CN113607947B - Method for detecting alpha fetoprotein by using aptamer based on aggregation-induced emission mark and azide functionalized single-walled carbon nanotube - Google Patents
Method for detecting alpha fetoprotein by using aptamer based on aggregation-induced emission mark and azide functionalized single-walled carbon nanotube Download PDFInfo
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- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57476—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving oncofetal proteins
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- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
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Abstract
The invention belongs to the technical fields of biochemistry, environment detection and food safety, and particularly relates to a protein detection method, in particular to a method for quenching fluorescence based on click reaction of azide functionalized single-walled carbon nanotubes SWNTs and fluorescent-labeled aptamer under the condition of no copper (I) catalysis. After adding alpha fetoprotein AFP, DNA preferentially binds specifically with the protein, so that the conformation of the aptamer is changed, and fluorescence is recovered. In addition, the nucleic acid aptamer with the end modified with octyne benzene ring DBCO can be specifically combined with AFP to induce conformational change, so that the detection sensitivity of the AFP is improved.
Description
Technical Field
The invention belongs to the technical fields of biochemistry, environment detection and food safety, and relates to a protein detection method, in particular to a detection method of alpha fetoprotein by using an aptamer based on aggregation-induced emission marks and an azide functionalized single-walled carbon nanotube.
Background
Alpha-fetoprotein (AFP) is a protein biological marker commonly used for liver cancer, and can diagnose related cancers more accurately. AFP is usually produced by the yolk sac of a 7-8 month fetus, and a plasma protein with a molecular weight of about 70kDa is secreted from the liver, and its content gradually decreases as the fetus is born and grows. In serum from healthy people, AFP concentrations below 25ng/mL were barely detectable, but AFP levels were statistically significantly elevated to 500ng/mL in nearly 75% of hepatocellular carcinoma (hepatocellular carcinoma, HCC) patients. High levels of AFP in adult blood may indicate the presence of certain types of cancer, in particular HCC, gastric cancer, pancreatic cancer, ovarian cancer or testicular cancer, and thus elevated AFP concentrations in adult serum are widely recognized clinically as early indicators of HCC or intradermal sinus tumors. Aggregation-induced emission (AIE) is a class of luminescent molecules that are non-emissive in dilute solutions, but when the molecule is in an aggregated state, the emission is greatly enhanced. Based on this characteristic feature, AIE can act as a fluorescent indicator of an aptamer biosensor, producing an "on" fluorescent signal of higher sensitivity and accuracy for the sensing platform. The combination of nanomaterials and biomolecules plays an increasingly important role in the field of biosensors, and Carbon Nanotubes (CNTs) as a novel nanomaterial have been widely studied and applied because of their unique photoelectric and mechanical properties. Furthermore, studies have shown that SWNTs are often used as good quenchers for constructing nanosensors because they are able to act as energy acceptors for FRET in fluorescent aptamer sensors.
Click chemistry is a new combinatorial chemistry approach based on carbon-heteroatom bond (C-X-C) synthesis and by means of these reactions to obtain molecular diversity simply and efficiently, which represents the reaction as copper-catalyzed azido-alkynyl husign cycloaddition. However, since the toxicity of copper (I) as a metal catalyst often results in degradation of part of the nucleotides and denaturation of proteins, and even limits to some extent practical applications in living cells or organisms, the drawbacks involved in CuAAC reactions can be overcome by reactions without copper variants, i.e. "copper-free click chemistry".
Disclosure of Invention
Aiming at the defects in the prior art, the reaction rate of the cyclooctyne reagent and the azide is relatively close to the metal catalysis rate, so that the DBCO modified nucleic acid aptamer and the azide functionalization of the SWNTs are used for carrying out copper-free click chemistry, the excellent performances of the FRET of AIE and the SWNTs are fully utilized, and the occurrence of false positive signals is reduced, so that the AFP is detected.
A method for detecting alpha fetoprotein based on aggregation-induced emission labeled aptamer and azide-functionalized single-walled carbon nanotubes, comprising the steps of:
(1) Modifying octyne benzene ring DBCO at the tail end of the DNA sequence, and dissolving the modified DNA sequence in ultrapure water;
(2)SWNTs-N 3 preparation of the conjugate:
uniformly dispersing SWNTs into a solution by using an ultrasonic cleaner, adding NaOH and chloroacetic acid into the initial SWNTs solution, performing ultrasonic treatment in a water bath, washing, centrifuging, purifying and drying to obtain carboxyl functionalized SWNTs, namely SWCNTs-COOH, after the-OH on the surface of the SWNTs is converted into-COOH;
dissolving the obtained SWCNTs-COOH in methanol solution, performing ultrasonic treatment, and mixing SWNTs-COOH and N 3 -PEG-NH 2 Adding into HEPES buffer solution, mixing thoroughly, reacting at room temperature, passing through-COOH and-NH 2 Covalent bonding to form azide-functionalized single-walled carbon nanotubes SWNTs-N 3 ；
(3) Fluorescence acquisition: adding the TPETA solution and the modified DNA in the step (1) into a brown centrifuge tube containing PBS to form a DNA sequence with fluorescence;
(4) Quenching: after incubating the DNA sequence with fluorescence of step (3) at room temperature, SWNTs-N prepared in step (2) is added 3 Reaction overnight, SWNTs-N 3 Click reaction is carried out on DBCO functional groups modified at the tail end of the DNA, and fluorescence is quenched through FRET;
(5) Obtaining the linear relation between AFP and its aptamer:
adding a series of AFP solutions with known concentration into the solution obtained in the step (4), reacting at normal temperature, wherein the aptamer, namely DNA in the solution obtained in the step (4), is subjected to specific binding with AFP to induce conformational change, so that the DNA part with TPETA fluorescence is far away from SWNTs-N 3 To recover fluorescence to a certain extent, and after the reaction is finished, measuring a fluorescence value, and preparing a corresponding linear relation graph according to the measured fluorescence value and AFP;
(6) AFP detection:
adding a certain amount of AFP with unknown concentration into the solution obtained in the step (4), reacting at normal temperature, measuring the fluorescence recovery value after the reaction is finished, and obtaining the concentration of the AFP according to the linear relation obtained in the step (5).
In step (1), the sequence of the DNA is: DNA:5'-DBCO-GTG ACG CTC CTA ACG CTG ACT CAG GTG CAG TTC TCG ACT CGG TCT TGA TGT GGG TCC TGT CCG TCC GAACCAATC-3'; the final concentration of the modified DNA sequence was 50nM after dissolving in ultrapure water.
In the step (2), the concentration of the initial SWNTs solution is 2mg/mL; the initial SWNTs solution, naOH and chloroacetic acid were used in a 5mL ratio: 0.6g:0.5g;
the volume percentage concentration of the methanol solution is 10%;
in the SWNTs-COOH methanol solution, the concentration of SWNTs-COOH is 2mg/mL;
SWNTs-COOH solution, N 3 -PEG-NH 2 And HEPES buffer solution at a dose ratio of 5mL:100mg:10mL, wherein the HEPES buffer solution was at a concentration of 0.1M, pH 7.4.
In the step (2), the reaction time at room temperature is 12h.
In step (3), the final concentration of TPETA in the DNA sequence with fluorescence was 10. Mu.M and the final concentration of DNA was 30nM.
In step (4), the incubation time was 15min and the final concentration of SWNTs-N3 was 20. Mu.g/mL; the reaction temperature is 4 ℃;
in step (5), the concentration of the AFP solution is 0.1-80 ng/mL. The reaction time at normal temperature is 30 minutes; the volume ratio of the solution obtained in the step (4) to the AFP solution was 300. Mu.L: 1.5 to 3 mu L.
In the step (6), the reaction time at normal temperature was 30 minutes.
The beneficial effects of the invention are as follows:
(1) The single-walled carbon nanotube nanomaterial is easy to obtain, and has the advantages of simple method, low cost and stable property.
(2) The copper-free click chemistry used in the invention can eliminate the toxicity generated by the metal catalyst, thereby avoiding the defects of degradation of partial nucleotide, protein denaturation, living cells or organisms and the like.
(3) The invention uses the tetraphenyl ethylene mono-quaternary ammonium salt (TPETA) active biological probe with AIE characteristic to react with the aptamer chain to form a DNA sequence (TPETA-DNA) with fluorescence, generates an 'open' fluorescence signal with higher sensitivity and accuracy for a sensing platform, and provides a new exploration direction for a label-free fluorescent aptamer sensor.
(4) The invention designs a nucleic acid aptamer with a tail end modified with octyne benzene ring (DBCO) and azide functionalized single-walled carbon nano (SWNTs-N) 3 ) Click reaction occurs and fluorescence quenching by FRETAnd (5) extinguishing. The principle is simple, the operation is convenient, the time and the labor are saved, and the DNA part with TPETA fluorescence is utilized to be far away from SWNTs-N 3 Thereby causing fluorescence to be recovered, to detect the AFP content.
Drawings
FIG. 1 is a technical scheme for detecting alpha fetoprotein according to the present invention;
fig. 2: kinetics of TPETA molecules. Variation of fluorescence intensity over the time range measured for different concentrations of TPETA molecules.
Fig. 3: optimal concentration selection map for SWNT-N3. Fig. 4: optimal concentration selection map of nucleic acid aptamer, fluorescence recovery intensity changes of different concentration nucleic acid aptamer solutions in reaction environment.
Fig. 5: sensitivity graph of aptamer, fluorescence intensity and fluorescence recovery (F/F) after addition of AFP at different concentrations to aptamer 0 -1) variation.
Fig. 6: selectivity profile of aptamer, change in fluorescence intensity after addition of different kinds of proteins to aptamer solution.
Detailed Description
The invention is further illustrated by the following examples, which are provided to illustrate the invention and are not intended to limit the scope of the invention.
Example 1:
(1) The synthesis of specific DNA sequences is shown below:
DNA：5'-DBCO-GTG ACG CTC CTA ACG CTG ACT CAG GTG CAG TTC TCG ACT CGG TCT TGATGT GGG TCC TGT CCG TCC GAACCAATC-3'
the 5' -end of the DNA sequence was modified with octyne benzene ring, and the final concentration of the modified DNA sequence was 50nM after dissolving in ultrapure water.
(2)SWNTs-N 3 Preparation of the conjugate:
SWNTs were first uniformly dispersed using an ultrasonic cleaner and formulated as a 2mg/mL solution. After adding 0.6g NaOH and 0.5g chloroacetic acid into 5mL initial SWNTs solution respectively, carrying out ultrasonic treatment in a water bath, and after the surface of the SWNTs is converted into-COOH, carrying out washing and centrifugation for multiple times to further purify, and drying the solution to obtain carboxyl functionalized SWNTs (SWCNTs-COOH).
The carboxyl functionalized single-walled carbon nanotubes obtained were dissolved in 10% methanol solution and sonicated, 5mL SWNTs-COOH (2 mg/mL) and 100mg N 3 -PEG-NH 2 Added into 10mL HEPES buffer solution (0.1M, pH 7.4) and mixed thoroughly, and reacted at room temperature for 12h through-COOH and-NH 2 Covalent bonding to form azide-functionalized single-walled carbon nanotubes (SWNTs-N 3 )。
(3) Fluorescence acquisition: adding the TPETA solution and the modified DNA in the step (1) into a brown centrifuge tube containing PBS to form a DNA sequence with fluorescence; TPETA final concentration was 10. Mu.M and DNA final concentration was 30nM;
(4) Quenching: after incubating the DNA sequence with fluorescence of step (3) at room temperature for 15min, SWNTs-N prepared in step (2) is added 3 Reaction at 4℃overnight, SWNTs-N 3 Click reaction is carried out on DBCO functional groups modified at the tail end of the DNA, and fluorescence is quenched through FRET; the final concentration of SWNTs-N3 was 20. Mu.g/mL;
(5) Obtaining the linear relation between AFP and its aptamer:
adding 1.5-3 mu L of a series of AFP solutions with known concentration into 300 mu L of the solution obtained in the step (4), reacting for 30 minutes at normal temperature, measuring a fluorescence value after the reaction is finished, and preparing a corresponding linear relation graph according to the measured fluorescence value and the AFP, wherein the concentration of the AFP solution is 0.1-80 ng/mL;
(6) AFP detection:
adding a certain amount of AFP with unknown concentration into the solution obtained in the step (4), reacting for 30 minutes at normal temperature, measuring the fluorescence recovery value after the reaction is finished, and obtaining the concentration of the AFP according to the linear relation obtained in the step (5).
SWNT-N 3 Selection of optimal concentration: adding nano material SWNTs-N with gradient concentration into 30nM DNA system 3 SWNTs-N with optimal quenching rate is selected 3 The concentration required is about 80% fluorescence quenching, where the final choice is 20. Mu.g/mL SWNT-N 3 。
Selection of optimal concentration of DNA solution: different concentrations of DNA (10, 20, 30, 40 and 50 nM) were added to the DNA containing 20. Mu.gSWNT-N/mL 3 In the system of (2), AFP was added at an equal concentration, and the result showed that the fluorescence increase rate caused by the solution added with 30nM DNA was highest.
AFP (0.1, 0.5, 0.8, 5, 8, 10, 20, 50 and 80 ng/mL) was added at various concentrations to a sample containing fluorescent molecule TPETA (10. Mu.M), nanomaterial SWNT-N 3 (20. Mu.g/mL) and aptamer (30 nM) in PBS buffer (10 mM, pH=7.4), the mixture was mixed well and incubated at room temperature for 30min to measure the fluorescence spectrum and record, and the recovery of fluorescence was examined.
Several analogues (BSA, CEA, HSA, igG and Thrombin) close to the AFP of the test object were selected to evaluate the selectivity of the sensor under the same system, and under the same conditions of (4).
FIG. 2 is a kinetic diagram of TPETA molecules. The fluorescent intensity of TPETA molecules with different concentrations in the measured time range has no obvious change, which indicates that the fluorescent molecules can keep good stability in a PBS buffer system.
FIG. 3 is a graph of optimal concentration selections for SWNT-N3. SWNT-N 3 The concentration was in the range of 5-20. Mu.g/mL with SWNT-N 3 Concentration increase, F/F 0 -1 is also increasing, but SWNT-N 3 The concentration is continuously increased on the basis of 20 mug/mL to obtain F/F 0 -1 in ever decreasing results, SWNT-N 3 The optimal concentration is 20. Mu.g/mL.
FIG. 4 is a graph showing the optimal concentration selection of aptamer DNA. The fluorescence recovery intensity of the aptamer solutions with different concentrations in the reaction environment is changed, and the optimal concentration is 30nM.
FIG. 5 is a plot of the sensitivity of nucleic acid aptamer DNA. Fluorescence intensity and fluorescence recovery (F/F) after adding AFP at different concentrations to the aptamer solution 0 -1) variation. The AFP concentration added was gradually increased from 0 to a final concentration of 80ng/mL.
FIG. 6 is a diagram showing the selectivity of nucleic acid aptamer DNA. The fluorescence intensity was varied after addition of different kinds of proteins to the aptamer solution, wherein the final concentration of each protein was 10ng/mL.
Claims (8)
1. Use of an aptamer based on aggregation-induced emission labeling and an azide-functionalized single-walled carbon nanotube in the preparation of a sensor for detecting alpha fetoprotein, characterized in that the detection method comprises the steps of:
(1) Modifying octyne benzene ring DBCO at the tail end of the DNA sequence, and dissolving the modified DNA sequence in ultrapure water;
(2)SWNTs-N 3 preparation of the conjugate:
uniformly dispersing SWNTs into a solution by using an ultrasonic cleaner, adding NaOH and chloroacetic acid into the initial SWNTs solution, performing ultrasonic treatment in a water bath, washing, centrifuging, purifying and drying to obtain carboxyl functionalized SWNTs, namely SWCNTs-COOH, after the-OH on the surface of the SWNTs is converted into-COOH;
dissolving the obtained SWCNTs-COOH in methanol solution, performing ultrasonic treatment, and mixing SWNTs-COOH and N 3 -PEG-NH 2 Adding into HEPES buffer solution, mixing thoroughly, reacting at room temperature, passing through-COOH and-NH 2 Covalent bonding to form azide-functionalized single-walled carbon nanotubes SWNTs-N 3 ；
(3) Fluorescence acquisition: adding the TPETA solution and the modified DNA in the step (1) into a brown centrifuge tube containing PBS to form a DNA sequence with fluorescence;
(4) Quenching: after incubating the DNA sequence with fluorescence of step (3) at room temperature, SWNTs-N prepared in step (2) is added 3 Reaction overnight, SWNTs-N 3 Click reaction is carried out on DBCO functional groups modified at the tail end of the DNA, and fluorescence is quenched through FRET;
(5) Obtaining the linear relation between AFP and its aptamer:
adding a series of AFP solutions with known concentrations into the solution obtained in the step (4), reacting at normal temperature, measuring a fluorescence value after the reaction is finished, and preparing a corresponding linear relation diagram according to the measured fluorescence value and the AFP;
(6) AFP detection:
adding a certain amount of AFP with unknown concentration into the solution obtained in the step (4), reacting at normal temperature, measuring the fluorescence recovery value after the reaction is finished, and obtaining the concentration of the AFP according to the linear relation obtained in the step (5).
2. The use according to claim 1, wherein in step (1), the DNA sequence is: DNA:5'-DBCO-GTG ACG CTC CTA ACG CTG ACT CAG GTG CAG TTC TCG ACT CGG TCT TGA TGT GGG TCC TGT CCG TCC GAA CCA ATC-3'; the final concentration of the modified DNA sequence was 50nM after dissolving in ultrapure water.
3. The method according to claim 1, wherein in step (2),
the concentration of the initial SWNTs solution was 2mg/mL;
the initial SWNTs solution, naOH and chloroacetic acid were used in a 5mL ratio: 0.6g:0.5g;
the volume percentage concentration of the methanol solution is 10%;
in the SWNTs-COOH methanol solution, the concentration of SWNTs-COOH is 2mg/mL;
SWNTs-COOH solution, N 3 -PEG-NH 2 And HEPES buffer solution at a dose ratio of 5mL:100mg:10mL, wherein the HEPES buffer solution was at a concentration of 0.1M, pH 7.4.
4. The method according to claim 1, wherein in step (2), the reaction time at room temperature is 12 hours.
5. The use according to claim 1, wherein in step (3) the final concentration of TPETA in the fluorescent DNA sequence is 10 μm and the final concentration of DNA is 30nM.
6. The use according to claim 1, wherein in step (4) the incubation time is 15min and the final concentration of swnts-N3 is 20 μg/mL; the reaction temperature was 4 ℃.
7. The use according to claim 1, wherein in step (5) the concentration of AFP solution is 0.1-80 ng/mL and the reaction time at ambient temperature is 30 minutes; the volume ratio of the solution obtained in the step (4) to the AFP solution was 300. Mu.L: 1.5 to 3 mu L.
8. The method according to claim 1, wherein in step (6), the reaction time at room temperature is 30 minutes.
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