CN110823977B - Hg detection method2+Preparation method of self-enhanced electrochemiluminescence aptamer sensor - Google Patents

Hg detection method2+Preparation method of self-enhanced electrochemiluminescence aptamer sensor Download PDF

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CN110823977B
CN110823977B CN201911031078.9A CN201911031078A CN110823977B CN 110823977 B CN110823977 B CN 110823977B CN 201911031078 A CN201911031078 A CN 201911031078A CN 110823977 B CN110823977 B CN 110823977B
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由天艳
陈柏年
李丽波
刘晓红
毕晓雅
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Abstract

The invention belongs to the technical field of biosensors, and relates to a method for detecting Hg2+The method for preparing the self-enhanced electrochemical luminescence aptamer sensor. The self-enhanced electrochemiluminescence is different from the traditional electrochemiluminescence, a luminophor and a coreactant are compounded in one molecule, in order to further improve the selectivity, an aptamer and a luminescent group are compounded, and T-Hg is utilized2+The characteristic structure of-T is firstly applied to Hg2+Detection of (3). In the present invention, Hg is introduced2+Aptamers, not only increasing Hg of sensors2+High specificity and can pass through T-Hg on the basis of self-enhancement2+And the T structure bends the adapter body, so that the self-reinforcing material connected with one end of the adapter body is close to the electrode, the electron transfer distance is shortened, and the ECL luminous efficiency is further improved. The constructed sensor has a detection linear range of 5.0 multiplied by 10‑11‑1.0×10‑6M, detection limit of 1.0X 10‑12And M. Through exploration, the sensor is simple in preparation method, high in selectivity, sensitivity and reproducibility, good in stability and capable of detecting Hg in actual samples2+Provides a good sensing platform.

Description

Hg detection method2+Preparation method of self-enhanced electrochemiluminescence aptamer sensor
Technical Field
The invention belongs to the technical field of biosensing detection, and relates to a method for detecting Hg2+In particular to a preparation method based on apt-NGQDs-NH2-Ru@SiO2A preparation method and application of a self-enhanced electrochemical luminescence aptamer sensor of a system.
Background
Mercury ion (Hg)2+) Is a heavy metal ion with high toxicity and easy accumulation. Hg is a mercury vapor2+Widely distributed in soil, crops, water bodies and atmosphere, and enter human bodies through biological chains. When Hg is in the ecological environment2+Too high content of the active ingredients can cause soil methylation, plant poisoning, water source pollution, damage aquatic organisms such as fishes, shrimps and the like, further enter human bodies through food chains, enrich the bodies, cause deformation of limbs and even cause feared water guarantee. Thus to Hg in water2+The content is necessary to be detected, and theoretical basis can be provided for water quality evaluation and subsequent treatment。
For Hg2+The detection of (A) is always a matter of delay, and Hg is 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. While these methods are accurate and efficient, they rely on specialized personnel and are expensive instruments. Electrochemical methods, Electrochemiluminescence (ECL), fluorescence methods, and the like, which are simple to operate and low in cost. The ECL has attracted much attention of scientists due to its characteristics of high sensitivity, good reproducibility, strong controllability, simple operation of instruments, etc. In order to further reduce the electron transfer distance, reduce the energy loss and improve the ECL luminous efficiency, the self-enhanced ECL gradually enters the sight of people and occupies a place in the detection field.
Disclosure of Invention
Aiming at the current detection of Hg2+The invention provides an ECL biosensor with low cost, high sensitivity and high luminous efficiency for detecting Hg2+。
Hg detection method2+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 obtainNH2-Ru@SiO2A dispersion liquid;
(3)NGQDs-NH2-Ru@SiO2preparing a composite material:
firstly, NH prepared in step (2)2-Ru@SiO2Mixing the dispersion with NGQDs, stirring to react, dissolving the obtained product with secondary water, centrifuging to obtain upper layer yellow clear liquid, i.e. NGQDs-NH2-Ru@SiO2；
(4)apt-NGQDs-NH2-Ru@SiO2Preparing a composite material:
first, NGQDs-NH prepared in step (3)2-Ru@SiO2Adding PBS buffer solution containing EDC and NHS, adding aptamer apt after reaction, and continuously stirring to obtain yellow apt-NGQDs-NH2-Ru@SiO2Standing at 4 deg.C;
(5) 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;
(6) soaking the modified electrode obtained in the step (5) in HAuCl4In the solution, electroplating is carried out under the potential from minus 0.25V to minus 0.1V, the time is controlled within 15s to 30s, and a layer of Au NPs is modified on the surface of the electrode and is used for connecting the material prepared in the step (4);
(7) the self-enhanced luminophore material apt-NGQDs-NH prepared in the step (4) is added2-Ru@SiO2Modifying the surface of the glassy carbon electrode prepared in the step (6), and fixing the glassy carbon electrode on the surface of the electrode through an Au-S covalent bond to form a self-assembled electrode;
(8) immersing the self-assembled electrode prepared in the step (7) into a bovine serum albumin BSA solution for incubation at a certain temperature to block the remaining non-specific binding sites, and preparing the electrode based on apt-NGQDs-NH2-Ru@SiO2The electrochemiluminescent aptamer sensor of (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, 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 reaction time is 24h, and centrifugal speed is 10000rpm, each time for 5min, obtaining 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 the step (4), NGQDs-NH2-Ru@SiO2PBS buffer, apt at a ratio of 800 μ L: 400 μ L: 800 μ L, PBS concentration of 0.01M, in which NHS concentration of 0.005M, EDC concentration of 0.01M, apt concentration of 2.5 μ M.
In the step (4), the sequence of the aptamer apt is as follows: 5' -NH2-TTGTTTGTCCCCTCTTTCTTA-(CH2)3-SH-3'。
The reaction time is 15 min; the time for continuing stirring after adding the aptamer is 2 h.
In the step (5), 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 (6), HAuCl4The mass percentage concentration of the solution is 1 percent.
In the step (7), apt-NGQDs-NH2-Ru@SiO2The dosage is 6 mu L; the reaction temperature is 4 ℃, and the reaction time is 8-12 h.
In the step (8), 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 × 10-12～5×10-6mol/L, after which the electrodes were washed with PBS (pH 7.0) solution. The prepared sensor is used as a working electrode, and a saturated Ag/AgCl electrode is used as a referenceElectrode, platinum electrode as counter electrode, in 0.1M PBS (pH 7.4) buffer solution for testing, Hg was obtained2+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 7.4) 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 apt-NGQDs-NH2-Ru@SiO2To the luminophor NH2-Ru@SiO2Is compounded with coreactant NGQDs to form a luminescent group (NGQDs-NH)2-Ru@SiO2) ECL signal strength may be enhanced. When the target Hg is2+After appearance, T-Hg formation with apt2+The specific structure of-T, making apt to bend, the luminophores NGQDs-NH2-Ru@SiO2The electron transfer distance is shortened near the electrode, the ECL signal intensity is further enhanced, and Hg is treated according to the change of the ECL signal intensity2+Detection of (3).
The invention has the beneficial effects that:
(1) the self-enhanced ECL biosensor provided by the invention is simple in preparation method, simplifies the complexity of experimental operation, and realizes simple, rapid and sensitive analysis and detection of a target object. Carbon dots as luminous bodies Ru (bpy) instead of toxic and volatile amines3 2+The coreactant not only can avoid the use of harmful coreactant, but also can improve the stability of the system and solve the problem of poor stability.
(2) Replaces the conventional ECL with a novel self-reinforcing ECL and is first applied to Hg2+The detection of (2) can obtain higher recovery rate in the detection of the actual sample.
(3) Construction of the inventionThe self-enhanced ECL aptamer sensor is used for Hg in a water body2+The specific detection has high sensitivity, good selectivity, good stability and wide linear range of 5 multiplied by 10-11-1×10-6mol L-1(ii) a Detection limit is as low as 1.0 multiplied by 10-12M。
Drawings
Fig. 1 is a diagram of a self-enhanced ECL aptamer sensor construction process.
FIG. 2(A) shows apt-NGQDs-NH2-Ru@SiO2Plot of incubation time versus ECL; (B) the incubation time of the target was plotted against ECL.
FIG. 3(A) shows Ru @ SiO2A TEM image of (B); (B) composite material NH2-Ru@SiO2A TEM image of (B); (C) TEM images of NGQDs; (D) composite material apt-NGQDs-NH2-Ru@SiO2A TEM image of (a).
FIG. 4(A) shows different Hg concentrations2+The corresponding ECL signal change: the concentration is 5 multiplied by 10 in sequence-11、2×10-10、1×10-9、 1×10-8、1×10-7、1×10-6mol/L; (B) different concentrations of Hg2+A linear relationship is constructed between the logarithm and the electrochemiluminescence signal.
Fig. 5(a) reproducibility of self-enhanced ECL aptamer sensors between different electrodes, (B) stable performance of the aptamer sensors for 7 consecutive days.
Figure 6 self-enhances the selectivity of ECL aptamer sensors. The interferents are respectively Mg2+、Fe2+、Fe3+、Na+、Ca2+、 Cd2+、Mn2+、Pb2+、Zn2+、Cu2+。
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)apt-NGQDs-NH2-Ru@SiO2preparing a composite material:
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) and 5mL (5mg/mL) of NGQDs are mixed and stirred for 12h to obtain a yellow solution NGQDs-NH2-Ru@SiO2。
In NGQDs-NH2-Ru@SiO2Adding 0.01M buffer solution (PBS, pH 7.4) containing 0.01M EDC and 0.005M NHS, stirring for 15min to allow amide reaction, adding 2.5 μ M aptamer apt after reaction, and stirring to obtain apt-NGQDs-NH2-Ru@SiO2And after the reaction is finished, centrifuging the obtained product for 5min at the centrifugal rotating speed of 10000rpm, and standing at 4 ℃ for later use.
(2) 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.
(3) Soaking the modified electrode obtained in the step (2) in 1% HAuCl4Electroplating at a potential of between-0.25V and-0.1V for 15 to 30s, and modifying a layer of Au nano-particles Au NPs on the surface of the electrode, wherein the sensor is represented as Au NPs/GCE;
(4) self-enhancing luminophore material apt-NGQDs-NH2-Ru@SiO2Modifying the surface of the glassy carbon electrode prepared in the step (3), apt-NGQDs-NH2-Ru@SiO2The dosage is 6 μ L, fixing on the electrode surface through Au-S covalent bond to form self-assembled electrode, placing at 4 deg.C, incubating for 8-12h, wherein the sensor is represented as apt-NGQDs-NH2-Ru@SiO2/Au NPs/GCE；
(5) At normal temperature, the steps(4) The prepared self-assembled electrode is washed with secondary water and incubated for 10-60min at 37 ℃ in 1% bovine serum albumin BSA solution to block the remaining non-specific binding sites, and the prepared ap-NGQDs-NH based electrode is prepared2-Ru@SiO2When the sensor is expressed as BSA/apt-NGQDs-NH2-Ru@SiO2/Au NPs/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 120min at room temperature, and then the electrode was washed with PBS (pH 7.0). The prepared sensor is used as a working electrode, a saturated Ag/AgCl electrode is used as a reference electrode, a platinum wire electrode is used as a counter electrode, and an MPI-EII electrochemiluminescence analyzer is used for recording ECL signals. 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 tube in the experiment is 650V.
FIG. 3(A) shows Ru @ SiO2The result shows Ru @ SiO2Is round and uniform in size; (B) composite material NH2-Ru@SiO2Representative TEM image of (A), observed when Ru @ SiO2After amination, the particle size becomes larger; (C) TEM image of NGQDs, the result shows that NGQDs are uniformly distributed in a spherical shape; (D) composite material NGQDs-NH2-Ru@SiO2TEM image of (A), it can be observed that NGQDs are successfully attached to NH2-Ru@SiO2Surface of (2) proves NGQDs-NH2-Ru@SiO2Successful synthesis of self-reinforcing materials.
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 left at 4 ℃ for 6, 8, 10, 12, 14h to detect ECL signals.
FIG. 2(A) shows apt-NGQDs-NH2-Ru@SiO2Plot of incubation time versus ECL; the incubation time was increased from 4h to 8h, and the ECL signal intensity gradually increased, reaching a plateau at 8 h. Therefore, 10h was chosen as the optimal incubation time for the aptamers.
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 40min to 70min, the ECL signal intensity is gradually increased, and the signal intensity is stabilized at 80min, and the target substance Hg is2+The incubation time on the electrode surface was 90 min.
Example 4
Detection of Hg by self-enhanced electrochemical luminescence aptamer sensor2+：
Respectively using 5X 10-11、2×10-10、1×10-9、1×10-8、1×10-7、1×10-6mol/L of Hg2+The best self-enhancing electrochemiluminescence aptamer sensor obtained in example 2 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.
Example 5
Self-enhanced electrochemiluminescence aptamer sensor selectivity analysis:
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)、Mn2+(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).
As can be seen from FIG. 4(A), as Hg is associated with2+Increase in concentration (concentration in the order of 5X 10-11、2×10-10、1×10-9、 1×10-8、1×10-7、1×10-6mol/L), the ECL signal gradually increases, which is attributed to Hg2+Form T-Hg with apt2+The specific structure of-T, allowing the apt to bend, thereby NGQDs-NH2-Ru@SiO2Close to the electrode surface.
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＝ 559lgCHg2++6323(R20.9959), linear range of 5.0 × 10-11-1.0×10-6M, detection limit of 1.0 × 10- 12M。
It can be seen from fig. 5(a) that the self-enhanced electrochemiluminescence aptamer sensor has good reproducibility.
It can be seen from fig. 5(B) that the self-enhanced electrochemiluminescence aptamer sensor has good stability.
It can be seen from fig. 6 that the change of ECL signal caused by other heavy metal ion interferents and the mixture of ten heavy metal ions is negligible, thus proving that the self-enhanced electrochemiluminescence aptamer sensor has good selectivity.

Claims (10)

1. Hg detection method2+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, and adding triton after the reaction is finishedThe bipyridyl ruthenium, tetraethyl silicate and ammonia water are continuously stirred for reaction, after the reaction is finished, the obtained product is sequentially dissolved in acetone, ethanol and secondary water, centrifuged and washed to obtain lower-layer precipitate, and vacuum dried 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)NGQDs-NH2-Ru@SiO2preparing a composite material:
firstly, NH prepared in step (2)2-Ru@SiO2Mixing the dispersion with NGQDs, stirring to react, dissolving the obtained product with secondary water, centrifuging to obtain upper layer yellow clear liquid, i.e. NGQDs-NH2-Ru@SiO2；
(4)apt-NGQDs-NH2-Ru@SiO2Preparing a composite material:
first, NGQDs-NH prepared in step (3)2-Ru@SiO2Adding PBS buffer solution containing EDC and NHS, adding aptamer apt after reaction, and continuously stirring to obtain yellow apt-NGQDs-NH2-Ru@SiO2Standing at 4 deg.C; the sequence of the aptamer apt is: 5' -NH2-TTGTTTGTCCCCTCTTTCTTA-(CH2)3-SH-3'；
(5) 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;
(6) soaking the electrode treated in the step (5) in HAuCl4In the solution, electroplating is carried out under the potential from minus 0.25V to minus 0.1V, the time is controlled within 15s to 30s, and a layer of Au NPs is modified on the surface of the electrode and is used for connecting the material prepared in the step (4);
(7) prepared by the step (4)Self-enhancing luminophore material apt-NGQDs-NH2-Ru@SiO2Modifying the surface of the glassy carbon electrode prepared in the step (6), and fixing the glassy carbon electrode on the surface of the electrode through an Au-S covalent bond to form a self-assembled electrode;
(8) immersing the self-assembled electrode prepared in the step (7) into a bovine serum albumin BSA solution for incubation at a certain temperature to block the remaining non-specific binding sites, and preparing the electrode based on apt-NGQDs-NH2-Ru@SiO2The electrochemiluminescent aptamer sensor of (1).
2. The method of claim 1 for detecting Hg2+The preparation method of the self-enhanced electrochemiluminescence aptamer sensor is characterized in that in the step (1), the dosage ratio 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。
3. The method of claim 1 for detecting Hg2+The preparation method of the self-enhanced electrochemiluminescence aptamer sensor is characterized in that 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。
4. The method of claim 1 for detecting Hg2+The method for preparing the self-enhanced electrochemiluminescence aptamer sensor is characterized in that in the step (3), NH is added2-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.
5. The method of claim 1 for detecting Hg2+FromThe preparation method of the enhanced electrochemiluminescence aptamer sensor is characterized in that in the step (4), NGQDs-NH2-Ru@SiO2PBS buffer, apt at a ratio of 800 μ L: 400 μ L: 800 μ L, PBS concentration of 0.01M, in which NHS concentration of 0.005M, EDC concentration of 0.01M, apt concentration of 2.5 μ M.
6. The method of claim 1 for detecting Hg2+The method for preparing the self-enhanced electrochemiluminescence aptamer sensor is characterized in that in the step (4),
the reaction time is 15 min; the time for continuing stirring after adding the aptamer is 2 h.
7. The method of claim 1 for detecting Hg2+The method for preparing the self-enhanced electrochemiluminescence aptamer sensor is characterized in that in the step (5), 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.
8. The method of claim 1 for detecting Hg2+The method for preparing the self-enhanced electrochemiluminescence aptamer sensor is characterized in that in the step (6), HAuCl4The mass percentage concentration of the solution is 1 percent.
9. The method of claim 1 for detecting Hg2+The method for preparing the self-enhanced electrochemiluminescence aptamer sensor is characterized in that in the step (7), apt-NGQDs-NH2-Ru@SiO2The dosage is 6 mu L; the reaction temperature is 4 ℃, and the reaction time is 8-12 h.
10. The method of claim 1 for detecting Hg2+The preparation method of the self-enhanced electrochemical luminescence aptamer sensor is characterized in that in the step (8), the mass percentage concentration of a bovine serum albumin BSA solution is 1%; the incubation temperature is 37 deg.C, and the incubation time is 10-60 min.
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