CN113607947A - Detection method of alpha fetoprotein by aptamer and azide functionalized single-walled carbon nanotube based on aggregation-induced emission marker - Google Patents
Detection method of alpha fetoprotein by aptamer and azide functionalized single-walled carbon nanotube based on aggregation-induced emission marker Download PDFInfo
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Abstract
The invention belongs to the technical field of biochemistry, environmental detection and food safety, and particularly relates to a protein detection method, in particular to a click reaction between azide-functionalized single-walled carbon nanotubes (SWNTs) and a fluorescence-labeled aptamer under the condition of no copper (I) catalysis, and fluorescence is quenched. After the alpha fetoprotein AFP is added, the DNA preferentially and specifically binds with the protein, so that the aptamer conformation is changed, and the fluorescence is recovered. In addition, the end of the aptamer modified with octyne benzene ring DBCO is used, and the aptamer can be specifically combined with AFP to induce the conformational change, so that the detection sensitivity of the AFP is improved.
Description
Technical Field
The invention belongs to the technical field of biochemistry, environmental detection and food safety, and relates to a protein detection method, in particular to a detection method of alpha fetoprotein based on an aptamer of an aggregation-induced emission marker and an azide-functionalized single-walled carbon nanotube.
Background
Alpha-fetoprotein (AFP) is a common protein biological marker for liver cancer, and can relatively accurately diagnose related cancers. AFP is normally produced by the yolk sac of a 7-8 month fetus, and is secreted from the liver as a plasma protein of about 70kDa in molecular weight, which gradually decreases as the fetus comes to birth and grows. In the serum of healthy people, AFP concentrations below 25ng/mL are barely detectable, but it is statistically significant that AFP levels rise to 500ng/mL in nearly 75% of patients with hepatocellular carcinoma (HCC). High levels of AFP in adult blood may indicate the presence of certain types of cancer, in particular HCC, gastric, pancreatic, ovarian or testicular cancer, and thus clinically elevated AFP concentrations in adult serum are widely considered as early indicators of HCC or intradermal sinus tumors. Aggregation-induced emission (AIE) is a class of luminescent molecules that are in a non-emissive state in dilute solution, but which, when in an aggregate state, provide a large degree of enhancement in emission. Based on the characteristic, the AIE can be used as a fluorescence indicator of an aptamer biosensor to generate a more sensitive and accurate 'turn-on' fluorescence signal for a sensing platform. The combination of nanomaterials and biomolecules plays an increasingly important role in the field of biosensors, and Carbon Nanotubes (CNTs), a novel nanomaterial, has been widely studied and applied because of its unique photoelectric and mechanical properties. In addition, studies have shown that SWNTs are often used as excellent quenchers for constructing nano-biosensors because they can act as energy acceptors for FRET in fluorescent aptamer sensors.
Click chemistry is a new combinatorial chemistry method based on carbon-heteroatom bond (C-X-C) synthesis, and molecular diversity is obtained simply and efficiently with the aid of these reactions, which is represented by copper-catalyzed azide-alkynyl Husigen cycloaddition. However, since the toxicity of copper (I) as a metal catalyst often causes partial nucleotide degradation and protein denaturation, and even limits the practical application of living cells or organisms, the CuAAC reaction has drawbacks that can be overcome by the copper-free variant reaction, 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 closer to the metal catalysis rate, so that copper-free click chemistry is carried out by utilizing the azide functionalization of the aptamer modified by DBCO and SWNTs, the excellent performances of FRET of AIE and SWNTs are fully utilized, and the occurrence of false positive signals is reduced, so that AFP is detected.
The method for detecting alpha fetoprotein based on the aggregation-induced emission labeled aptamer and the azide-functionalized single-walled carbon nanotube comprises the following steps:
(1) modifying a octyne benzene ring DBCO at the tail end of the DNA sequence, and dissolving the modified DNA sequence in ultrapure water;
(2)SWNTs-N3preparation of the conjugate:
firstly, uniformly dispersing SWNTs by using an ultrasonic cleaning machine to prepare a solution, adding NaOH and chloroacetic acid into the initial SWNTs solution, carrying out water bath ultrasonic treatment, washing, centrifuging, purifying and drying after-OH on the surface of the SWNTs is converted into-COOH, so as to obtain carboxyl functionalized SWNTs, namely SWCNTs-COOH;
dissolving the obtained SWCNTs-COOH in a methanol solution for ultrasonic treatment, and mixing the SWNTs-COOH and N3-PEG-NH2Adding into HEPES buffer solution, mixing, reacting at room temperature, passing through-COOH and-NH2Covalent bonding of (A) to form an azide-functionalized single-walled carbon nanotube SWNTs-N3；
(3) Fluorescence acquisition: adding the TPETA solution and the DNA modified in the step (1) into a brown centrifugal tube containing PBS to form a DNA sequence with fluorescence;
(4) quenching: after the DNA sequence with fluorescence obtained in step (3) is incubated at room temperature, the SWNTs-N obtained in step (2) is added3Reaction overnight, SWNTs-N3Performing click reaction with a DBCO functional group modified at the tail end of the DNA, and quenching fluorescence through FRET action;
(5) obtaining linear relationship between AFP and its nucleic acid aptamer:
adding a series of AFP solutions with known concentration into the solution obtained in the step (4), reacting at normal temperature, and allowing the aptamer, namely the DNA in the solution obtained in the step (4), to specifically bind with the AFP to induce the conformational change, so that the DNA part with TPETA fluorescence is far away from SWNTs-N3The fluorescence is recovered to a certain degree, after the reaction is finished, the fluorescence value is measured, and a corresponding linear relation graph is prepared according to the measured fluorescence value and AFP;
(6) AFP detection:
and (4) 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 the step (1), the sequence of the DNA is as follows: 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 in ultrapure water.
In the step (2), the concentration of the initial SWNTs solution is 2 mg/mL; the initial SWNTs solution, NaOH and chloroacetic acid were used in a ratio of 5 mL: 0.6 g: 0.5 g;
the volume percentage concentration of the methanol solution is 10 percent;
in the methanol solution of SWNTs-COOH, the concentration of SWNTs-COOH is 2 mg/mL;
SWNTs-COOH solution, N3-PEG-NH2And the dosage ratio of the HEPES buffer solution is 5 mL: 100 mg: 10mL, wherein the concentration of HEPES buffer solution is 0.1M, pH 7.4.
In the step (2), the reaction time is 12h at room temperature.
In the step (3), in the DNA sequence with fluorescence, the final concentration of TPETA is 10 mu M, and the final concentration of DNA is 30 nM.
In the step (4), the incubation time is 15min, and the final concentration of SWNTs-N3 is 20 mu g/mL; the reaction temperature is 4 ℃;
in the 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 is 300 mu L: 1.5-3 μ L.
In the step (6), the reaction time at normal temperature was 30 minutes.
The invention has the beneficial effects that:
(1) the single-walled carbon nanotube nano-material is easy to obtain, and the method is simple, low in cost and stable in property.
(2) The copper-free click chemistry used in the invention can eliminate the toxicity generated by a metal catalyst, thereby avoiding the defects of partial nucleotide degradation, protein denaturation, even living cells or organisms and the like.
(3) The invention uses the tetraphenyl ethylene single 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, thereby generating a 'turn-on' fluorescence signal with higher sensitivity and accuracy for a sensing platform and providing a new exploration direction for a non-labeled fluorescent aptamer sensor.
(4) The invention designs an aptamer with a decorated octyne benzene ring (DBCO) at the tail end and azide functionalized single-walled carbon nano (SWNTs-N)3) A click reaction occurs and fluorescence is quenched by FRET. Simple principle, convenient operation, time and labor saving, and the DNA part with TPETA fluorescence is used to be far away from SWNTs-N3The surface of the strain leads to the recovery of fluorescence, so as to detect the content of AFP and Hg in the fields of environment, food safety and the like2+Great convenience is brought.
Drawings
FIG. 1 is a technical scheme for the detection of alpha-fetoprotein according to the present invention;
FIG. 2: kinetic profiles of the TPETA molecules. Change in fluorescence intensity over the time range measured for different concentrations of the TPETA molecules.
FIG. 3: optimal concentration selection plot for SWNT-N3.
FIG. 4: the optimal concentration of the aptamer is selected as a graph, and the fluorescence recovery intensity of solutions of different concentrations of the aptamer in a reaction environment is changed.
FIG. 5: sensitivity profiles of aptamers with fluorescence intensity and fluorescence recovery (F/F) after addition of varying concentrations of AFP to aptamers0-1) of a change.
FIG. 6: a selectivity profile of aptamers, the change in fluorescence intensity upon addition of different types of proteins to a solution of aptamers.
Detailed Description
The present invention is further illustrated by the following examples, which are intended to be illustrative of the present invention and are not intended to limit the scope of the present 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 TGA TGT GGG TCC TGT CCG TCC GAA CCA ATC-3'
the 5' end of the DNA sequence is modified with octyne benzene ring, and the final concentration of the modified DNA sequence is 50nM after being dissolved in ultrapure water.
(2)SWNTs-N3Preparation of the conjugate:
the SWNTs were first dispersed uniformly using an ultrasonic cleaner and formulated into a 2mg/mL solution. After 0.6g NaOH and 0.5g chloroacetic acid are respectively added into 5mL of initial SWNTs solution, ultrasonic treatment is carried out in a water bath, after-OH on the surface of the SWNTs is converted into-COOH, the SWNTs are further purified by washing and centrifuging for multiple times, and the solution is dried to obtain carboxyl functionalized SWNTs (SWCNTs-COOH).
Dissolving the obtained carboxyl functionalized single-walled carbon nanotube in 10% methanol solution, performing ultrasonic treatment, and mixing 5mL SWNTs-COOH (2mg/mL) and 100mg N3-PEG-NH2Adding into 10mL HEPES buffer solution (0.1M, pH 7.4) and mixing well, reacting at room temperature for 12h, passing through-COOH and-NH2Forming azide-functionalized single-walled carbon nanotubes (SWNTs-N)3)。
(3) Fluorescence acquisition: adding the TPETA solution and the DNA modified in the step (1) into a brown centrifugal tube containing PBS to form a DNA sequence with fluorescence; TPETA final concentration of 10. mu.M, DNA final concentration of 30 nM;
(4) quenching: incubating the DNA sequence with fluorescence of step (3) at room temperature for 15min, adding the SWNTs-N prepared in step (2)3Reaction overnight at 4 ℃ with SWNTs-N3Performing click reaction with a DBCO functional group modified at the tail end of the DNA, and quenching fluorescence through FRET action; the final concentration of SWNTs-N3 was 20. mu.g/mL;
(5) obtaining linear relationship between AFP and its nucleic acid aptamer:
adding 1.5-3 mul of a series of AFP solution with known concentration into 300 mul of the solution obtained in the step (4), wherein the concentration of the AFP solution is 0.1-80 ng/mL, 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 AFP;
(6) AFP detection:
and (4) 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-N3Selection of optimal concentration: adding the nano material SWNTs-N with gradient concentration into a 30nM DNA system3Selecting the best quenching rate of SWNTs-N3Concentration, requiring fluorescence quenching rates of around 80%, where the final choice is SWNT-N at 20. mu.g/mL3。
Selection of the optimal concentration of the solution of DNA: different concentrations of DNA (10, 20, 30, 40 and 50nM) were added to SWNT-N containing 20. mu.g/mL3The result of adding an AFP at an equal concentration in the system of (1) shows that the fluorescence increase rate is the highest due to the addition of a solution of 30nM DNA.
Different concentrations of AFP (0.1, 0.5, 0.8, 5, 8, 10, 20, 50 and 80ng/mL) were added to the nano-material SWNT-N containing fluorescent molecule TPETA (10. mu.M)3(20. mu.g/mL) and an aptamer (30nM) in PBS buffer (10mM, pH 7.4), the mixture was mixed well and incubated at room temperature for 30min to measure and record the fluorescence spectrum, and the recovery of fluorescence was examined.
Several analogues (BSA, CEA, HSA, IgG and Thrombin) similar to the AFP to be tested were selected to evaluate the selectivity of the sensor under the same system and under the same conditions of (4).
Figure 2 is a kinetic diagram of the TPETA molecule. The fluorescence intensity of different concentrations of the TPETA molecules in the measured time range has no obvious change, which indicates that the fluorescent molecules can maintain good stability in a PBS buffer system.
FIG. 3 is a graph showing the selection of the optimal concentration of SWNT-N3. SWNT-N3Concentration in the range of 5-20. mu.g/mL with SWNT-N3Increase in concentration, F/F0-1 is also increasing, but SWNT-N3The concentration is increased continuously on the basis of 20 mu g/mL to obtain F/F01 as a result of the decreasing so SWNT-N3Optimization ofThe concentration was 20. mu.g/mL.
FIG. 4 is a diagram showing the selection of the optimum concentration of the aptamer DNA. The fluorescence recovery intensity of the aptamer solutions with different concentrations in the reaction environment is changed, and the optimal concentration is 30 nM.
FIG. 5 is a graph showing the sensitivity of aptamer DNA. Fluorescence intensity and fluorescence recovery (F/F) after adding AFP with different concentrations into aptamer solution0-1) of a change. The concentration of AFP added was gradually increased from 0 to a final concentration of 80 ng/mL.
FIG. 6 is a graph showing the selectivity of aptamer DNA. The change in fluorescence intensity after addition of different types of proteins to the aptamer solution, wherein the final concentration of each protein is 10 ng/mL.
Claims (8)
1. The detection method of alpha fetoprotein based on the aptamer of aggregation-induced emission marker and the azide-functionalized single-walled carbon nanotube is characterized by comprising the following steps:
(1) modifying a octyne benzene ring DBCO at the tail end of the DNA sequence, and dissolving the modified DNA sequence in ultrapure water;
(2)SWNTs-N3preparation of the conjugate:
firstly, uniformly dispersing SWNTs by using an ultrasonic cleaning machine to prepare a solution, adding NaOH and chloroacetic acid into the initial SWNTs solution, carrying out water bath ultrasonic treatment, washing, centrifuging, purifying and drying after-OH on the surface of the SWNTs is converted into-COOH, so as to obtain carboxyl functionalized SWNTs, namely SWCNTs-COOH;
dissolving the obtained SWCNTs-COOH in a methanol solution for ultrasonic treatment, and mixing the SWNTs-COOH and N3-PEG-NH2Adding into HEPES buffer solution, mixing, reacting at room temperature, passing through-COOH and-NH2Covalent bonding of (A) to form an azide-functionalized single-walled carbon nanotube SWNTs-N3；
(3) Fluorescence acquisition: adding the TPETA solution and the DNA modified in the step (1) into a brown centrifugal tube containing PBS to form a DNA sequence with fluorescence;
(4) quenching: after the DNA sequence with fluorescence obtained in step (3) is incubated at room temperature, the SWNTs-N obtained in step (2) is added3On the contraryAt night, SWNTs-N3Performing click reaction with a DBCO functional group modified at the tail end of the DNA, and quenching fluorescence through FRET action;
(5) obtaining linear relationship between AFP and its nucleic acid aptamer:
adding a series of AFP solutions with known concentration 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 graph according to the measured fluorescence value and AFP;
(6) AFP detection:
and (4) 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 detection method according to claim 1, wherein in the 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 GAA CCA ATC-3'. The final concentration of the modified DNA sequence was 50nM in ultrapure water.
3. The detection method according to claim 1, wherein, in the step (2),
the initial concentration of SWNTs solution was 2 mg/mL;
the initial SWNTs solution, NaOH and chloroacetic acid were used in a ratio of 5 mL: 0.6 g: 0.5 g;
the volume percentage concentration of the methanol solution is 10 percent;
in the methanol solution of SWNTs-COOH, the concentration of SWNTs-COOH is 2 mg/mL;
SWNTs-COOH solution, N3-PEG-NH2And the dosage ratio of the HEPES buffer solution is 5 mL: 100 mg: 10mL, wherein the concentration of HEPES buffer solution is 0.1M, pH 7.4.
4. The detection method according to claim 1, wherein in the step (2), the reaction time is 12 hours at room temperature.
5. The detection method according to claim 1, wherein in the step (3), the final concentration of TPETA is 10 μ M and the final concentration of DNA is 30nM in the DNA sequence having fluorescence.
6. The assay of claim 1, wherein in step (4), the incubation time is 15min and the final concentration of SWNTs-N3 is 20 μ g/mL; the reaction temperature was 4 ℃.
7. The detection method according to claim 1, wherein in the step (5), the concentration of the AFP solution is 0.1-80 ng/mL, and the reaction time at normal temperature is 30 minutes; the volume ratio of the solution obtained in the step (4) to the AFP solution is 300 mu L: 1.5-3 μ L.
8. The detection method according to claim 1, wherein in the step (6), the reaction time at normal temperature is 30 minutes.
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