuridine as a pale yellow solid foam (650mg, 64%).
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 and reacted at room temperature for 6 h. 5mL of methanol was added and stirring was continued for 0.5 h. Concentration of the reaction productPost column Chromatography (CH)2Cl2MeOH in a 30:1 volume ratio) to give, after isolation and purification, 5 '-DMT-5-tripropargylamine-2' -deoxyuridine as a pale yellow solid foam (600mg, 51%).
The resulting pale yellow solid foam, 5 '-DMT-5-tripropargylamine-2' -deoxyuridine (600mg, 0.91mmol), was dissolved in 10mL anhydrous CH under nitrogen2Cl2In, add (iPr)2EtN (300. mu.L, 1.8mmol) and 2-cyanoethyl-N, N-diisopropylphosphoramidite chloride (378. mu.L, 1.62mmol) were reacted at room temperature for 1 h. Then the reaction system uses CH2Cl2Diluted (50mL), and 50mL of Na was added2CO3Extracting the solution (5 wt%), and extracting the organic layer with anhydrous Na2SO4Drying, concentrating, and performing column Chromatography (CH)2Cl2MeOH in a volume ratio of 40:1) to yield, after isolation and purification, a foamed uracil phosphate derivative as a white solid (650mg, 85%).31P NMR(CDCl3):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 NaBH4And 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 phosphoramide, DMT-dG (dmf) -CE phosphoramide, DMT-dC (Ac) -CE phosphoramide and DMT-dT-CE phosphoramide, all available from Dironaceae Biotech Co., Ltd., Hebei) in accordance with 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 (1mL), sealed, and aminolyzed at 55 ℃ for 14 h. 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 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 to dryness. Dissolving in 0.5mL of distilled water, desalting with High Performance Liquid Chromatography (HPLC) to obtain DNA sequence with 5' -DMT removed, i.e. GDNA-1：TTT TTU1 GGG TTA GGG TTA GGG TTA GGG-3'。
4. Benzopyrene modified G-quadruplex DNA sequence (G)* DNA-1) Synthesis of (2)
Form GDNA-1(5260units) in a centrifuge tube, 50. mu.L of freshly prepared CuSO was added sequentially4TBTA (1: 1 by mass) solution (20mM solute in H solvent by volume)2O DMSO/t-BuOH 4:3:1 mixed solution), 60 μ L of 1- (azidomethyl) pyrene solution (solute 20mM, solvent in volume ratio H)2O dioxane (DMSO: 1: 1), 50 μ L of a newly prepared aqueous solution of tris (2-carboxyethyl) phosphine (solute 20mM), and 50 μ L of NaHCO3Aqueous solution (solute: 100mM), 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 DNA sequence modified by benzopyrene, namely G* DNA-1：5'-TTT TTU1 *GGG TTA GGG TTA GGG TTA GGG-3'。
5. Benzopyrene-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 (1mL) and incubated at room temperature for 0.5 h; 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 benzopyrene modified G-four-chain fluorescent probe which is marked as rGO/G* DNA-1。
G-four-chain fluorescent probe (rGO/G) modified by benzopyrene with different rGO concentrations* DNA-1) Fluorescence intensity measurements were performed.
Example 2
Benzopyrene-modified G-four-chain 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 phosphoramide, DMT-dG (dmf) -CE phosphoramide, DMT-dC (Ac) -CE phosphoramide and DMT-dT-CE phosphoramide, all available from Dironaceae Biotech Co., Ltd., Hebei) in accordance with 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 (1mL), sealed, and aminolyzed at 55 ℃ for 14 h. 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. GDNA-2：5'-TTT TU1U1 GGG TTA GGG TTA GGG TTA GGG-3'。
2. Benzopyrene modified G-quadruplex DNA sequence (G)* DNA-2) Synthesis of (2)
Form GDNA-2(5260units) were placed in centrifuge tubes, and 50. mu.L of freshly prepared CuSO was added to each tube4TBTA (1: 1 by mass) solution (20mM in H2O DMSO/t-BuOH 4:3: 1), 60 μ L of 1- (azidomethyl) pyrene solution (20mM, solvent in volume ratio H)2Dioxane (DMSO) ═ 1:1: 1), 50 μ L of newly formulated aqueous tris (2-carboxyethyl) phosphine (solute 20mM), 50 μ L of NaHCO (NaHCO)3Aqueous solution (solute: 100mM), 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 U1 *U1 *GGG TTA GGG TTA GGG TTA GGG-3'。
3. Synthesis of benzopyrene-modified G-four-chain fluorescent probe
G is to be* DNA-2(1. mu. mol) was added to 20mM PBS buffer (1mL) and incubated at room temperature for 0.5 h; 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 benzopyrene modified G-four-chain fluorescent probe which is marked as rGO/G* DNA-2。
Benzopyrene 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-2Detection of Bovine Serum Albumin (BSA)
In the presence of rGO/G * DNA2 to 1mL of 20mM PBS buffer, solutions of bovine serum albumin (0units/mL, 50units/mL, 75units/mL, 100units/mL, 125units/mL, 250units/mL, 375units/mL, 500units/mL) at various concentrations were added, and the mixture was incubated at room temperature for 0.5h with thrombin. Finally, the G-four-stranded fluorescent probes (rGO/G) modified by benzopyrene 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 the DNA sequence modified by benzopyrene. In FIGS. 2 and 3, the fluorescence quenching effect on benzopyrene-modified DNA sequences was enhanced with increasing rGO content.
Differential Thrombin concentration vs rGO/G* DNA-2The response of the fluorescence signal is shown in FIG. 5. In FIG. 5, the fluorescence curve is from bottom to top, and the concentration of thrombin is from 0units/mL to 500units/mL, and it can be seen from the graph that when the G-quadruplex DNA sequence is combined with thrombin in the presence of thrombin, the adsorption capacity of the G-quadruplex DNA sequence modified by benzopyrene on the surface of the reduced graphene oxide is reduced, and the fluorescence is enhanced.
In conclusion, the invention provides a benzopyrene modified G-four-chain fluorescent probe and a preparation method and application thereof, the benzopyrene-modified G-quadruplex fluorescent probe comprises reduced graphene oxide and a benzopyrene-modified G-quadruplex DNA sequence adsorbed on the surface of the reduced graphene oxide, since the G-quadruplex DNA sequence modified by benzopyrene is adsorbed on the surface of the reduced graphene oxide, so that the fluorescence of the G-quadruplex DNA sequence modified by benzopyrene is quenched, when the G-quadruplex DNA sequence is combined with thrombin, the adsorption capacity of the G-quadruplex DNA sequence modified by benzopyrene on the surface of the reduced graphene oxide is weakened, the fluorescence is enhanced, and the interference of a background signal is well avoided by the design of the G-quadruplex fluorescent probe modified by benzopyrene. 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 (10)
1. A G-quadruplex fluorescent probe modified by benzopyrene is characterized by comprising reduced graphene oxide and a G-quadruplex DNA sequence modified by benzopyrene adsorbed on the surface of the reduced graphene oxide.
2. The benzopyrene-modified G-quadruplex fluorescent probe according to claim 1, wherein the G-quadruplex DNA isThe base sequence of the sequence is 5' -TTT TTU1GGG TTA GGG TTA GGG TTA GGG-3 'or 5' -TTT TU1U1GGG TTA GGG TTA GGG TTA GGG-3', wherein,
3. the benzopyrene-modified G-quadruplex fluorescent probe according to claim 1, characterized in that the base sequence of the benzopyrene-modified G-quadruplex DNA sequence is 5' -TTT TTU1 *GGG TTA GGG TTA GGG TTA GGG-3 'or 5' -TTT U1 *U1 *GGG TTA GGG TTA GGG TTA GGG-3'，
Wherein,
4. the method for preparing the benzopyrene-modified G-four-strand fluorescent probe according to any one of claims 1 to 3, 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 benzopyrene modified G-quadruplex DNA sequence by taking the G-quadruplex DNA sequence as a template through a click reaction;
and incubating the reduced graphene oxide and the G-quadruplex DNA sequence modified by the benzopyrene to obtain the G-quadruplex fluorescent probe modified by the benzopyrene.
5. The method for preparing a benzopyrene-modified G-four-strand fluorescent probe according to claim 4, wherein the step of preparing the uracil phosphate derivative comprises:
providing 5-tripropargylamine-2 '-deoxyuridine, 4' -bismethoxytrityl chloride, 2-cyanoethyl-N, N-diisopropylphosphoroamidite;
reacting the 5-tripropargylamine-2 '-deoxyuridine with the 4,4' -bismethoxytrityl 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.
6. The method for preparing a benzopyrene-modified G-four-chain fluorescent probe according to claim 5, wherein the 5-tripropargylamine-2 '-deoxyuridine is prepared from 5-iodo-2' -deoxyuridine and tripropargylamine.
7. The method for preparing a benzopyrene-modified G-four-chain fluorescent probe according to claim 4, wherein the step of preparing the reduced graphene oxide comprises the steps of:
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.
8. The method for preparing the benzopyrene-modified G-quadruplex fluorescent probe according to claim 4, wherein the step of preparing the benzopyrene-modified G-quadruplex DNA sequence by using the G-quadruplex DNA sequence as a template and using a click reaction comprises the following steps:
combining the G-quadruplex DNA sequence with CuSO4TBTA solution, 1- (azidomethyl) pyrene solution, NaHCO3And mixing the aqueous solution and dimethyl sulfoxide, carrying out a light-resistant reaction, and separating and purifying to obtain the benzopyrene modified G-quadruplex DNA sequence.
9. The method for preparing the benzopyrene-modified G-quadruplex fluorescent probe according to claim 4, wherein the step of incubating the reduced graphene oxide with the benzopyrene-modified G-quadruplex DNA sequence to obtain the benzopyrene-modified G-quadruplex fluorescent probe comprises:
adding the benzopyrene modified G-quadruplex DNA sequence 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 benzopyrene modified G-four-chain fluorescent probe.
10. Use of the benzopyrene-modified G-quadruplex fluorescent probe according to any one of claims 1 to 3 for detecting thrombin.
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| CN114540012A (en) * | 2022-02-23 | 2022-05-27 | 华南师范大学 | Fluorescent molecular probe and application thereof in preparation of cancer cell detection reagent |
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| CN102864143A (en) * | 2012-09-26 | 2013-01-09 | 北京科技大学 | Pyrene-marked single-chain DNA (deoxyribonucleic acid) fluorescent probe and preparation method thereof |
| CN110257482A (en) * | 2019-07-16 | 2019-09-20 | 上海纳米技术及应用国家工程研究中心有限公司 | A kind of blood coagulation enzyme assay method based on aptamer and telomere enzymatic amplification |
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| CN114540012A (en) * | 2022-02-23 | 2022-05-27 | 华南师范大学 | Fluorescent molecular probe and application thereof in preparation of cancer cell detection reagent |
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