CN114636746B - Pb detection2+Carboxyl ligand-induced annihilation ratio electrochemiluminescence aptamer sensing method - Google Patents
Pb detection2+Carboxyl ligand-induced annihilation ratio electrochemiluminescence aptamer sensing method Download PDFInfo
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Abstract
The invention belongs to the field of biosensing detection, and relates to an annihilation ratio electrochemiluminescence aptamer sensing method for detecting Pb 2+ by using a carboxyl ligand. According to the detection method, carboxyl terpyridine ruthenium with strong cathode-anode signal intensity is used as a luminophor, the resonance energy transfer receptor gold nanoparticle of the carboxyl terpyridine ruthenium is fixed by utilizing the hybridization of the Pb 2+ aptamer and complementary DNA thereof, and the annihilation type ratio electrochemiluminescence aptamer detection method for detecting Pb 2+ is developed, because the carboxyl terpyridine ruthenium with excellent annihilation performance and the resonance energy transfer receptor gold nanoparticle which can be distinguished at the cathode and anode are combined, the reliability of the annihilation type ratio electrochemiluminescence method is improved; because the aptamer of the specific recognition element Pb 2+ is introduced, the selectivity of an electrochemiluminescence method is improved, and the specific analysis of Pb 2+ is realized; the method has high sensitivity, good selectivity and high reliability.
Description
Technical Field
The invention belongs to the field of biosensing detection, and particularly relates to an annihilation ratio electrochemiluminescence aptamer sensing method for detecting Pb 2+ by using a carboxyl ligand.
Background
The problem of heavy metal pollution in soil has attracted great attention from various governments and related international organizations. Pb 2+ is used as common heavy metal ions in farmland soil, and has the characteristics of difficult decomposition, high toxicity, easy enrichment in organisms and the like. They can contaminate farmland soil, reduce the fertility of tillable soil, resulting in reduced crop yields; and the quality and safety of agricultural products can be influenced by absorption and accumulation in plants, so that the human health is threatened. Currently, the detection and analysis methods of Pb 2+ mainly include atomic absorption spectrometry, flame atomic absorption spectrometry, inductively coupled plasma-mass spectrometry, inductively coupled plasma-emission spectrometry, electrochemical methods, fluorescence methods, photoelectrochemical methods, electrochemical luminescence methods (ECL), and the like. The analysis methods have good sensing value in detection of Pb 2+.
Among them, the ECL method is widely used for detecting Pb 2+ due to its advantages of high sensitivity, short response time, wide dynamic range, good selectivity, and the like. Among the numerous luminophores, tris (2, 2' -bipyridine) ruthenium (II) dichloride (Ru (bpy) 3 2+) has been widely used because of its advantages of high luminescence efficiency, good chemical stability and good solubility in aqueous and organic solutions. Currently, most Ru (bpy) 3 2+ -based annihilation reactions are highly dependent on the organic environment in which the supporting electrolyte is required. However, the analytical environment of this organic phase limits the application of Ru (bpy) 3 2+ -based annihilation ECL in the field of ECL biosensing with aqueous phase as the core. Ru (bpy) 3 2+ in aqueous media has been reported to exhibit a relatively good anodic ECL signal, whereas its cathodic ECL emission is very weak due to the unusual instability of the reduced form of Ru (bpy) 3 2+. Although a single luminophore Ru (bpy) 3 2+ may directly emit two ECL signals, the mismatch between its anodic and cathodic signal intensities is detrimental to the construction and development of rate sensing strategies. To solve this problem, researchers have achieved well matched anode and cathode signals by introducing Ru (bpy) 3 2+ cathode co-reactant. However, in some applications, the introduction of co-reactants may interfere with the detection environment and affect the stability of ECL systems, resulting in measurement errors. Therefore, it is critical to develop an annihilation luminophore that produces strong ECL emissions without any co-reactants to meet the needs of a ratiometric electrochemiluminescence sensing strategy to detect Pb 2+.
Disclosure of Invention
Aiming at the problems existing in the prior art, the invention takes carboxyl terpyridine ruthenium with strong cathode-anode signal intensity as a luminophor, utilizes the hybridization of Pb 2+ aptamer and complementary DNA (cDNA) thereof to fix the resonance energy transfer receptor gold nano particles (AuNPs) of the carboxyl terpyridine ruthenium, and develops a carboxyl ligand-induced annihilation ratio electrochemiluminescence aptamer detection method for detecting Pb 2+.
The invention is realized by the following technical scheme:
A carboxyl ligand-induced annihilation ratio electrochemiluminescence aptamer detection method for detecting Pb 2+, comprising the steps of:
(1) Preparation of AuNPs and AuNPs-cDNA;
Dissolving HAuCl 4·3H2 O solution in ultrapure water, stirring and dissolving to obtain a mixed solution; then heating the mixed solution to boiling at a certain temperature, and adding sodium citrate solution to obtain red solution; then, standing for reacting for a period of time, and cooling to room temperature to obtain AuNPs solution;
In the preparation of AuNPs-cDNA, firstly, cDNA solution with mercapto group at the end and tris (2-carbonyl ethyl) phosphorus hydrochloride (TCEP) solution are mixed and stood for a period of time to obtain activated cDNA solution; then mixing the activated cDNA solution with the AuNPs solution, shaking and incubating, and centrifugally collecting supernatant after incubating to obtain the AuNPs-cDNA solution;
(2) Sequentially polishing a Glassy Carbon Electrode (GCE) with aluminum oxide powder with different particle sizes, sequentially performing ultrasonic treatment in ethanol and water, and finally drying in air to obtain a pretreated electrode;
(3) Immersing the electrode pretreated in the step (2) in HAuCl 4·3H2 O solution to electrodeposit gold particles (AuPs), and obtaining an electrodeposited electrode after the electrodepositing treatment;
(4) Modifying Pb 2+ aptamer (Apt) solution on the electrodeposited electrode prepared in the step (3), and continuously reacting for a period of time under a certain temperature condition to obtain an aptamer electrode;
(5) Dropwise adding a mercapto hexanol (MCH) solution on the aptamer electrode prepared in the step (4), and incubating at room temperature to obtain an incubated electrode;
(6) Dropwise adding the AuNPs-cDNA solution prepared in the step (1) on the electrode after incubation in the step (5); reacting for a period of time under a certain temperature condition, and obtaining a sensor after the reaction, wherein the sensor is marked as AuNPs-cDNA/MCH/Apt/AuPs/GCE;
(7) Placing the sensors obtained in the step (6) in Pb 2+ standard solutions with different concentrations for binding for a certain time, wherein one Pb 2+ standard solution with one concentration corresponds to one sensor, and the concentrations and the sensors are in one-to-one correspondence; naturally airing at room temperature after washing with ultrapure water; the electrode thus prepared was designated Pb 2+/AuNPs-cDNA/MCH/Apt/AuPs/GCE-1; the electrode is used as a working electrode, a saturated Ag/AgCl electrode is used as a reference electrode, and a platinum wire electrode is used as a counter electrode; testing the three-electrode system in a buffer solution containing carboxyl terpyridyl ruthenium, and detecting an electrochemiluminescence signal; recording and detecting an electrochemiluminescence signal by an MPI-EII electrochemiluminescence analyzer; by monitoring the change of the ECL signal ratio (I Yin type vagina /I Yang (Yang) ) of the cathode and the anode of carboxyl terpyridyl ruthenium, a standard linear curve of the corresponding relation between Pb 2+ solution concentration and electrochemiluminescence signals is established;
(8) Detection of Pb 2+ concentration in sample to be measured:
Placing the sensor obtained in the step (6) into a solution to be detected of Pb 2, flushing with ultrapure water, naturally airing at room temperature, and marking as Pb 2+/AuNPs-cDNA/MCH/Apt/AuPs/GCE-2; pb 2+/AuNPs-cDNA/MCH/Apt/AuPs/GCE-2 is used as a working electrode, a saturated Ag/AgCl electrode is used as a reference electrode, and a platinum wire electrode is used as a counter electrode; testing the three-electrode system in a buffer solution containing carboxyl terpyridyl ruthenium, and detecting an electrochemiluminescence signal; substituting the monitored change of the I Yin type vagina /I Yang (Yang) of the carboxyl terpyridine ruthenium into the standard linear curve established in the step (7) to obtain the concentration of Pb 2+ in the solution to be detected, thereby realizing high-sensitivity detection of Pb 2+.
Further, in the step (1), the concentration of the HAuCl 4·3H2 O solution is 0.1M, and the certain temperature is 150 ℃; the concentration of the sodium citrate solution is 100 mg/mL -1, and the volume ratio of the HAuCl 4·3H2 O solution to the ultrapure water to the sodium citrate solution is 0.2:25:0.25; the standing reaction is carried out for 15-20 min;
The concentration of the cDNA solution with the mercapto group at the tail end is 100 mu M; the tris (2-carboxyethyl) phosphonium chloride (TCEP) solution has a concentration of 10mM; the volume ratio of the cDNA with the sulfhydryl group at the tail end to the TCEP solution is 0.5:1; and standing for 1h.
The volume ratio of the activated cDNA solution to AuNPs solution is 150. Mu.L: 1850 μl; the shaking incubation time was 12h.
Further, in the step (2), the diameter of the GCE is 3mm; the particle sizes of the aluminum oxide powder with different particle sizes are 0.3 μm and 0.05 μm in sequence.
Further, in the step (3), the mass concentration of HAuCl 4·3H2 O is 0.1%; the potential of the electrodeposition was-0.2V, and the time of the electrodeposition was 45s.
Further, in the step (4), the concentration of the Pb 2+ aptamer solution is 0.8 mu M, and the amount of modification on the electrode is 6 mu L; the certain temperature is 4 ℃, and the reaction time is 12 hours.
Further, in step (5), the concentration of the MCH solution is 1mM; the amount of the solution to be added dropwise to the electrode was 6. Mu.L; the incubation time was 1h.
Further, in the step (6), the concentration of the AuNPs-cDNA solution is 0.2-5.2 nM; the drop dosage is 6 mu L; the certain temperature condition is 37 ℃ and the reaction time is 2 hours.
Further, in the step (7), the concentration of the Pb 2+ solution is 1.0×10 -12～3.0×10-8 M; the binding time is 40min; the buffer solution is phosphate buffer solution, the concentration is 0.1M, and the pH value is 7.5; the carboxyl terpyridyl ruthenium is one of tris (2, 2' -bipyridyl) ruthenium (II) dichloride hexahydrate (Ru (bpy) 3 2+), bis (2, 2' -bipyridyl) (4-carboxyl-2, 2' -bipyridyl) ruthenium (II) dichloride (Ru (cbpy) 3 2+) or tris (4, 4' -dicarboxylic acid-2, 2' -bipyridyl) ruthenium (II) dichloride (Ru (dcbpy) 3 2+), and the concentration of the carboxyl terpyridyl ruthenium in the buffer solution is 0.5mM. The parameters adopted in the test are as follows: the voltage is-1.8V- +1.35V, the scanning speed is 0.1V/s, and the high voltage of the photomultiplier is 700V.
Further, in the step (8), the binding is performed for 40min; the buffer solution is phosphate buffer solution, the concentration is 0.1M, and the pH value is 7.5; the carboxyl terpyridine ruthenium is one of Ru (bpy) 3 2+、Ru(cbpy)3 2+ and Ru (dcbpy) 3 2+, and the concentration of the carboxyl terpyridine ruthenium in the buffer solution is 0.5mM. The parameters adopted in the test are as follows: the voltage is-1.8V- +1.35V, the scanning speed is 0.1V/s, and the high voltage of the photomultiplier is 700V.
The working principle of the carboxyl ligand-induced annihilation ratio electrochemiluminescence aptamer sensor is as follows:
It has been found that ruthenium carboxyterpyridine in an aqueous medium can undergo electrochemical oxidation and reduction reactions on the electrode surface, resulting in simultaneous anode and cathode ECL emissions. The mechanism research shows that the efficient annihilation ECL luminescence of carboxyl terpyridine ruthenium depends on carboxyl serving as an auxiliary ligand. The carboxyl has excellent stabilization effect on free radical ions of carboxyl terpyridine ruthenium, and can effectively promote transfer in a ligand and charge transfer from metal to the ligand, thereby generating more emission excited states. Carboxyl terpyridine ruthenium based on double emission signals is used for constructing an annihilation ratio ECL aptamer sensor for Pb 2+ analysis by introducing energy transfer resonance receptors AuNPs which can be distinguished by the carboxyl terpyridine ruthenium at an anode and a cathode, so that sensitive and reliable analysis of Pb 2+ in a soil sample is realized.
The invention has the beneficial effects that:
(1) According to the invention, through examining the ECL signal intensity of the cathode and anode of the carboxyl terpyridine ruthenium in the PBS buffer solution, the electrochemical and electrochemical luminescence properties of the annihilation type carboxyl terpyridine ruthenium are obtained, excellent cathode and anode signals are obtained, and finally, the controllable and simultaneous acquisition of the cathode and anode signals of the carboxyl terpyridine ruthenium is realized, thus providing a foundation for the construction of annihilation type ratio ECL aptamer sensors.
(2) The auxiliary ligand carboxyl based on carboxyl terpyridine ruthenium has excellent stabilization effect on free radical ions, and promotion effect on ligand internal transfer and metal-to-ligand charge transfer, so that the stability of the annihilation type electrochemiluminescence sensing method is improved.
(2) According to the invention, auNPs are used as Resonance Energy Transfer (RET) receptors of carboxyl terpyridine ruthenium distinguishable at an anode and a cathode, distinguishable signal intensity is obtained to complete the acquisition of the signal ratio of the anode and the cathode, an annihilation ratio ECL aptamer sensor is constructed, and the reliability of an electrochemiluminescence sensing method is improved.
(2) The invention utilizes the specific binding of Pb 2+ and the aptamer thereof to trigger the AuNPs-cDNA to be released from the electrode interface, so as to obtain electrochemiluminescence signals with different signal amplification degrees at the cathode and the anode, thereby improving the sensitivity of the electrochemiluminescence sensing method.
(5) According to the invention, the aptamer of the specific recognition element Pb 2+ is introduced, so that the selectivity of the ratio electrochemiluminescence aptamer sensing method is improved, the potential interference of other ions existing in an actual sample is reduced, and the Pb 2+ selective analysis is realized.
(6) The annihilation ratio electrochemiluminescence aptamer sensing method constructed by the invention is used for detecting Pb 2+, and has the advantages of high sensitivity, good reliability, good selectivity and good stability.
(7) The annihilation ratio electrochemiluminescence aptamer sensing method constructed by the invention is used for detecting Pb 2+ in a soil sample, so that satisfactory recovery rate is obtained, and meanwhile, the result is basically consistent with that of a standard method, so that the reliability of the electrochemiluminescence aptamer sensing method is shown.
Drawings
FIG. 1 (A) shows an annihilation electrochemiluminescence mechanism of ruthenium carboxyterpyridine, wherein the graph is an ECL-potential diagram of ruthenium carboxyterpyridine in PBS; (B) A mechanical plot of detection Pb 2+ for annihilation-ratio electrochemiluminescent aptamer, wherein the inset is the ECL-potential plot of the sensor before and after Pb 2+ is present.
FIG. 2 shows the structural formula of Ru (bpy) 3 2+,Ru(cbpy)3 2+,Ru(dcbpy)3 2+.
FIG. 3 shows the UV-visible absorption spectrum of AuNPs and the ECL spectrum of Ru (dcbpy) 3 2+.
Fig. 4 (a) shows ECL signals corresponding to the logarithm of Pb 2+ at different concentrations of ECL aptamer sensor, pb 2+ concentration: the concentrations from left to right were 1.0×10-12M,3.0×10-11M,1.0×10-10M,3.0×10-10M,1.0×10-9M,3.0×10-9M,1.0×10-8M and 3.0X10 -8 M in order, and the corresponding working curve (B).
FIG. 5 is a selection performance of ECL aptamer sensing: the interfering substances in the figure are K+,Na+,Ca2+,Mg2+,Fe2+,Fe3+,Cd2 +, Cu2+,Hg2+,Ni2+,Mn2+,Zn2+ and mixtures (Mix) of the above ions.
FIG. 6 is a graph of the reproducibility of ECL aptamer sensors within and between lots.
Detailed Description
The invention is further elucidated below in connection with specific embodiments and with the accompanying drawing. In the present invention, ECL annihilation of ruthenium carboxyterpyridine in aqueous medium is disclosed. Ruthenium carboxyterpyridine can undergo electrochemical oxidation and reduction reactions, resulting in simultaneous anode and cathode ECL emissions. Based on carboxyl terpyridine ruthenium of double emission signals, a ratio ECL aptamer sensor for Pb 2+ analysis is constructed by introducing resonance energy transfer acceptors AuNPs which can be distinguished by carboxyl terpyridine ruthenium at an anode and a cathode.
In the present invention, cDNA and Pb 2+ aptamer having a thiol group at the terminal are purchased from Shanghai, inc. of Biotechnology. The sequence is as follows: cDNA:5'-SH-AAC CAC ACC AA-3'
An aptamer: 5'-SH- (CH 2)6 -GGT TGG TGT GGT TGG-3'
Example 1:
the preparation process is as described in fig. 1:
(1) Preparation of AuNPs and AuNPs-cDNA
0.2ML of a 0.1M HAuCl 4·3H2 O solution was dissolved in 25mL of ultrapure water, and the solution was stirred and dissolved to obtain a mixed solution. Then heating the mixed solution to boiling at 150 ℃; after the solution is completely boiled, 0.25mL of 100 mg/mL -1 sodium citrate solution is rapidly added to obtain a red solution; then, the solution is cooled to room temperature after the reaction is continued for 15min, and AuNPs solution is obtained; the AuNPs solution is stored in a refrigerator at 4 ℃ for later use in a dark place;
Preparation of AuNPs-cDNA: firstly, 50 mu L of cDNA solution with thiol at 100 mu M end and 100 mu L of 10mM tris (2-carbonyl ethyl) phosphonium hydrochloride (TCEP) solution are mixed, and after standing for 1h, activated cDNA solution is obtained; then, 150. Mu.L of the activated cDNA solution and 1850. Mu.L of AuNPs solution were mixed, incubated with shaking for 12 hours, centrifuged after incubation to remove insoluble impurities, and the supernatant was collected as AuNPs-cDNA solution and stored in a refrigerator at 4℃in the absence of light for use.
(2) Sequentially polishing GCE with 0.3 μm and 0.05 μm aluminum oxide powder, and drying in air after ultrasonic treatment in ethanol and water, wherein the diameter d=3mm of the GCE;
(3) Immersing the electrode in the step (2) in HAuCl 4·3H2 O, electroplating for 45s at the potential of-0.2V, and forming a layer AuPs on the surface of the electrode to obtain an electrodeposited electrode;
(4) Modifying 6 mu L of the Apt solution of 0.8 mu M Pb 2+ on the surface of the electrodeposited electrode prepared in the step (3), and reacting for 12 hours at 4 ℃ to obtain an aptamer electrode;
(5) Dropwise adding 6 mu L of 1mM MCH solution on the aptamer electrode prepared in the step (4), and incubating for 1h at room temperature to block the non-specific binding site, so as to obtain an incubated electrode;
(6) Dropwise adding 6 mu L of AuNPs-cDNA solution on the electrode after incubation in the step (5), and reacting for 2 hours at 37 ℃; obtaining a sensor which is marked as AuNPs-cDNA/MCH/Apt/AuPs/GCE; namely the ECL aptamer sensor;
(7) Placing the sensor obtained in the step (6) in Pb 2+ standard solutions with different concentrations, wherein the concentration of Pb 2+ is 1.0×10-12M,3.0×10-11M,1.0×10-10M,3.0×10-10M,1.0×10-9M,3.0×10-9M,1.0×10-8M and 3.0X10 -8 M in sequence, and naturally airing the sensor at room temperature after washing with ultrapure water; the electrode prepared at this time is named Pb 2+/AuNPs-cDNA/MCH/Apt/AuPs/GCE-1, the electrode is used as a working electrode, a saturated Ag/AgCl electrode is used as a reference electrode, and a platinum wire electrode is used as a counter electrode. The three electrode system was tested in a buffer solution containing Ru (dcbpy) 3 2+ at a concentration of 0.5mM Ru (dcbpy) 3 2+; detecting an electrochemiluminescence signal; by monitoring the change of I Yin type vagina /I Yang (Yang) of Ru (dcbpy) 3 2+, a standard linear curve of the corresponding relation between Pb 2+ solution concentration and electrochemiluminescence signals is established;
(8) Placing the sensor obtained in the step (6) into a solution to be detected of Pb 2, flushing with ultrapure water, naturally airing at room temperature, and marking as Pb 2+/AuNPs-cDNA/MCH/Apt/AuPs/GCE-2; pb 2+/AuNPs-cDNA/MCH/Apt/AuPs/GCE-2 is used as a working electrode, a saturated Ag/AgCl electrode is used as a reference electrode, and a platinum wire electrode is used as a counter electrode. The three electrode system was tested in a buffer solution of Ru (dcbpy) 3 2+ at a concentration of 0.5mM Ru (dcbpy) 3 2+; detecting an electrochemiluminescence signal; substituting the monitored change of I Yin type vagina /I Yang (Yang) of Ru (dcbpy) 3 2+ into the standard linear curve established in the step (7) to obtain the concentration of Pb 2+ in the solution to be detected, so as to realize high-sensitivity detection of Pb 2+.
From FIG. 2, it can be seen that Ru (bpy) 3 2+ does not contain a carboxyl functionality, ru (cbpy) 3 2+ contains 1 carboxyl functionality, and Ru (dcbpy) 3 2+ contains 6 carboxyl functionalities.
From fig. 3, it can be seen that the uv-visible absorption peak of AuNPs at 520nm has good spectral overlap with ECL spectrum of the anode at 650nm and ECL spectrum of the cathode at 700nm of Ru (dcbpy) 3 2+, respectively, indicating that AuNPs can be used as Ru (dcbpy) 3 2+ interaction probe. More importantly, the ECL spectrum of Ru (dcbpy) 3 2+ anode and the uv absorption peak of AuNPs have a tighter spectral overlap than the ECL spectrum of Ru (dcbpy) 3 2+ cathode, and therefore the RET performance of AuNPs to Ru (dcbpy) 3 2+ in the anode is higher than that of the cathode.
From fig. 4 (a), it can be seen that the emission signals of both the anode and cathode ECL gradually increase with increasing Pb 2+ concentration, and the anode emission increases to a significantly higher extent than the cathode, which verifies that the resonance energy transfer capability of AuNPs to Ru (dcbpy) 3 2+ is different. The logarithmic concentrations of I Yin type vagina /I Yang (Yang) and Pb 2+ between 3.0×10 -12-1.0×10-8 M are well-linear fig. 4 (B). The linear regression equation was I Yin type vagina /I Yang (Yang) ＝-1.469-0.343logCPb 2+, the correlation coefficient (R 2) was 0.998, and the detection limit was 1.3X10 -13 M.
Example 2:
using the sensor prepared in example 1 to investigate the selectivity of annihilation ratio electrochemiluminescence aptamer sensors;
When the ECL aptamer sensor is placed in an aqueous solution, the obtained I Yin type vagina /I Yang (Yang) is a blank signal; when the blank was replaced with different ions (K+, Na+,Ca2+,Mg2+,Fe2+,Fe3+,Cd2+,Cu2+,Hg2+,Ni2+,Mn2+,Zn2+,Mix,Pb2+ and Pb 2+ +mix), respectively, I Yin type vagina /I Yang (Yang) obtained was the test signal; the absolute value of the difference between the test signal and the blank signal is recorded as |DeltaI Yin type vagina /I Yang (Yang) |, and the selection performance is examined.
FIG. 5 shows the selective performance of the sensor: the blank is an aqueous solution, which is defined as a blank ;K+,Na+,Ca2+,Mg2+, Fe2+,Fe3+,Cd2+,Cu2+,Hg2+,Ni2+,Mn2+,Zn2+,Mix as an interfering substance, and Mix is a mixed solution of the above 12 interfering ions.
Example 3:
Using the sensor prepared in example 1, the reproducibility of annihilation ratio electrochemiluminescence aptamer sensors was examined;
When 5 ECL aptamer sensors prepared in parallel in the same batch were placed in 5 Pb 2 + solutions containing 1.0×10 -9 M, respectively, I Yin type vagina /I Yang (Yang) in the batch was recorded; when 5 ECL aptamer sensors prepared in different batches were placed in 5 Pb 2+ solutions containing 1.0×10 -9 M, respectively, I Yin type vagina /I Yang (Yang) was recorded between batches.
From fig. 6, it can be seen that the ECL signal Relative Standard Deviation (RSD) of the 5 parallel aptamer sensors in the batch was 2.86% and 2.62% between batches, demonstrating good reproducibility of the sensors.
Example 4:
the sensor and the detection method prepared in example 1 were used as an actual detection model to detect the farmland soil extract.
(1) Collecting and treating farmland soil
Farmland soil was collected in the north of the lake and treated according to industry standard (HJ 803-2016). And (5) air-drying the obtained soil sample, and finely grinding the soil sample to a nylon sieve of 200 meshes. A mixed solution of 0.1g of soil sample and 6mL of nitric acid/hydrochloride (HCl/HNO 3, v/v, 3/1) was gradually added to the Erlenmeyer flask and heat digestion was performed on an electric heating plate. After digestion, impurities were removed by filtration through a 0.22 μm cellulose membrane and the pH of the extract was adjusted to 7.0 with 1M NaOH. Finally, the solution was further diluted to the desired concentration for further use.
(2) And (3) adding a part of the extracting solution to prepare, so that the concentration of Pb 2+ in the farmland soil extracting solution is 0,5 multiplied by 10 -11M,5×10-10 M and 5 multiplied by 10 -9 M respectively, and obtaining 4 samples to be tested in total.
(3) Placing the 4 electrodes obtained in the step (6) in the example 1 into 4 Pb 2+ solutions to be tested respectively, flushing with ultrapure water, naturally airing at room temperature, and marking as Pb 2+/AuNPs-cDNA/MCH/Apt/AuPs/GCE-2; the electrode Pb 2+/AuNPs-cDNA/MCH/Apt/AuPs/GCE-2 prepared at this time is used as a working electrode, a saturated Ag/AgCl electrode is used as a reference electrode, and a platinum wire electrode is used as a counter electrode. Testing the three-electrode system in a buffer solution containing Ru (dcbpy) 3 2+, detecting the cathode and anode ECL signals of Ru (dcbpy) 3 2+, and performing ratio, repeating the operation for 3 times, and taking an average value; substituting the Pb 2+ into the standard linear curve established in the step (7) to obtain the concentration of Pb 2+ in the solution to be detected.
(3) The reliability of the developed electrochemical luminescence aptamer sensing method was verified by standard methods (inductively coupled plasma mass spectrometry, ICP-MS); and measuring the concentration of Pb 2+ in the soil sample using the developed electrochemiluminescence aptamer sensing method (n=3) and ICP-MS method, the results are shown in table 1;
Table 1: detection of Pb 2+ concentration in soil sample
From table 1, it can be seen that the background concentration of Pb 2+ was detected as 2.40×10 -12 M using the aptamer sensor (table 1). The recovery rate obtained by adding Pb 2+ with different concentrations is between 93.6% and 103%, and RSD is lower than 5.54%. The recovery rate obtained by the ICP-MS method was between 78.2% and 101%, and the results verify the reliability of the aptamer sensor. More importantly, ECL aptamer sensors were able to detect 2.40×10 -12 M Pb 2+, whereas ICP-MS was able to detect only 6.28×10 -10 M Pb 2+. The aptamer sensor exhibits higher sensitivity and comparable reliability in analysis of actual samples relative to ICP-MS methods.
Description: the above embodiments are only for illustrating the present invention and not for limiting the technical solution described in the present invention; thus, while the invention has been described in detail with reference to the various embodiments described above, it will be understood by those skilled in the art that the invention may be modified or equivalents; all technical solutions and modifications thereof that do not depart from the spirit and scope of the present invention are intended to be included in the scope of the appended claims.
Claims (7)
1. A carboxyl ligand-induced annihilation ratio electrochemiluminescence aptamer sensing method for detecting Pb 2+ is characterized by comprising the following steps:
(1) Preparation of AuNPs and AuNPs-cDNA;
Dissolving HAuCl 4·3H2 O solution in ultrapure water, stirring and dissolving to obtain a mixed solution; then heating the mixed solution to boiling at 150 ℃, and adding sodium citrate solution with the concentration of 100 mg.mL -1 to obtain red solution; then, standing for 15-20 min, and cooling to room temperature to obtain AuNPs solution; the HAuCl 4·3H2 O solution had a concentration of 0.1M; the volume ratio of the HAuCl 4·3H2 O solution to the ultrapure water to the sodium citrate solution is 0.2:25:0.25;
In the process of preparing AuNPs-cDNA, firstly, cDNA solution with sulfhydryl group at the end and tri (2-carbonyl ethyl) phosphorus hydrochloride solution are mixed, and the mixture is stood for 1 h to obtain activated cDNA solution; then mixing the activated cDNA solution with the AuNPs solution, shaking and incubating, and centrifugally collecting supernatant after incubating to obtain the AuNPs-cDNA solution; the concentration of the cDNA solution with the mercapto group at the tail end is 100 mu M; the concentration of the tris (2-carboxyethyl) phosphorus hydrochloride solution is 10 mM; the volume ratio of the cDNA with the mercapto group at the tail end to the tris (2-carbonyl ethyl) phosphorus hydrochloride solution is 0.5:1; the volume ratio of the activated cDNA solution to AuNPs solution is 150. Mu.L: 1850. mu L; the shaking incubation time is 12 h;
(2) Sequentially polishing the glassy carbon electrode with aluminum oxide powder with different particle sizes, sequentially performing ultrasonic treatment in ethanol and water, and finally drying in air to obtain a pretreated electrode;
(3) Immersing the electrode pretreated in the step (2) in HAuCl 4·3H2 O solution to electrodeposit gold particles, and obtaining an electrodeposited electrode after the electrodepositing treatment;
(4) Modifying Pb 2+ aptamer solution on the electrodeposited electrode prepared in the step (3), and continuing to react at the temperature of 4 ℃ for 12 h to obtain an aptamer electrode; the concentration of the Pb 2+ aptamer solution is 0.8 mu M, and the modification dosage on the electrode is 6 mu L;
(5) Dropwise adding a mercapto hexanol solution on the aptamer electrode prepared in the step (4), and incubating at room temperature to obtain an incubated electrode; the concentration of the mercapto hexanol solution is 1 mM; the amount of the solution to be added dropwise to the electrode was 6. Mu.L; the incubation time is 1 h;
(6) Dropwise adding the AuNPs-cDNA solution prepared in the step (1) on the electrode after incubation in the step (5); reacting at 37 ℃ for 2h ℃ to obtain a sensor which is marked as AuNPs-cDNA/MCH/Apt/AuPs/GCE; the concentration of the AuNPs-cDNA solution is 0.2-5.2 nM; the drop dosage is 6 mu L;
(7) Placing the sensor obtained in the step (6) in Pb 2+ standard solutions with different concentrations to bind 40min, wherein one Pb 2+ standard solution with one concentration corresponds to one sensor, and the concentrations and the sensors are in one-to-one correspondence; naturally airing at room temperature after washing with ultrapure water; the electrode thus prepared was designated Pb 2+/AuNPs-cDNA/MCH/Apt/AuPs/GCE-1; the electrode is used as a working electrode, a saturated Ag/AgCl electrode is used as a reference electrode, and a platinum wire electrode is used as a counter electrode; testing the three-electrode system in a buffer solution containing carboxyl terpyridyl ruthenium, and detecting an electrochemiluminescence signal; recording and detecting an electrochemiluminescence signal by an MPI-EII electrochemiluminescence analyzer; by monitoring the change of ECL signal ratio of a cathode and an anode of carboxyl terpyridyl ruthenium, a standard linear curve of the corresponding relation between Pb 2+ solution concentration and electrochemiluminescence signals is established; the concentration of the Pb 2+ solution is 1.0' -10 -12 ~ 3.0´10-8 M; the buffer solution is phosphate buffer solution, the concentration is 0.1M, and the pH value is 7.5; the carboxyl terpyridyl ruthenium is one of tris (2, 2' -bipyridyl) ruthenium (II) dichloride hexahydrate, bis (2, 2' -bipyridyl) (4-carboxyl-2, 2' -bipyridyl) ruthenium (II) dichloride or tris (4, 4' -dicarboxylic acid-2, 2' -bipyridyl) ruthenium (II) dichloride, and the concentration of the carboxyl terpyridyl ruthenium in a buffer solution is 0.5 mM; the parameters adopted in the test are as follows: the voltage is-1.8V to +1.35V, the scanning speed is 0.1V/s, and the high voltage of the photomultiplier is 700V;
(8) Detection of Pb 2+ concentration in sample to be measured:
Placing the sensor obtained in the step (6) into a solution to be detected of Pb 2+, binding 40 min, flushing with ultrapure water, naturally airing at room temperature, and marking as Pb 2+/AuNPs-cDNA/MCH/Apt/AuPs/GCE-2 after airing; then Pb 2+/AuNPs-cDNA/MCH/Apt/AuPs/GCE-2 is used as a working electrode, a saturated Ag/AgCl electrode is used as a reference electrode, and a platinum wire electrode is used as a counter electrode; testing the three-electrode system in a buffer solution containing carboxyl terpyridyl ruthenium, and detecting an electrochemiluminescence signal; substituting the monitored change of the I Yin type vagina /I Yang (Yang) of the carboxyl terpyridyl ruthenium into the standard linear curve established in the step (7) to obtain the concentration of Pb 2+ in the solution to be detected, so as to realize high-sensitivity detection of Pb 2+; the concentration of the carboxyl terpyridyl ruthenium in the buffer solution is 0.5 mM; the parameters adopted in the test are as follows: the voltage is minus 1.8V to plus 1.35V, the scanning speed is 0.1V/s, and the high voltage of the photomultiplier tube is 700V.
2. The carboxyl ligand-induced annihilation ratio electrochemiluminescence aptamer sensing method for detecting Pb 2+ according to claim 1, wherein in step (2), the diameter of the glassy carbon electrode is 3 mm.
3. The method for sensing carboxyl ligand-induced annihilation ratio electrochemiluminescence aptamer of Pb 2+ according to claim 1, wherein in step (2), the particle sizes of the aluminum oxide powders of different particle sizes are 0.3 mm and 0.05 mm in order.
4. The carboxyl ligand-induced annihilation ratio electrochemiluminescence aptamer sensing method for detecting Pb 2+ according to claim 1, wherein in step (3), the mass concentration of HAuCl 4·3H2 O is 0.1%.
5. The carboxyl ligand-induced annihilation type ratio electrochemiluminescence aptamer sensing method for detecting Pb 2+ according to claim 1, wherein in step (3), the electric potential of the electrodeposition is-0.2V, and the time of the electrodeposition is 45 s.
6. The method for detecting carboxyl ligand-induced annihilation ratio electrochemiluminescence aptamer of Pb 2+ as claimed in claim 1, wherein in step (8), the buffer solution is phosphate buffer solution with a concentration of 0.1m and a ph value of 7.5.
7. The method for detecting the carboxyl ligand-induced annihilation type ratio electrochemiluminescence aptamer of Pb 2+ as claimed in claim 1, wherein in step (8), the carboxyl terpyridyl ruthenium is one of tris (2, 2' -bipyridyl) ruthenium (II) dichloride hexahydrate, bis (2, 2' -bipyridyl) (4-carboxyl-2, 2' -bipyridyl) ruthenium (II) dichloride, and tris (4, 4' -dicarboxylic acid-2, 2' -bipyridyl) ruthenium (II) dichloride.
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