A method for detecting Cd 2+ Preparation method and application of electrochemiluminescence aptamer sensor

Abstract

The application belongs to the field of biosensing detection, and relates to a preparation method of an electrochemiluminescence aptamer sensor for detecting Cd 2+ and application thereof. The application first prepares two materials of PTCA-COF and AuNCs, and modifies the two materials to the surface of a treated glassy carbon electrode in a layer-by-layer manner. Meanwhile, a specific recognition element aptamer for Cd 2+ is introduced, and an electrochemiluminescence aptamer sensor based on PTCA-COF / AuNCs is constructed based on the quenching effect of Fc connected to the aptamer on the electrochemiluminescence signal. The electrochemiluminescence aptamer sensor takes PTCA-COF as a base material and enhances the electrochemiluminescence signal of the luminescent body AuNCs. The constructed sensor has excellent detection and analysis performance on the target Cd 2+ , and has a linear range of 1.0*10 ‑12 -5.0*10 ‑9 M, a detection limit of 6.6*10 ‑13 M, and advantages of high sensitivity, selectivity and reliability.

Description

Technical Field

[0001] This invention belongs to the field of biosensor technology, specifically relating to a method for detecting Cd. 2+ Preparation method and application of electrochemiluminescence aptamer sensor. Background Technology

[0002] Heavy metal pollution is a significant environmental problem in water bodies. Cadmium ions (Cd) 2+ Cadmium ions (Cd) are among the most common toxic heavy metal ions in water bodies, characterized by their long half-life, high mobility, high toxicity, and difficulty in degradation. With the rapid development of modern industry and the widespread use of cadmium-containing dyes, the concentration of Cd in water bodies has increased significantly. 2+ Pollution is becoming increasingly serious. Among the pollutants is Cd in water bodies. 2+ It accumulates in the soil, enters the food chain through food crops, and can build up in the human body, causing diseases such as diabetes, heart disease, and cancer. It can also damage kidney and liver function, harming human health. Therefore, achieving Cd control is crucial. 2+ Sensitive and specific detection is very important.

[0003] Electrochemiluminescence (ECL) is a technique that combines electrochemistry and chemiluminescence. It boasts advantages such as high sensitivity, wide linear range, low detection limit, minimal background interference, and ease of operation, leading to its rapid development in environmental monitoring, drug detection, and food analysis. In current ECL systems, the luminescent material is crucial to the analytical performance. Gold nanoclusters (AuNCs), widely used in ECL, offer advantages such as high stability and good optical properties. However, compared to organic-phase ECL, the aqueous ECL efficiency of AuNCs is lower due to the movement of surface ligands and the valence state changes of the internal inorganic gold nucleus.

[0004] To further improve the ECL luminescence performance of AuNCs, it is crucial to seek effective strategies to enhance their ECL efficiency. Starting with surface ligands, introducing loading materials to construct rigid structures has been proven effective in improving the ECL efficiency of AuNCs. However, the loading materials currently used for AuNCs are mainly chitosan nanogels and metal-organic frameworks, which suffer from poor environmental stability. Therefore, it is desirable to develop an electrochemiluminescence aptamer sensor to achieve the detection of Cd... 2+ Sensitive and specific detection. Summary of the Invention

[0005] To address the problems existing in the prior art, this invention aims to introduce the supporting material PTCA-COF to enhance the electrochemiluminescence signal of AuNCs, and combine it with a specific recognition element aptamer to construct a method for detecting Cd. 2+ An electrochemiluminescence aptamer sensor was developed to enable the detection of Cd. 2+Sensitive and accurate detection.

[0006] To achieve the above technical objectives, the present invention adopts the following technical solution.

[0007] A method for detecting Cd 2+ The method for preparing an electrochemiluminescence aptamer sensor includes the following steps:

[0008] (1) Preparation of PTCA-COF:

[0009] First, perylene tetracarboxylic dianhydride (PTCDA) and melamine (MA) are mixed and ground to obtain a grinding mixture. The resulting grinding mixture is calcined, then cooled to room temperature. The calcined product is taken out, washed, centrifuged, and the precipitate is collected and dried to obtain a dried solid powder, denoted as PTCA-COF.

[0010] (2) Preparation of AuNCs:

[0011] First, 6-aza-2-thiothymidine (ATT) and sodium hydroxide (NaOH) were weighed and dissolved in ultrapure water to obtain an ATT solution containing NaOH. Then, chloroauric acid (HAuCl4) was weighed and dissolved in ultrapure water to obtain an HAuCl4 solution. The ATT solution containing NaOH and the HAuCl4 solution were mixed, stirred evenly, and then subjected to a light-protected reaction to obtain a yellow solution. The yellow solution was then dialyzed, and the dialyzed solution was freeze-dried. The resulting powdered solid was AuNCs.

[0012] (3) Preparation of DNA double strands (Fc-Apt-CP):

[0013] Cd modified with Fc 2+ The aptamer solution is denoted as Fc-Apt, Cd 2+ The aptamer complementary strand solution is denoted as CP; Fc-Apt is mixed with CP, and the mixed solution is denatured and annealed to synthesize DNA double strands. The resulting product is labeled as Fc-Apt-CP.

[0014] (4) The glassy carbon electrode (GCE) was successively ground and polished with aluminum oxide powder of different particle sizes, and then ultrasonically cleaned in water, ethanol, acetone and water and air-dried to obtain the treated glassy carbon electrode.

[0015] (5) Disperse the PTCA-COF prepared in step (1) in ultrapure water to obtain a PTCA-COF solution. Then, drop the PTCA-COF solution onto the surface of the treated glassy carbon electrode and dry it at room temperature. The product is labeled as PTCA-COF / GCE.

[0016] (6) Dissolve the AuNCs prepared in step (2) in ultrapure water to obtain an AuNCs solution; and drop the AuNCs solution onto the surface of the PTCA-COF / GCE obtained in step (5), and dry it at room temperature. The resulting product is labeled as AuNCs / PTCA-COF / GCE.

[0017] (7) The Fc-Apt-CP prepared in step (3) is dropped onto the surface of AuNCs / PTCA-COF / GCE obtained in step (6), and after incubation, it is rinsed with Tris-HCl buffer solution. The product obtained after rinsing is labeled as Fc-Apt-CP / AuNCs / PTCA-COF / GCE.

[0018] (8) The short-chain DNA (ssDNA) solution modified on the surface of the Fc-Apt-CP / AuNCs / PTCA-COF / GCE electrode prepared in step (7) is incubated and then rinsed with Tris-HCl buffer solution. The result after rinsing is the electrochemiluminescence aptamer sensor, labeled as ssDNA / Fc-Apt-CP / AuNCs / PTCA-COF / GCE.

[0019] Preferably, in step (1), the molar ratio of perylene tetracarboxylic dianhydride (PTCDA) to melamine (MA) is 1:1; the calcination temperature is 325℃, the reaction time is 4h, and the heating rate is 5℃ / min; the mixing and grinding time is 30-40min; and the drying conditions are 60-80℃ for 6-12h.

[0020] Preferably, in step (2), the ratio of 6-aza-2-thiothymidine (ATT), sodium hydroxide (NaOH), and ultrapure water A is 0.1718 g: 0.12 g: 15 mL; the ratio of chloroauric acid (HAuCl4) to ultrapure water is 0.15 g: 15 mL; the volume ratio of the NaOH solution of ATT to the HAuCl4 solution is 1:1; the light-protected reaction time is 1 h; the molecular cutoff of the dialysis bag is 50 kDa, and the dialysis time is 24 h; the freeze-drying temperature is -60 °C, and the drying time is 72 h.

[0021] Preferably, in step (3), the concentrations of Fc-Apt and CP are both 1.8-3.0 μM, and the volume ratio of Fc-Apt to CP is 1:1; the denaturation annealing conditions are: denaturation temperature of 95℃, held for 3 min, and then annealed at 37℃ for 3 min.

[0022] Preferably, in step (4), the diameter of the glassy carbon electrode is 3 mm; the particle sizes of the different aluminum oxide powders used are 0.3 μm and 0.05 μm, respectively; in step (5), the concentration of the PTCA-COF solution is 0.2 mg / mL, and the amount of PTCA-COF solution used for modification is 6 μL; in step (6), the concentration of the AuNCs solution is 1 mg / mL, and the amount of AuNCs solution used for modification is 6 μL.

[0023] Preferably, in step (7), the concentration of Fc-Apt-CP is 1.8-3 μM, the amount of Fc-Apt-CP modification is 6 μL, the incubation temperature is 4℃, the incubation time is 10-12 h, and the pH value of the buffer solution is 7.4.

[0024] Preferably, in step (8), the concentration of the short-chain DNA solution is 2 μM; the amount of short-chain DNA solution used for modification is 6 μL; the incubation temperature is 37°C; and the incubation time is 60 min.

[0025] The prepared electrochemiluminescence aptamer sensor was used to detect Cd. 2+ The purpose and steps are as follows:

[0026] S1. Different concentrations of Cd were modified onto the surface of the obtained sensor ssDNA / Fc-Apt-CP / AuNCs / PTCA-COF / GCE. 2+ Standard solutions containing the sensor ssDNA / Fc-Apt-CP / AuNCs / PTCA-COF / GCE and Cd 2+ The concentrations of the standard solutions are in a one-to-one correspondence, meaning one sensor corresponds to one concentration of Cd. 2+ Standard solution; then incubate at room temperature and rinse the electrode with ultrapure water. The resulting product is labeled Cd. 2+ / ssDNA / Fc-Apt-CP / AuNCs / PTCA-COF / GCE; and using this electrode as the working electrode in a three-electrode system, with an Ag / AgCl (saturated KCl) electrode as the reference electrode and a platinum disk electrode as the counter electrode, the test was performed in a buffer solution containing triethylamine (TEA), and the electrochemiluminescence signal was detected; the electrochemiluminescence signal was used as the ordinate, and Cd was used as the symmetric axis. 2+ The lg value of the standard solution concentration is used as the x-axis to establish Cd. 2+ Standard linear curves showing the correlation between solution concentration and electrochemiluminescence signal;

[0027] S2, Actual sample Cd 2+ Detection:

[0028] First, a sample solution was obtained and then modified onto the surface of the sensor ssDNA / Fc-Apt-CP / AuNCs / PTCA-COF / GCE. After incubation at room temperature, the electrode was rinsed with ultrapure water and air-dried at room temperature. The dried electrode was used as the working electrode in a three-electrode system, with an Ag / AgCl (saturated KCl) electrode as the reference electrode and a platinum disk electrode as the counter electrode. The test was performed in a buffer solution containing triethylamine (TEA), and the electrochemiluminescence signal was recorded. The recorded electrochemiluminescence signal was then substituted into the standard linear curve equation established in S1 to calculate Cd. 2+ The concentration of Cd in actual samples was determined. 2+ The detection.

[0029] Preferably, the Cd mentioned in step S1 2+ The concentration of the standard solution is 5.0 × 10⁻⁶. -13 -5.0×10 -9 M; the different concentrations of Cd 2+ The standard solution modification volume was 6 μL; the incubation time was 60 min; the buffer solution was PBS with a concentration of 0.1 M and a pH of 7.5; the final concentration of triethylamine (TEA) contained was 0.05 M; the electrochemical detection instrument was ECL-MPI EII, and the parameters used for the test were: scanning voltage range of 0-1.4 V, and photomultiplier tube set to 500 V.

[0030] Preferably, the amount of sample solution modified in step S2 is 6 μL; the incubation time is 60 min; the buffer solution is PBS with a concentration of 0.1 M and a pH of 7.5; the final concentration of triethylamine (TEA) is 0.05 M; the electrochemical detection instrument is ECL-MPI EII, and the parameters used for the test are: scanning voltage range of 0-1.4 V, and photomultiplier tube set to 500 V.

[0031] The beneficial effects of this invention are as follows:

[0032] (1) The sensor constructed in this invention uses porous material PTCA-COF as a substrate, which increases the loading of AuNCs and promotes the construction of rigid AuNCs structure, exhibiting a strong AIE effect, thereby enhancing ECL luminescence.

[0033] (2) The sensor constructed in this invention improves the selectivity of the sensor by introducing a specific recognition element aptamer, and constructs a corresponding electrochemiluminescence aptamer sensor by quenching the luminescent AuNCs by the Fc group connected to one end of the aptamer.

[0034] (3) The electrochemiluminescence aptamer sensor constructed in this invention is used for Cd 2+The detection method exhibits high sensitivity and a wide linear range, with a detection limit of 0.66 pM and a linear range of 1 pM–5 nM. Furthermore, the results obtained from this invention in actual sample testing are largely consistent with those of standard methods, demonstrating high accuracy and reliability. Attached Figure Description

[0035] Figure 1 This is a schematic diagram of the construction process of the electrochemiluminescence aptamer sensor of the present invention; in the inset, Ⅰ is a schematic diagram of the basic signal of the ECL system, Ⅱ is a schematic diagram of the ECL signal change after the introduction of the double-stranded aptamer, and Ⅲ is a schematic diagram of the introduction of the target analyte Cd. 2+ A schematic diagram of the changes in the ECL signal afterward.

[0036] Figure 2 This is a diagram showing the optimal concentration of double-stranded DNA (Fc-Apt-CP).

[0037] Figure 3 (A) shows the ECL aptamer sensor at different Cd concentrations. 2+ The ECL signal corresponding to the logarithm, Cd 2+ Concentration: The concentrations are 1.0 × 10⁻⁶. -12 M, 5.0×10 -12 M, 1.0×10 -11 M, 5.0×10 -11 M, 1.0×10 -10 M, 5.0×10 -10 M, 1.0×10 -9 M, 5.0×10 -9 M; (B) is the corresponding working curve.

[0038] Figure 4 In the figure, (A) represents the selectivity of the electrochemiluminescence aptamer sensor; and (B) represents the reproducibility of the electrochemiluminescence aptamer sensor. Detailed Implementation

[0039] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0040] It should be understood that the terminology used herein is merely for describing particular embodiments and is not intended to limit the invention. Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0041] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be readily apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0042] The Cd used in this invention 2+ The aptamers (Apt), complementary strands (CP), and short DNA strands (ssDNA) were purchased from Sangon Biotech (Shanghai) Co., Ltd. These are standard primers and do not involve any sequence listing invention.

[0043] Its sequence is as follows:

[0044] Apt:5'-CTC AGG ACG ACG GGT TCA CAG TCC GTT GTC-Fc-3';

[0045] CP:5'-SH-(CH2)3-ACT AAT GAC AAC GGA CTG TGA ACC CGT CGT CCT GAG-3';

[0046] ssDNA:5'-SH-(CH2)3-CCCCCC-3'.

[0047] Example 1:

[0048] according to Figure 1 The sensor construction diagram shown illustrates the following steps in its fabrication:

[0049] (1) Preparation of PTCA-COF:

[0050] 0.3923 g of perylene tetracarboxylic dianhydride (PTCDA) and 0.1261 g of melamine (MA) were weighed and mixed evenly in an agate mortar and ground for 30 min. Then, the resulting mixture was transferred from the agate mortar to an alumina crucible and calcined in a tube furnace at 325 °C for 4 h at a heating rate of 5 °C / min. Subsequently, the calcined product was cooled to room temperature, washed with ultrapure water, and centrifuged. The precipitate obtained after washing was dried, and the resulting solid powder product was designated as PTCA-COF.

[0051] (2) Preparation of AuNCs:

[0052] Weigh 0.1718 g of 6-aza-2-thiothymidine (ATT) and 0.12 g of sodium hydroxide (NaOH) into a beaker, mix them, and then add 15 mL of ultrapure water to dissolve them, obtaining an ATT solution containing NaOH. Weigh 0.15 g of chloroauric acid (HAuCl4) and dissolve it in 15 mL of ultrapure water to obtain an HAuCl4 solution. Add the ATT solution containing NaOH to the HAuCl4 solution, stir and mix well, and react in the dark for 1 h to obtain a yellow solution. Then pour the yellow solution obtained from the reaction into a 50 kDa dialysis bag for dialyzing. After dialysis, freeze the solution and place it in a vacuum freezer at -60℃ for 72 h. The resulting powdered solid is AuNCs, which should be stored at 4℃ in the dark for later use.

[0053] (3) Preparation of DNA double strands (Fc-Apt-CP):

[0054] Take 3.6 μL, 4.0 μL, 4.4 μL, 4.8 μL, 5.2 μL, 5.6 μL, and 6 μL of 100 μM Fc-modified Cd, respectively. 2+ Aptamer solution (Fc-Apt) with 3.6 μL, 4.0 μL, 4.4 μL, 4.8 μL, 5.2 μL, 5.6 μL and 6 μL of 100 μM Cd 2+ The aptamer complementary strand (CP) solution was mixed, and then 192.8 μL, 192 μL, 191.2 μL, 190.4 μL, 189.6 μL, 188.8 μL, and 188.0 μL of Tris-HCl (pH = 7.4) were added accordingly to obtain seven mixed solutions, each with a total volume of 200 μL. The resulting mixed solutions were then denatured at 95 °C for 3 min and annealed at 37 °C for 3 min in a metal bath to synthesize DNA double strands. The resulting products were labeled Fc-Apt-CP (concentrations of 1.8, 2.0, 2.2, 2.4, 2.6, 2.8, and 3.0 μM, respectively).

[0055] (4) The glassy carbon electrode (GCE) with d=3mm was polished and cleaned by grinding with 0.30μm and 0.05μm aluminum oxide powder respectively. Then it was ultrasonically cleaned in water, ethanol, acetone and water respectively and dried at room temperature to obtain the treated glassy carbon electrode.

[0056] Then the powdered PTCA-COF prepared in step (1) was dispersed in ultrapure water to obtain a PTCA-COF solution with a concentration of 0.2 mg / mL; 6 μL of PTCA-COF solution was applied to the surface of the treated glassy carbon electrode and dried at room temperature. The resulting product was labeled as PTCA-COF / GCE.

[0057] (5) Dissolve the powdered AuNCs prepared in step (2) in ultrapure water to obtain an AuNCs solution with a concentration of 1 mg / mL; modify the surface of the treated glassy carbon electrode with 6 μL of AuNCs solution and air dry at room temperature. The product is labeled as AuNCs / PTCA-COF / GCE.

[0058] (6) Take 6 μL of the DNA double-stranded solution (Fc-Apt-CP) prepared in step (3) and modify it onto the surface of AuNCs / PTCA-COF / GCE obtained in step (5). Under the action of Au-S, the DNA double strands are fixed on the electrode surface. Incubate at 4℃ for 10-12 h, then rinse with Tris-HCl (pH=7.4), and dry at room temperature. The product is labeled as Fc-Apt-CP / AuNCs / PTCA-COF / GCE.

[0059] (7) Take 6 μL of 2.0 μM short-chain DNA solution to modify the surface of Fc-Apt-CP / AuNCs / PTCA-COF / GCE obtained in step (6), incubate at room temperature for 60 min, and then rinse with Tris-HCl (pH=7.4). The resulting product is labeled as ssDNA / Fc-Apt-CP / AuNCs / PTCA-COF / GCE.

[0060] Figure 2 For double-chain concentration and ΔI (ΔI = I AuNCs / PTCA-COF / GCE -I Fc-Apt-CP / AuNCs / PTCA-COF / GCE The relationship between the concentrations of the DNA double-stranded solution (Fc-Apt-CP) and the concentration range (1.8-3.0 μM) is shown in the graph. It can be seen that the electrochemiluminescence quenching signal first increases and then decreases with increasing double-stranded concentration, reaching a maximum at 2.6 μM. Therefore, 2.6 μM was selected as the optimal concentration of the DNA double-stranded solution (Fc-Apt-CP), and the sensor (ssDNA / Fc-Apt-CP / AuNCs / PTCA-COF / GCE) obtained under this concentration condition was used as the final sensor for subsequent detection.

[0061] Example 2:

[0062] Take 6 μL of Cd at different concentrations 2+ The standard solution was applied to the surface of the sensor ssDNA / Fc-Apt-CP / AuNCs / GCE finally prepared in Example 1, incubated at room temperature for 60 min, then rinsed with ultrapure water, and dried at room temperature. The resulting sensor was labeled Cd. 2+ / ssDNA / Fc-Apt-CP / AuNCs / GCE; where Cd 2+ The standard solution concentrations were 5.0 × 10⁻⁶. -13 M, 1.0×10 -12 M, 5.0×10 -12 M, 1.0×10 -11 M, 5.0×10 -11 M, 1.0×10 -10 M, 5.0×10 -10 M, 1.0×10 -9 M, 5.0×10 -9 M.

[0063] Electrochemiluminescence signal testing was performed using a three-electrode system, wherein the sensor (Cd) prepared above was used. 2+ The working electrode was a ssDNA / Fc-Apt-CP / AuNCs / GCE electrode, the reference electrode was an Ag / AgCl (saturated KCl) electrode, and the counter electrode was a platinum disk electrode. The test solution was prepared in 0.05M TEA-containing PBS (0.1M concentration, pH 7.5). The scan potential was 0-1.4V, and the scan rate was 0.1V / s. -1 The photomultiplier tube was set to 500V. The electrochemiluminescence signal was detected, and a correlation was established between the electrochemiluminescence signal and the corresponding Cd. 2+ A standard curve showing the correspondence between the concentrations of standard solutions.

[0064] from Figure 3 As can be seen from (A), with Cd 2+ With increasing concentration, the AuNCs signal gradually increases, which is due to Cd 2+ The specific binding of the aptamer to the aptamer causes the Fc on the aptamer, which quenches the luminescent signal, to move away from the electrode surface, thus increasing the electrochemiluminescence signal. Specifically, the electrochemiluminescence signal (I) is related to Cd. 2+ Logarithm of concentration (lgC) Cd 2+ The standard curve is plotted as I = 1250.151 + 3493.545lgC. Cd 2+ (R 2=0.996), the linear range is 1pM-5nM, and the detection limit is 0.66pM.

[0065] Example 3:

[0066] Performance analysis of the electrochemiluminescence biosensor finally prepared in Example 1:

[0067] Examining the selectivity of the sensor: Cd 2+ The target analyte has a corresponding concentration of 100 pM; Cu was selected. 2+ Hg 2+ ,Pb 2+ ,Zn 2 + Ca 2+ ,K + ,Fe 3+ ,Mn 2+ Al 3+ Ag + Mg 2+ ,Cl - SO4 2- NO3 - As interfering agents, their corresponding concentrations were all 1 nM; the Mix was Cd. 2+ A mixed solution with interfering substances, in which Cd 2+ The concentration was 100 pM, and the concentration of the interfering substance was 1 nM.

[0068] Target Cd 2+ and interfering substance Cu 2+ Hg 2+ ,Pb 2+ ,Zn 2+ Ca 2+ ,K + ,Fe 3+ ,Mn 2+ Al 3+ Ag + Mg 2+ ,Cl - SO4 2- NO3 - The target and interfering agent mixture (Mix) were incubated with the sensor separately. Results are as follows: Figure 4 As shown in (A), the target object Cd 2+ The electrochemiluminescence signal response of the target analyte was significantly higher than that of other interfering substances. The electrochemiluminescence signal response of a mixed solution containing both the target analyte and interfering substances was significantly higher for pure Cd. 2+ The solution response was largely consistent with that of the sensor. This indicates that the sensor exhibits good selectivity.

[0069] at the same time Figure 4(B) shows the reproducibility of the electrochemiluminescence aptamer sensor. As can be seen from the figure, the electrochemiluminescence aptamer sensor has good reproducibility.

[0070] Example 4:

[0071] The electrochemiluminescence aptamer sensor constructed in Example 1 was used to detect Cd in farmland irrigation water. 2+ Content monitoring:

[0072] (1) The actual sample was taken from irrigation water in a farmland near a farmland in Dantu District, Zhenjiang City, Jiangsu Province;

[0073] (2) The actual water sample was pretreated by filtering the water sample through a 0.22μm filter membrane to remove suspended solids and impurities in the water to obtain the sample solution;

[0074] (3) Using the standard addition method, add 0 μg L to the sample solution respectively. -1 0.1 μg L -1 and 0.5 μg L -1 Cd 2+ Standard solution was used to obtain 3 test samples;

[0075] (4) The constructed electrochemiluminescence aptamer sensor was used to detect three samples, among which the water sample was a positive sample, and Cd was detected. 2+ The content is 0.0068 μg / L. -1 Meanwhile, calculations showed that adding 0.1 μg / L... -1 and 0.5 μg / L -1 The recovery rates of farmland irrigation water were 92.70% and 101.43%, respectively, with relative standard deviations (RSDs) of 5.01% and 0.92%, respectively, indicating that the sensor can be used for actual sample detection.

[0076] (5) Furthermore, in order to verify the reliability of the sensor, the detection results of the constructed sensor were verified by the standard method—inductively coupled plasma mass spectrometry (ICP-MS). The recovery rates of the spiked actual samples obtained by this method were 92.30% and 99.96%, respectively, indicating that the constructed sensor has good reliability in the detection of actual farmland irrigation water samples.

[0077] Table 1. Detection results of cadmium ions in farmland irrigation water

[0078]

[0079] Note: The above embodiments are only used to illustrate the present invention and are not intended to limit the technical solutions described in the present invention. Therefore, although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the present invention. All technical solutions and improvements that do not depart from the spirit and scope of the present invention should be covered within the scope of the claims of the present invention.

Claims

1. A method for detecting Cd 2+ The method for preparing an electrochemiluminescence aptamer sensor is characterized by, The steps are as follows: (1) Preparation of PTCA-COF: First, perylene tetracarboxylic acid dianhydride and melamine are mixed and ground to obtain a grinding mixture. The grinding mixture is then calcined, cooled to room temperature, and the calcined product is taken out. After washing and centrifugation, the precipitate is collected and dried to obtain a dried solid powder, denoted as PTCA-COF. (2) Preparation of AuNCs: First, 6-aza-2-thiothymidine and sodium hydroxide were weighed and dissolved in ultrapure water to obtain an ATT solution containing NaOH. Then, chloroauric acid was weighed and dissolved in ultrapure water to obtain a HAuCl4 solution. The ATT solution containing NaOH and the HAuCl4 solution were mixed, stirred evenly, and then subjected to a light-protected reaction to obtain a yellow solution. The yellow solution was then dialyzed, and the dialyzed solution was freeze-dried. The powdered solid obtained after freeze-drying is AuNCs. (3) Preparation of Fc-Apt-CP: Cd modified with Fc 2+ The aptamer solution is denoted as Fc-Apt, Cd 2+ The aptamer complementary strand solution is denoted as CP; Fc-Apt is mixed with CP, and the mixed solution is denatured and annealed to synthesize DNA double strands. The resulting product is labeled as Fc-Apt-CP. (4) The glassy carbon electrode was successively ground and polished with aluminum oxide powder of different particle sizes, and then ultrasonically cleaned in water, ethanol, acetone and water and air-dried to obtain the treated glassy carbon electrode. (5) Disperse the PTCA-COF prepared in step (1) in ultrapure water to obtain a PTCA-COF solution. Then, drop the PTCA-COF solution onto the surface of the treated glassy carbon electrode and dry it at room temperature. The resulting product is labeled as PTCA-COF / GCE. (6) Dissolve the AuNCs prepared in step (2) in ultrapure water to obtain an AuNCs solution; The AuNCs solution was then dropped onto the surface of the PTCA-COF / GCE obtained in step (5), and dried at room temperature. The resulting product was labeled as AuNCs / PTCA-COF / GCE. (7) The Fc-Apt-CP prepared in step (3) is dropped onto the surface of AuNCs / PTCA-COF / GCE obtained in step (6), and after incubation, it is rinsed with Tris-HCl buffer solution. The product obtained after rinsing is labeled as Fc-Apt-CP / AuNCs / PTCA-COF / GCE. (8) The short-chain DNA solution modified on the surface of the Fc-Apt-CP / AuNCs / PTCA-COF / GCE electrode prepared in step (7) is incubated and then rinsed with Tris-HCl buffer solution. The result after rinsing is the electrochemiluminescence aptamer sensor, labeled as ssDNA / Fc-Apt-CP / AuNCs / PTCA-COF / GCE.

2. A method for detecting Cd according to claim 1 2+ The method for preparing an electrochemiluminescence aptamer sensor is characterized by, In step (1), the molar ratio of perylene tetracarboxylic dianhydride and melamine is 1:1; the calcination temperature is 325 ℃, the reaction time is 4 h, and the heating rate is 5 ℃ / min; the mixing and grinding time is 30-40 min; and the drying conditions are 60-80 ℃ for 6-12 h.

3. A method for detecting Cd according to claim 1 2+ The method for preparing an electrochemiluminescence aptamer sensor is characterized by, In step (2), the ratio of 6-aza-2-thiothymidine, sodium hydroxide, and ultrapure water A is 0.1718 g: 0.12 g: 15 mL; the ratio of chloroauric acid to ultrapure water is 0.15 g: 15 mL; the volume ratio of the NaOH solution and HAuCl4 solution of ATT is 1:1; the reaction time in the dark is 1 h; the molecular cutoff of the dialysis bag is 50 kDa, and the dialysis time is 24 h; the freeze-drying temperature is -60 ℃, and the drying time is 72 h.

4. A method for detecting Cd according to claim 1 2+ The method for preparing an electrochemiluminescence aptamer sensor is characterized by, In step (3), the concentrations of Fc-Apt and CP are both 1.8-3.0 μM, and the volume ratio of Fc-Apt to CP is 1:1; the conditions for denaturation annealing are: denaturation temperature of 95℃, held for 3 min, and then annealed at 37℃ for 3 min.

5. A method for detecting Cd according to claim 1 2+ The method for preparing an electrochemiluminescence aptamer sensor is characterized by, In step (4), the diameter of the glassy carbon electrode is 3 mm; the particle sizes of the different aluminum oxide powders used are 0.3 μm and 0.05 μm, respectively; in step (5), the concentration of the PTCA-COF solution is 0.2 mg / mL, and the amount of PTCA-COF solution used for modification is 6 μL; in step (6), the concentration of the AuNCs solution is 1 mg / mL, and the amount of AuNCs solution used for modification is 6 μL.

6. A method for detecting Cd according to claim 1 2+ The method for preparing an electrochemiluminescence aptamer sensor is characterized by, In step (7), the concentration of Fc-Apt-CP is 1.8-3 mM, the amount of Fc-Apt-CP modification is 6 μL, the incubation temperature is 4 ℃, the incubation time is 10-12 h, and the pH value of the buffer solution is 7.

4.

7. A method for detecting Cd according to claim 1 2+ The method for preparing an electrochemiluminescence aptamer sensor is characterized by, In step (8), the concentration of the short-chain DNA solution is 2 mM; the amount of short-chain DNA solution used for modification is 6 μL; the incubation temperature is 37 ℃ and the time is 60 min.

8. An electrochemiluminescence aptamer sensor prepared according to any one of claims 1-7 for detecting Cd. 2+ Its uses, characterized in that, The steps are as follows: S1. Different concentrations of Cd were modified onto the surface of the obtained sensor ssDNA / Fc-Apt-CP / AuNCs / PTCA-COF / GCE. 2+ Standard solutions containing the sensor ssDNA / Fc-Apt-CP / AuNCs / PTCA-COF / GCE and Cd 2+ The concentrations of the standard solutions have a one-to-one correspondence, meaning one sensor corresponds to one concentration of Cd. 2+ Standard solution; then incubate at room temperature and rinse the electrode with ultrapure water. The resulting product is labeled Cd. 2+ / ssDNA / Fc-Apt-CP / AuNCs / PTCA-COF / GCE; and this electrode was used as the working electrode in the three-electrode system, with the Ag / AgCl electrode as the reference electrode and the platinum disk electrode as the counter electrode. The test was carried out in a buffer solution containing triethylamine to detect the electrochemiluminescence signal. And the electrochemiluminescence signal was used as the ordinate, and Cd was used as the ordinate. 2+ The lg value of the standard solution concentration is used as the x-axis to establish Cd. 2+ Standard linear curves showing the correlation between solution concentration and electrochemiluminescence signal; S2, Actual sample Cd 2+ Detection: First, a sample solution was obtained and then modified onto the surface of the sensor ssDNA / Fc-Apt-CP / AuNCs / PTCA-COF / GCE. After incubation at room temperature, the electrode was rinsed with ultrapure water and air-dried at room temperature. The dried electrode was used as the working electrode in a three-electrode system, with the Ag / AgCl electrode as the reference electrode and the platinum disk electrode as the counter electrode. The test was performed in a buffer solution containing triethylamine, and the electrochemiluminescence signal was recorded. The recorded electrochemiluminescence signal was then substituted into the standard linear curve equation established in S1 to calculate Cd. 2+ The concentration of Cd in actual samples was determined. 2+ The detection.

9. The use according to claim 8, characterized in that, The Cd mentioned in step S1 2+ The concentration of the standard solution is 5.0 × 10⁻⁶. -13 -5.0×10 -9 M; the different concentrations of Cd 2+ The standard solution modification volume was 6 μL; the incubation time was 60 min; the buffer solution was PBS with a concentration of 0.1 M and a pH of 7.5; the final concentration of triethylamine contained was 0.05 M; the electrochemical detection instrument was ECL-MPI EII, and the parameters used for the test were: scanning voltage range of 0-1.4 V, and photomultiplier tube set to 500 V.

10. The use according to claim 8, characterized in that, The amount of sample solution modified in step S2 is 6 μL; the incubation time is 60 min; the buffer solution is PBS with a concentration of 0.1 M and a pH of 7.5; the final concentration of triethylamine is 0.05 M; the electrochemical detection instrument is ECL-MPI EII, and the parameters used for the test are: scanning voltage range of 0-1.4 V, and photomultiplier tube set to 500 V.

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