CN111220672B - Detection of Hg based on energy resonance transfer2+Preparation method of self-enhanced electrochemiluminescence aptamer sensor - Google Patents

Detection of Hg based on energy resonance transfer2+Preparation method of self-enhanced electrochemiluminescence aptamer sensor Download PDF

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CN111220672B
CN111220672B CN202010094283.6A CN202010094283A CN111220672B CN 111220672 B CN111220672 B CN 111220672B CN 202010094283 A CN202010094283 A CN 202010094283A CN 111220672 B CN111220672 B CN 111220672B
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由天艳
陈柏年
李丽波
罗莉君
毕晓雅
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Abstract

Hair brushBelongs to the technical field of biosensors, and relates to a preparation method of a self-enhanced electrochemical luminescence aptamer sensor based on energy resonance transfer and Hg detection2+The use of (1). First, self-enhanced electrochemiluminescence, unlike traditional electrochemiluminescence, complexes a luminophore with a co-reactant in one molecule. Next, the gold nanoparticles are complexed with the aptamer in order to bind to the DNA associated with the luminescent complex. The spectrum overlapping of the ultraviolet absorption peak of the gold nanoparticles and the ECL emission peak of the luminescent compound is utilized, energy resonance transfer occurs between the ultraviolet absorption peak and the ECL emission peak, and the ECL signal is reduced. When the target mercury ion exists, the target mercury ion is specifically combined with the aptamer to form T-Hg2+-structure of T, keeping the attached AuNPs away from the electrode surface, ECL signal recovery. The linear detection range of the sensor is 10‑14‑10‑6M, detection limit of 3.33X 10‑15And M. The sensor has the advantages of simple preparation method, high selectivity, high sensitivity, good reproducibility and good stability, and is used for detecting Hg in actual samples2+Provides a good sensing platform.

Description

Detection of Hg based on energy resonance transfer2+Preparation method of self-enhanced electrochemiluminescence aptamer sensor
Technical Field
The invention belongs to the technical field of biosensing detection, and relates to a preparation method of a self-enhanced electrochemical luminescence aptamer sensor based on an energy resonance transfer system and a sensor in Hg2+The method is applied to detection.
Background
Mercury is the only heavy metal present in the liquid state. As is well known, Hg2+Is the most widely distributed and stable existing form. Hg is a mercury vapor2+Is a highly toxic substance, poses great threat to the ecological environment, and even in very low concentration, can damage the human body, such as destroying the function of the human body, leading to irreversible consequences of skin ulceration, brain injury and the like.
The rapid and simple method for developing the Hg is realized2+The detection of (a) is not trivial. Hg commonly used at present2+The analytical measurement methods include atomic absorption spectrometry (AFS), inductively coupled plasma mass spectrometry (ICP-MS), atomic fluorescence spectrometry, and the like. Although these methods are accurate, they require professional technical support and are expensive instruments. Electrochemical method (EC), electrochemiluminescence method (ECL), fluorescenceMethods (FL) and the like, which are simple to operate, less costly and highly reliable. The ECL has attracted much attention of scientists due to its features such as strong controllability and stable performance. To further improve ECL luminous efficiency, self-enhancing ECLs are gradually brought into the line of sight of people. Meanwhile, in order to make the result more accurate, energy resonance transfer is introduced, and signal change brought by the energy resonance transfer is applied to Hg2+The detection of (2) is more sensitive.
Disclosure of Invention
Aiming at the current detection of Hg2+The present invention provides an ECL biosensor for detecting Hg with low cost, high sensitivity and high luminescence efficiency2+。
Hg detection based on energy resonance transfer2+The preparation method of the self-enhanced electrochemical luminescence aptamer sensor comprises the following steps:
(1)Ru@SiO2preparation of the material:
firstly, adding triton, cyclohexane and 1-hexanol into secondary water, obtaining microemulsion through a reverse microemulsion method, adding terpyridyl ruthenium, tetraethyl silicate and ammonia water to continue stirring and reacting after the reaction is finished, dissolving the obtained product into acetone, ethanol and secondary water in sequence after the reaction is finished, centrifugally washing to obtain lower-layer precipitate, and drying in vacuum to obtain Ru @ SiO2(ii) a Dissolving in ultrapure water to obtain Ru @ SiO2A dispersion liquid;
(2)NH2-Ru@SiO2preparing a composite material:
firstly, Ru @ SiO prepared in step (1)2Mixing the dispersion with APTES, stirring for reaction, dissolving the obtained product with ethanol, centrifuging to obtain lower precipitate, and vacuum drying to obtain NH2-Ru@SiO2(ii) a Dissolving in ethanol to obtain NH2-Ru@SiO2A dispersion liquid;
(3)NH2-Ru@SiO2preparation of NGQDs composites:
firstly, NH prepared in step (2)2-Ru@SiO2Mixing the dispersion liquid with NGQDs, and after the stirring reaction is finished, using the obtained product twiceDissolving in water, centrifuging to obtain supernatant as NH2-Ru@SiO2-NGQDs；
(4)NH2-Ru@SiO2-preparation of NGQDs-DNA composites:
first, NH prepared in step (3)2-Ru@SiO2Adding PBS buffer solution containing EDC and NHS into-NGQDs, adding DNA after reaction, and stirring to obtain yellow NH2-Ru@SiO2-NGQDs-DNA, left at 4 ℃ for future use;
(5) preparing gold nanoparticle solution Au NPs:
first, a chloroauric acid solution (HAuCl)4·3H2O) and secondary water are mixed, stirred and reacted under the oil bath reflux device until the mixture is boiled, trisodium citrate solution is added, the mixture is continuously stirred until the color is changed into mauve, and the mixture is placed at 4 ℃ for standby after the reaction is finished;
(6) preparation of Au NPs-apt:
firstly, mixing the gold nanoparticle solution Au NPs prepared in the step (5) with the aptamer solution adapted to the DNA in the step (4), standing for 1 hour in the dark, then stirring for 16 hours at normal temperature, after the reaction is finished, centrifuging, and standing the obtained upper layer solution at 4 ℃ for later use;
(7) sequentially polishing glassy carbon electrodes by using aluminium oxide powder with different particle sizes, performing ultrasonic treatment in ethanol and water, and drying in the air;
(8) adding the self-enhanced luminophore material NH prepared in the step (4)2-Ru@SiO2Modifying NGQDs-DNA on the surface of the glassy carbon electrode prepared in the step (7), and drying at 37 ℃;
(9) dripping the Au NPs-apt prepared in the step (6) on the surface of the self-assembled electrode prepared in the step (8), and carrying out base pairing with NH at 37 DEG C2-Ru@SiO2-NGQDs-DNA binding to form self-assembled electrodes;
(10) immersing the self-assembled electrode prepared in the step (9) into a bovine serum albumin BSA solution for incubation at a certain temperature so as to block the remaining non-specific binding sites, thus preparing the NH-based self-assembled electrode2-Ru@SiO2-electrochemiluminescent aptamer sensors of NGQDs.
In the step (1), the dosage proportion of the triton, the cyclohexane, the 1-hexanol and the secondary water is 1.77 mL: 7.5 mL: 1.8 mL: 340 mu L, and the reaction time is 15 min; the dosage proportion of the terpyridyl ruthenium, the tetraethyl silicate and the ammonia water is 80 mu L: 100 μ L of: 60 mu L of the solution; stirring and reacting for 24h, centrifuging at 10000rpm for 5min each time to obtain Ru @ SiO2The concentration of the dispersion was 2mg/mL-1。
In the step (2), Ru @ SiO2The dosage ratio of the dispersion liquid to the APTES is 1 mL: 400 mu L, stirring the two for reaction for 4h, centrifuging the ethanol for three times at the centrifugal speed of 10000rpm for 5min each time to obtain NH2-Ru@SiO2The concentration of the dispersion was 1mg/mL-1。
In step (3), NH2-Ru@SiO2The dosage ratio of the dispersion liquid to the NGQDs is 1 mL: 5mL, the concentration of NGQDs is 5mg/mL-1The time for stirring the two materials for reaction is 12 h.
In step (4), NH2-Ru@SiO2-NGQDs-DNA, PBS buffered, DNA dose ratio 1000 μ L: 500. mu.L: 1000 μ L, PBS concentration of 0.01M, in which NHS concentration is 0.005M, EDC concentration is 0.01M, DNA concentration is 3 μ M.
In the step (4), the sequence of the DNA is as follows: 5' -TAA GAA AGA GGG GAC AAA- (CH)2)3-NH2-3'。
The reaction time is 15 min; the time for continuing stirring after adding the aptamer is 2 h.
In the step (5), the dosage proportion of the chloroauric acid solution, the secondary water and the trisodium citrate is 200 mu L: 25mL of: 250 mu L, the first two are stirred for 10-30min, the last reaction time is 15min, wherein the concentration of the chloroauric acid solution is 0.1M, and the concentration of the trisodium citrate solution is 100mg mL-1。
In the step (6), the dosage ratio of Au NPs, apt is 1950 muL: 50 mu L, standing for 1h, reaction time for 16h, centrifugal speed of 10000rpm, and time for 15min, wherein the concentration of Au NPs is 5nM, and the concentration of apt is 25 mu M.
The sequence of apt is: 5' -TTG TTT GTC CCC TCT TTC TTA- (CH)2)3-SH-3'；
In the step (7), the diameter d of the glassy carbon electrode is 3 mm; the grain sizes of the aluminum oxide powders used are 0.3 μm and 0.05 μm in this order.
In step (8), NH2-Ru@SiO2The amount of NGQDs-DNA was 6. mu.L; the reaction temperature is 37 ℃ and the reaction time is 5-10 min.
In the step (9), the dosage of Au NPs-apt is 6 mu L; the reaction temperature is 37 ℃ and the reaction time is 10-70 min.
In the step (10), the mass percentage concentration of the bovine serum albumin BSA solution is 1%; the incubation temperature is 37 deg.C, and the incubation time is 10-60 min.
The biosensor prepared by the invention is sequentially soaked in Hg with different concentrations2+In the solution, the binding time is 1-2h in a dark place at room temperature, Hg2+The concentration range is 1 × 10-14～1×10-6mol/L, after which the electrodes were washed with PBS (pH 7.0) solution. The prepared sensor was used as a working electrode, a saturated Ag/AgCl electrode as a reference electrode, a platinum electrode as a counter electrode, and the measurement was performed in 0.1M PBS (pH 7.5) buffer solution to obtain Hg2+Linear relationship between concentration and ECL signal intensity.
The biosensor prepared by the invention is soaked in unknown Hg2+In the solution to be measured with the concentration, the binding time is 1-2h at the dark room temperature, and Hg is2+The concentration was unknown, and then the electrodes were washed with a PBS (pH 7.0) solution. The prepared sensor is used as a working electrode, a saturated Ag/AgCl electrode is used as a reference electrode, a platinum electrode is used as a counter electrode, the test is carried out in 0.1M PBS (pH 7.5) buffer solution, and the obtained ECL signal is substituted into the obtained linear relation, so that Hg in the solution to be tested can be obtained2+The concentration of (c).
The working principle of the ECL biosensor is as follows:
composite luminescent material NH2-Ru@SiO2-NGQDs-DNA luminophores NH2-Ru@SiO2Is compounded with coreactant NGQDs to form a luminescent group (NH)2-Ru@SiO2NGQDs-DNA) that can enhance ECL signal intensity. When Au NPs-apt exists, apt is coupled with DNA pair, and then DNA is connectedBonded NH2-Ru@SiO2The energy resonance transfer of NGQDs and the apt-connected Au NPs occurs, and the ECL signal is reduced. When the target Hg is2+After appearance, it binds to apt at T-Hg2+Au NPs-apt and NH under structural force of-T2-Ru@SiO2-NGQDs-DNA isolation, ECL signal recovery. According to the change of ECL signal intensity, the Hg is achieved2 +Detection of (3).
The invention has the beneficial effects that:
(1) the self-enhanced ECL biosensor has simple preparation method, combines the scientific law of energy resonance transfer, ensures that the biosensor is more reliable, and provides a new method in the aspect of material application.
(2) Replaces the conventional ECL with a novel self-reinforcing ECL and is first applied to Hg2+The detection of (2) has lower detection limit, large order span of 8 orders of magnitude, and obtains higher recovery rate in the detection of actual samples.
(3) The self-enhanced ECL aptamer sensor constructed by the invention is used for Hg in a water body2+The specific detection has high sensitivity, good selectivity, good stability and wide linear range of 10-14-10-6mol L-1(ii) a Detection limit is as low as 3.33 multiplied by 10-15M。
Drawings
Fig. 1 is a diagram of a self-enhanced ECL aptamer sensor construction process.
FIG. 2(A) is NH2-Ru@SiO2-NGQDs-DNA incubation time vs ECL plot; (B) the incubation time of the target was plotted against ECL.
FIG. 3(A) is a TEM image of Au NPs; (B) ultraviolet spectrograms of Au NPs and Au NPs-apt;
FIG. 4(A) shows different Hg concentrations2+The corresponding ECL signal change: the concentration is blank (a), 1 × 10-14(b)、1× 10-13(c)、1×10-12(d)、1×10-11(e)、10-10(f)、5×10-10(g)、2×10-9(h)、5×10-8(i)、2×10-7(j)、 1×10-6mol/L (k); (B) different concentrations of Hg2+A linear relationship is constructed between the logarithm and the electrochemiluminescence signal.
FIG. 5(A) Selectivity of self-enhancing ECL aptamer sensors with respective interferents Mg2+、Fe2+、Fe3+、Na+、 Ca2 +、Cd2+、Mn2+、Pb2+、Zn2+、Cu2+. (B) The aptamer sensor has stable performance after being continuously placed for 15 days.
Detailed Description
The invention is further elucidated with reference to the embodiments and the drawings of the description.
Example 1
The preparation process according to the figure 1:
(1)NH2-Ru@SiO2-preparation of NGQDs-DNA composites:
firstly, mixing 1.77mL of triton, 7.5mL of cyclohexane, 1.8mL of 1-hexanol and 340 μ L of secondary water together, stirring for 15min, adding 80 μ L of terpyridyl ruthenium, 100 μ L of tetraethyl silicate and 60 μ L of ammonia water, continuing stirring for 24h, after stirring, centrifuging acetone and ethanol, washing for three times respectively, wherein the centrifugal rotation speed is 10000rpm, and each time is 5min, and finally dissolving the obtained precipitate in ultrapure water for later use.
Ru @ SiO obtained by the method2Mixing (2mg/mL)4mL with 1600 μ L APTES, stirring for 4h, dissolving the obtained product with ethanol after reaction, centrifuging for three times at 10000rpm for 5min, collecting the lower layer precipitate, and vacuum drying to obtain NH2-Ru@SiO2。
Reacting NH2-Ru@SiO21mL (1mg/mL) of the solution was mixed with 5mL of NGQDs (5mg/mL) and stirred for 12 hours to obtain a yellow solution NH2-Ru@SiO2-NGQDs。
At NH2-Ru@SiO2Adding 0.01M buffer solution (PBS, pH 7.4) containing 0.01M EDC and 0.005M NHS into NGQDs, stirring for 15min to allow amide reaction, adding 3 μ M DNA after reaction, and stirring to obtain NH2-Ru@SiO2NGQDs-DNA, centrifuging the obtained product for 5min after the reaction is finished, wherein the centrifugation rotation speed is 10000rpm, and standing at 4 ℃ for later use。
(2) Preparation of Au NPs-apt
First, 200. mu.L of chloroauric acid solution (0.1M) and 25mL of secondary water were mixed, stirred under reflux of an oil bath for 10-30min and then boiled, followed by addition of 250. mu.L of trisodium citrate (100mg mL)-1) And (3) continuously stirring and reacting for 15min, standing to room temperature after the reaction is finished, and standing at 4 ℃ for later use to obtain an Au NPs solution.
mu.L of Au NPs solution (5nM) and 50. mu.L of apt (25. mu.M) were mixed, left to stand in the dark for 1 hour, and then stirred for 16 hours, after the reaction was completed, the resultant was centrifuged at 10000rpm for 15 minutes to obtain Au NPs-apt solution.
(3) Glassy carbon electrodes (d ═ 3mm GCE) were polished with 0.3 μm and 0.05 μm aluminum oxide powders in this order, sonicated in ethanol and water and dried in air.
(4) Modifying the self-enhanced luminescent material prepared in the step (1) on the glassy carbon electrode prepared in the step (3) to obtain NH2-Ru@SiO2The amount of-NGQDs-DNA was 6. mu.L, in which case the sensor was indicated as NH2-Ru@SiO2-NGQDs-DNA/GCE；
(5) Modifying the Au NPs-apt obtained in the step (2) to the surface of the electrode prepared in the step (4), wherein the dosage of the Au NPs-apt is 6 mu L, and combining the Au NPs-apt with the material prepared in the step (1) on the surface of the electrode through base complementary pairing to form a self-assembly electrode Au NPs-apt/NH2-Ru@SiO2-NGQDs-DNA/GCE；
(6) Washing the self-assembled electrode prepared in the step (5) with secondary water at normal temperature, and soaking the self-assembled electrode in 1% bovine serum albumin BSA solution at 37 ℃ for incubation for 10-60min to block the remaining non-specific binding sites to prepare the electrode based on apt-NGQDs-NH2-Ru@SiO2In this case the sensor is expressed as BSA/Au NPs-apt/NH2-Ru@SiO2-NGQDs-DNA/GCE。
The sensor prepared above was immersed in 200. mu.L of 1X 10-6moL·L-1Hg2+In the solution, the binding time was 60 to 100min at room temperature, and then the electrode was washed with PBS (pH 7.0). The prepared sensor is used as a working electrode and is saturated Ag/AgCl electrode is reference electrode, platinum wire electrode is counter electrode, and MPI-EII electrochemiluminescence analyzer is used to record ECL signal. The test was performed in 0.1M PBS (pH 6.0-8.5) buffer. The scanning voltage range is 0.2-1.25V, the scanning speed is 0.1V s, and the high voltage of the photomultiplier in the experiment is 800V.
FIG. 3(A) is a TEM image of Au NPs, which shows that the Au NPs are uniformly distributed in spherical form; (B) the ultraviolet spectrograms of Au NPs and Au NPs-apt can observe that when an aptamer is compounded with the Au NPs, a 260nm basic group characteristic peak and a 519nm Au NPs characteristic absorption peak appear simultaneously, and the successful synthesis of the Au NPs-apt material is proved.
Example 2
Method for detecting Hg by using prepared self-enhanced electrochemiluminescence aptamer sensor2+Optimizing the experimental conditions:
the biosensor prepared in example 1 was placed at 37 ℃ for 40, 50, 60, 70, 80, 90min to detect ECL signals.
FIG. 2(A) is a graph of Au NPs-apt incubation time versus ECL; the incubation time was increased from 40min to 90min, and the ECL signal intensity gradually increased to plateau at 80 min. Therefore, 90min was chosen as the optimal incubation time for the aptamer.
Example 3
Method for detecting Hg by using prepared self-enhanced electrochemiluminescence aptamer sensor2+Optimizing the experimental conditions:
based on the self-enhanced electrochemical luminescence aptamer sensor with optimal conditions obtained in example 2, the sensor surface is modified with target Hg2+FIG. 2(B) is a graph showing the relationship between the incubation time of the target substance and ECL, wherein the incubation time is increased from 50min to 100min, the ECL signal intensity gradually increases, and reaches a plateau at 70min, and the target substance Hg is present2+The incubation time on the electrode surface was 80 min.
Example 4
Detection of Hg by self-enhanced electrochemical luminescence aptamer sensor2+：
Respectively using 1X 10-14、1×10-13、1×10-12、1×10-11、5×10-10、2×10-9、5×10-8、2×10-7、1 ×10-6mol/L of Hg2+The best self-enhancing electrochemiluminescence aptamer sensor obtained in example 1 was modified and ECL signals were recorded with MPI-EII electrochemiluminescence analyzer according to the best experimental conditions obtained in example 3. With Hg2+The concentration is increased, the ECL signal is increased and the ECL intensity and Hg are within a certain range2+The logarithm of the concentration is linear.
As can be seen from FIG. 4(A), as Hg is associated with2+Increase in concentration (concentration in the order of 1X 10-14、1×10-13、1×10-12、 1×10-11、5×10-10、2×10-9、5×10-8、2×10-7、1×10-6mol/L), the ECL signal gradually increases due to the greater concentration, the stronger the force with the apt, the more distant from the electrode surface, and the more pronounced the signal recovery.
As can be seen from FIG. 4(B), ECL signal and Hg2+Logarithmic value of concentration (logC)Hg 2+) Drawing a standard curve of IECL＝ 255lgCHg2++4490(R20.9978) linear range of 10-14-10-6M, detection limit of 3.33 × 10-15M。
Example 5
Self-enhanced electrochemiluminescence aptamer sensor selectivity analysis:
it can be seen from fig. 5(a) that the self-enhanced electrochemiluminescence aptamer sensor has good selectivity. Experimental conditions for the best self-enhancing electrochemiluminescence aptamer sensor, perturber Mg, explored in example 32+(5×10-7M)、Fe2+(5 ×10-7M)、Fe3+(5×10-7M)、Na+(5×10-7M)、Ca2+(5×10-7M)、Cd2+(5×10-7M)、K+ (5×10- 7M)、Pb2+(5×10-7M)、Zn2+(5×10-7M)、Cu2+(5×10-7M) incubation with the respective sensors, the ECL response results were essentially the same as the blank, however, 10-8M Hg2+After incubation with the sensor, ECL signal response results were significantly higher than blank. When the biosensor is 10-8M Hg2+And 10 interferents (5X 10)-7M) when incubated with a mixture of Hg alone, the response2+The response is substantially unchanged compared to the previous response. The result shows that the electrochemical biosensor has good specificity and can be used for Hg2+Detection of (3).
It can be seen from fig. 5(B) that the self-enhanced electrochemiluminescence aptamer sensor has good stability.

Claims (9)

1. Hg detection based on energy resonance transfer2+The preparation method of the self-enhanced electrochemiluminescence aptamer sensor is characterized by comprising the following steps:
(1)Ru@SiO2preparation of the material:
firstly, adding triton, cyclohexane and 1-hexanol into secondary water, obtaining microemulsion through a reverse microemulsion method, adding terpyridyl ruthenium, tetraethyl silicate and ammonia water to continue stirring and reacting after the reaction is finished, dissolving the obtained product into acetone, ethanol and secondary water in sequence after the reaction is finished, centrifugally washing to obtain lower-layer precipitate, and drying in vacuum to obtain Ru @ SiO2(ii) a Dissolving in ultrapure water to obtain Ru @ SiO2A dispersion liquid;
(2)NH2-Ru@SiO2preparing a composite material:
firstly, Ru @ SiO prepared in step (1)2Mixing the dispersion with APTES, stirring for reaction, dissolving the obtained product with ethanol, centrifuging to obtain lower precipitate, and vacuum drying to obtain NH2-Ru@SiO2(ii) a Dissolving in ethanol to obtain NH2-Ru @ SiO2A dispersion liquid;
(3)NH2-Ru@SiO2preparation of NGQDs composites:
firstly, NH prepared in step (2)2-Ru@SiO2Mixing the dispersion with NGQDs, stirring, dissolving the obtained product with secondary water, centrifuging to obtain upper layer yellow clear liquid, i.e. NH2-Ru@SiO2-NGQDs；
(4)NH2-Ru@SiO2-preparation of NGQDs-DNA composites:
first, NH prepared in step (3)2-Ru@SiO2Adding PBS buffer solution containing EDC and NHS into-NGQDs, adding DNA after reaction, and stirring to obtain yellow NH2-Ru@SiO2-NGQDs-DNA, left for use;
(5) preparing gold nanoparticle solution Au NPs:
first, a chloroauric acid solution (HAuCl)4·3H2O) and secondary water are mixed, stirred and reacted under the oil bath reflux device until the mixture is boiled, trisodium citrate solution is added, the mixture is continuously stirred until the color is changed into mauve, and the mixture is placed for standby after the reaction is finished;
(6) preparation of Au NPs-apt:
firstly, mixing the gold nanoparticle solution Au NPs prepared in the step (5) with the aptamer solution adapted to the DNA prepared in the step (4), standing in the dark, then stirring at normal temperature, and after the reaction is finished, centrifuging to obtain an upper solution for later use;
the sequence of the aptamer matched with the DNA in the step (4) is as follows: 5' -TTG TTT GTC CCC TCT TTC TTA- (CH)2)3-SH-3'；
(7) Sequentially polishing glassy carbon electrodes by using aluminium oxide powder with different particle sizes, performing ultrasonic treatment in ethanol and water, and drying in the air;
(8) adding the self-enhanced luminophore material NH prepared in the step (4)2-Ru@SiO2Modifying NGQDs-DNA on the surface of the glassy carbon electrode prepared in the step (7), and drying;
(9) dripping the Au NPs-apt prepared in the step (6) on the surface of the self-assembled electrode prepared in the step (8), and carrying out base pairing with NH2-Ru@SiO2-NGQDs-DNA binding to form self-assembled electrodes;
(10) immersing the self-assembled electrode prepared in the step (9) into a bovine serum albumin BSA solution for incubation at a certain temperature so as to block the remaining non-specific binding sites, thus preparing the NH-based self-assembled electrode2-Ru@SiO2-electrochemiluminescent aptamer sensors of NGQDs.
2. The production method according to claim 1,
in the step (1), the dosage proportion of the triton, the cyclohexane, the 1-hexanol and the secondary water is 1.77 mL: 7.5 mL: 1.8 mL: 340 mu L, the reaction time is 15min, and the proportion of the usage of the ruthenium terpyridyl, the tetraethyl silicate and the ammonia water is 80 mu L: 100 μ L of: 60 μ L of: stirring and reacting for 24h, centrifuging at 10000rpm for 5min each time to obtain Ru @ SiO2The concentration of the dispersion was 2mg/mL-1；
In the step (2), Ru @ SiO2The dosage ratio of the dispersion liquid to the APTES is 1 mL: 400 mu L, stirring the two for reaction for 4h, centrifuging the ethanol for three times at the centrifugal speed of 10000rpm for 5min each time to obtain NH2-Ru@SiO2The concentration of the dispersion was 1mg/mL-1；
In step (3), NH2-Ru@SiO2The dosage ratio of the dispersion liquid to the NGQDs is 1 mL: 5mL, the concentration of NGQDs is 5mg/mL-1The time for stirring the two materials for reaction is 12 h.
3. The method according to claim 1, wherein in the step (4), NH is added2-Ru@SiO2NGQDs, PBS buffer, in a DNA dose ratio of 1000. mu.L: 500. mu.L: 1000 μ L, PBS concentration of 0.01M, wherein NHS concentration of 0.005M, EDC concentration of 0.01M, DNA concentration of 3 μ M;
the sequence of the DNA is: 5' -TAA GAA AGA GGG GAC AAA- (CH)2)3-NH2-3'；
The reaction time is 15 min; after the aptamer is added, stirring is continued for 2 hours, and the standing temperature is 4 ℃.
4. The preparation method according to claim 1, wherein in the step (5), the ratio of the amount of the chloroauric acid solution, the secondary water and the trisodium citrate is 200 μ L: 25mL of: 250 mu L, the first two are stirred for 10-30min, the last reaction time is 15min, wherein the concentration of the chloroauric acid solution is 0.1M, and the concentration of the trisodium citrate solution is 100mg mL-1The standing temperature was 4 ℃.
5. The method according to claim 1, wherein in the step (6), the Au NPs, apt is used in a proportion of 1950 μ L: 50 mu L, standing for 1h, reaction time for 16h, centrifugal speed of 10000rpm, time for 15min, wherein the concentration of Au NPs is 5nM, the concentration of apt is 25 mu M, and the standing temperature is 4 ℃.
6. The production method according to claim 1, wherein in the step (7), the diameter d of the glassy carbon electrode is 3 mm; the grain sizes of the aluminum oxide powders used are 0.3 μm and 0.05 μm in this order.
7. The method according to claim 1, wherein in the step (8), NH is added2-Ru@SiO2The amount of NGQDs-DNA was 6. mu.L; the reaction temperature is 37 ℃ and the reaction time is 5-10 min.
8. The production method according to claim 1, wherein in the step (9), the amount of Au NPs-apt is 6 μ L; the reaction temperature is 37 ℃ and the reaction time is 10-70 min.
9. The method according to claim 1, wherein in the step (10), the bovine serum albumin BSA solution is 1% by mass; the incubation temperature is 37 deg.C, and the incubation time is 10-60 min.
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