Target cycle fluorescence-electrochemical double-signal aptamer sensor for detecting lead ions as well as preparation method and application of target cycle fluorescence-electrochemical double-signal aptamer sensor

Abstract

The invention belongs to the technical field of fluorescence and electrochemistry, and particularly discloses a target cycle fluorescence-electrochemical double-signal aptamer sensor for detecting lead ions and a preparation method and application thereof.The preparation method of the aptamer sensor comprises the steps that fluorescent nanoclusters and gold magnetic nanoparticles are prepared and coupled with Apt and ssDNA respectively, and the aptamer sensor is obtained; constructing a fluorescence detection system; synthesizing Au-MoOx as an enhancement substrate for modifying the surface of the working electrode, amplifying an electrochemical signal and providing a coupling site for eDNA; and synthesizing an S1-S2 cross-linked substance, H1-Ag NCs and H2-Ag NCs, and constructing an electrochemical detection system with the modified electrode. In the aptamer sensor, Apt is separated from double strands by specifically capturing Pb < 2 + >, and RecJf enzymolysis is triggered, so that Pb < 2 + > is recycled and fluorescent substances are released; after magnetic separation, ssDNA separated from double strands is hybridized with S1, released S2 serves as a secondary target substitute and enters an electrochemical detection system, and DPV signals provided by DNA-Ag NCs synthesized in advance are further amplified in the HCR process; pb < 2 + > detection is realized according to changes of a fluorescence signal and an electrochemical signal, and specific, sensitive and rapid detection of Pb < 2 + > can be effectively realized.

Description

Technical Field

[0001] The present invention belongs to the field of fluorescence and electrochemical sensing technology, and specifically relates to a target cycle fluorescence-electrochemical dual signal aptamer sensor for detecting lead ions and a preparation method and application thereof, that is, using an aptamer sensor based on metal cluster fluorescence signal and electrochemical signal to detect Pb in food. 2+ application. Background Art

[0002] In response to the current demand for high-sensitivity and high-specificity detection of heavy metal ions, researchers are committed to building biosensors with a variety of different signal modes. Single-signal mode biosensors use specific biomolecules to selectively bind to targets and detect signal changes through various transduction techniques such as electrochemical or optical methods. In contrast, multi-signal mode biosensors use two or more different recognition elements to determine the content of the target. This method combines multiple sensing mechanisms at the same time, which can effectively improve the sensitivity and selectivity of the detection system. In a signal mode biosensor, one recognition element can specifically interact with the target to produce a signal, while the other element responds to another signal change caused by the presence of the target. By analyzing the above two signals simultaneously, the target can be effectively quantitatively analyzed and the accuracy and reliability of the biosensor can be improved.

[0003] As a key technology in the field of sensing, signal amplification can overcome the limitations of narrow practical application range or limited sensitivity of detection methods by amplifying signal intensity, thereby achieving quantitative analysis of low-concentration targets. Based on DNA assembly amplification technology, enzyme catalysis technology can degrade nucleotides in nucleic acid sequences, causing the complementary chains on the programmed oligonucleotide track to move progressively and undergo repeated pairing, and release the target into the previous reaction cycle. The number of target analytes is indirectly increased with the target release and recovery process, thereby achieving secondary amplification of the signal. In addition, autocatalytic biosensors have the advantages of simple operation and reaction at room temperature. Therefore, in order to meet the requirements of convenient detection program design, the development of a simple, rapid, and isothermal autocatalytic amplification analysis method is an ideal choice for pollutant determination. At present, the fluorescence-electrochemical dual-signal aptamer sensor based on dual signal amplification of target cycle and hybridization chain reaction is widely used for the detection of Pb 2+ There are few related studies. Summary of the invention

[0004] In view of the deficiencies in the prior art, the first aspect of the present invention provides a method for preparing a target cycle fluorescence-electrochemical dual-signal aptamer sensor for detecting lead ions, wherein gold-silver bimetallic nanoclusters and silver nanoclusters are used as fluorescence signal and electrochemical signal labels, respectively, and aptamers are used as specific recognition biological molecules. Secondary signal amplification is achieved through enzyme-assisted target cycle and hybridization chain reaction, thereby constructing a target cycle fluorescence-electrochemical dual-signal aptamer sensor for detecting lead ions.

[0005] The second aspect of the present invention provides a target cycle fluorescence-electrochemical dual-signal aptamer sensor for detecting lead ions, which is obtained by the above-mentioned preparation method.

[0006] The third aspect of the present invention provides a method for detecting lead ions using the target cycle fluorescence-electrochemical dual-signal aptamer sensor for detecting lead ions.

[0007] The fourth aspect of the present invention provides the application of the above-mentioned target cycle fluorescence-electrochemical dual-signal aptamer sensor for detecting lead ions.

[0008] To achieve the above object, the technical solution of the present invention is:

[0009] The first aspect of the present invention provides a method for preparing a target cycle fluorescence-electrochemical dual-signal aptasensor for detecting lead ions, comprising the following steps:

[0010] (1) Preparation of fluorescent nanomaterials Au NPs@BSA@AuAg NCs and magnetic nanomaterials Au-MnFe2O4;

[0011] (2) Apt and ssDNA were coupled with the fluorescent nanomaterial Au NPs@BSA@AuAg NCs and the magnetic nanomaterial Au-MnFe2O4, respectively, and complemented to form double-stranded DNA to construct a fluorescence detection system;

[0012] (3) During the electrochemical detection process, the biochemical reaction is carried out on a screen-printed electrode, the electrode is based on polyethylene terephthalate, and a working electrode, a counter electrode and a reference electrode are fixed on the substrate by screen printing, wherein the working electrode and the counter electrode are composed of graphite and a polyacrylic acid derivative, and the reference electrode is composed of silver / silver chloride paste;

[0013] (4) In situ generation of gold nanoparticles on the surface of molybdenum oxide nanosheets to obtain the composite Au-MoO x ;

[0014] (5) Preparation of Au-MoO x Modification of screen-printed electrodes to improve conductivity and specific surface area to obtain AuNPs-MoO x modifying an electrode, and coupling the modified electrode to eDNA;

[0015] (6) Prepare S1-S2 crosslinked products, H1-Ag NCs and H2-Ag NCs required for HCR reaction, and construct an electrochemical detection system with the screen-printed electrode treated in step (5).

[0016] The mechanism of the target cycle fluorescence-electrochemical dual signal aptamer sensor prepared by the present invention for detecting lead ions is as follows:

[0017] When the sample contains Pb 2+ When Apt specifically captures Pb 2+ The double-stranded structure is separated, triggering the RecJf enzyme to hydrolyze Pb 2+ After magnetic separation, the supernatant contains fluorescent nanoclusters and the precipitate contains Au-MnFe2O4 coupled with ssDNA. The fluorescence signal of the supernatant is detected to realize the Pb 2+ The Au-MnFe2O4 precipitate containing coupled ssDNA hybridizes with the S1 chain in the S1-S2 crosslinker, and the released S2 chain undergoes a hybridization chain reaction with the eDNA coupled to the electrode surface as well as H1-Ag NCs and H2-AgNCs. The DPV signal provided by the silver nanoclusters is measured to detect Pb 2+ Electrochemical detection.

[0018] In some embodiments, in step (1), the preparation method of the fluorescent nanomaterial Au NPs@BSA@AuAg NCs includes: mixing Au NPs with a BSA solution, stirring and reacting to obtain Au NPs@BSA. After Au NPs@BSA is uniformly mixed with a mixed solution of HAuCl4 and AgNO3, an appropriate amount of NaOH is added to adjust the pH of the solution system, and the stirring reaction is continued to obtain Au NPs@BSA@AuAg NCs.

[0019] In some embodiments, in step (1), the amount of Au NPs added is 2-3 mL, preferably 2.5 mL.

[0020] In some embodiments, in step (1), the amount of BSA solution added is 2-3 mL, preferably 2.5 mL; the concentration is 50-60 mg / mL, preferably 50 mg / mL.

[0021] In some embodiments, in step (1), the reaction temperature for preparing Au NPs@BSA is 30-40°C, preferably 37°C; and the reaction time is 4-6h, preferably 5h.

[0022] In some embodiments, in step (1), the amount of Au NPs@BSA added to the mixed solution of HAuCl4 and AgNO3 is 4-5 mL, preferably 4 mL.

[0023] In some embodiments, in step (1), the amount of the mixed solution of HAuCl4 and AgNO3 added is 1-1.5 mL, preferably 1 mL; wherein the concentration of HAuCl4 is 8-12 mM, preferably 10 mM; the concentration of AgNO3 is 8-12 mM, preferably 10 mM; and the molar ratio of Au to Ag is 19:1-17:3, preferably 18:2.

[0024] In some embodiments, in step (1), the concentration of NaOH is 0.5-1M, preferably 1M; the pH range of the system is 12-13, preferably 12.

[0025] In some embodiments, in step (1), the reaction temperature for preparing the Au NPs@BSA@AuAg NCs is 30-40°C, preferably 37°C; and the reaction time is 4-6h, preferably 5h.

[0026] In some embodiments, in step (1), the preparation method of the magnetic nanomaterial Au-MnFe2O4 includes: FeCl3·6H2O, MnCl2·4H2O and PVP are dissolved in ethylene glycol / diethylene glycol (V1:V2=1:1), stirred to react until the solution is transparent, and sodium acetate is added and stirred. Then the above solution is transferred to a reactor for reaction. After the reaction is completed, magnetic separation and washing are performed, and vacuum drying is performed overnight to obtain MnFe2O4 powder. HAuCl4, trisodium citrate and NaBH4 are added to ultrapure water in turn, stirred until the color of the solution changes to orange, and continued to stir and react at room temperature to obtain gold seeds. MnFe2O4 powder is added to PEI solution, and magnetic separation and washing are performed after ultrasonic reaction to obtain PEI-MnFe2O4. The above solid is dispersed in the gold seed solution for ultrasonic reaction, and after magnetic separation and washing, it is dispersed in 100 mL of ultrapure water to obtain Au NPs-MnFe2O4. Au NPs-MnFe2O4 was dispersed in 150 mL ultrapure water, and PVP was added. After complete dissolution, hydroxylamine hydrochloride was added. Finally, HAuCl4 was added, and Au-MnFe2O4 was obtained by magnetic separation and washing after ultrasonic reaction.

[0027] In some embodiments, in step (1), the amount of FeCl3·6H2O added is 350-400 mg, preferably 360 mg; the amount of MnCl2·4H2O added is 130-135 mg, preferably 132 mg; and the amount of PVP added is 1.5-2 g, preferably 2 g.

[0028] In some embodiments, in step (1), the volume of the ethylene glycol / diethylene glycol solution is 18-20 mL, preferably 20 mL.

[0029] In some embodiments, in step (1), the stirring reaction temperature of the solution containing FeCl3·6H2O, MnCl2·4H2O and PVP is 100-120°C, preferably 120°C; and the reaction time is 8-15 min, preferably 10 min.

[0030] In some embodiments, in step (1), the amount of sodium acetate added is 1.2-1.5 g, preferably 1.5 g; and the stirring time is 20-40 min, preferably 30 min.

[0031] In some embodiments, in step (1), the reaction temperature in the reactor is 190-210° C., preferably 200° C.; the reaction time is 9-10 h, preferably 10 h.

[0032] In some embodiments, in step (1), the vacuum drying temperature is 50-60°C, preferably 60°C.

[0033] In some embodiments, in step (1), the amount of HAuCl4 added is 8-10 mL, preferably 10 mL; the concentration is 4-6 mM, preferably 5 mM; the amount of trisodium citrate added is 8-10 mL, preferably 10 mL; the concentration is 4-6 mM, preferably 5 mM; the amount of NaBH4 added is 4-6 mL, preferably 5 mL; the concentration is 0.1-0.15 M, preferably 0.1 M; the volume of ultrapure water is 180-190 mL, preferably 180 mL.

[0034] In some embodiments, in step (1), the gold seed preparation reaction time is 4-6 hours, preferably 4 hours; the particle size is 3-5 nm, preferably 4 nm.

[0035] In some embodiments, in step (1), the amount of MnFe2O4 powder added is 80-100 mg, preferably 100 mg; the amount of PEI solution added is 45-50 mL, preferably 50 mL; the concentration is 9-10 mg / mL, preferably 10 mg / mL; and the ultrasonic reaction time is 20-30 min, preferably 20 min.

[0036] In some embodiments, in step (1), the volume of the gold seed solution used to disperse PEI-MnFe2O4 is 180-200 mL, preferably 200 mL; and the ultrasonic reaction time is 1-1.5 h, preferably 1 h.

[0037] In some embodiments, in step (1), the amount of Au NPs-MnFe2O4 added is 45-50 mL, preferably 50 mL; the amount of PVP added is 2.5-3 g, preferably 2.5 g; the amount of hydroxylamine hydrochloride added is 2-2.5 mL, preferably 2 mL; and the concentration is 45-50 mg / mL, preferably 50 mg / mL.

[0038] In some embodiments, in step (1), the amount of HAuCl4 added is 1.2-1.5 mL, preferably 1.5 mL; the concentration is 20-25 mM, preferably 24 mM; and the ultrasonic reaction time is 5-8 min, preferably 5 min.

[0039] In some embodiments, in step (2), the preparation method of coupling Apt with fluorescent nanomaterial Au NPs@BSA@AuAg NCs includes: dispersing Au NPs@BSA@AuAg NCs in PBS (10mM, pH 7.0), adding EDC and NHS in sequence, and continuing the reaction at 37° C. Adding amino-modified Apt to the above system to react to obtain Apt / Au NPs@BSA@AuAgNCs.

[0040] In some embodiments, in step (2), the amount of Au NPs@BSA@AuAg NCs added is 40-50 μL, preferably 40 μL.

[0041] In some embodiments, in step (2), the volume of the PBS solution is 150-160 μL, preferably 160 μL.

[0042] In some embodiments, in step (2), the amount of EDC added is 4-4.5 mg, preferably 4.48 mg; the amount of NHS added is 2-2.5 mg, preferably 2.5 mg; and the reaction time is 20-40 min, preferably 30 min.

[0043] In some embodiments, in step (2), the amount of amino-modified Apt added is 10-15 μL, preferably 10 μL; the concentration is 40-50 μM, preferably 50 μM; the reaction temperature is 35-40°C, preferably 37°C; and the reaction time is 4-5h, preferably 4h.

[0044] In some embodiments, in step (2), the preparation method of coupling ssDNA with magnetic nanomaterial Au-MnFe2O4 includes: reducing ssDNA modified with thiol by TCEP, mixing with Au-MnFe2O4 and stirring overnight. After the reaction is completed, the mixture is washed by magnetic separation and redissolved in 500 μL ultrapure water to obtain ssDNA / Au-MnFe2O4.

[0045] In some embodiments, in step (2), the amount of thiol-modified ssDNA added is 45-50 μL, preferably 50 μL; the concentration is 50-80 μM, preferably 50 μM.

[0046] In some embodiments, in step (2), the amount of Au-MnFe2O4 added is 4-6 mg, preferably 5 mg; the reaction temperature is 25-30°C, preferably 25°C.

[0047] In some embodiments, in step (3), the working electrode diameter of the screen-printed electrode is 3.5 mm.

[0048] In some embodiments, in step (4), the Au-MoO x The preparation method of the composite material includes: adding molybdenum powder to 8 mL ultrapure water, ultrasonicating at room temperature until uniformly dispersed, slowly adding 12 mL H2O2, diluting with water to a total volume of 20 mL after complete dissolution, and then adding polyethylene glycol 2000, continuously and rapidly stirring with a magnetic stirrer to form a transparent yellow solution, and transferring it to a 50 mL reactor for reaction. After the reaction is completed, the blue MoO x The nanosheet precipitate was redissolved in 20 mL of water. 50 μL HAuCl4 and 100 μL trisodium citrate were added to 3.5 mL of ultrapure water in sequence, and 100 μL NaBH4 was added while stirring. The gold seeds were obtained by vigorous stirring. 110 mL CTAB and 100 μL NaI were fully mixed and divided into three parts of different volumes: A (10 mL), B (10 mL), and C (90 mL). 1.2 mL HAuCl4, 0.5 mL ascorbic acid, and 0.5 mL NaOH were fully mixed and added to systems A and B respectively, and 1.2 mL HAuCl4 was added to system C. Then, 1 mL of the gold seed solution was added to system A, mixed for 10 seconds, and 1 mL of the mixed solution was added to system B and mixed for 10 seconds. Finally, all the solutions in system B were mixed with 500 μL of the MoO prepared above. x The solution was added to system C and stirred until the solution turned blue-purple. The above solution was placed in a light-proof environment for aging. After the reaction was completed, it was centrifuged and washed, and finally dispersed in 10 mL of ultrapure water to obtain Au-MoO x .

[0049] In some embodiments, in step (4), the amount of molybdenum powder added is 1.8-2g, preferably 1.96g; the ultrasonic time is 40-60min, preferably 60min; the amount of polyethylene glycol 2000 added is 8-10g, preferably 10g; the reaction temperature in the reactor is 75-85°C, preferably 80°C; the reaction time is 10-12h, preferably 12h.

[0050] In some embodiments, in step (4), the HAuCl4 concentration is 20-25 mM, preferably 24 mM; the trisodium citrate concentration is 8-10 mM, preferably 10 mM; the NaBH4 concentration is 0.1-0.15 M, preferably 0.1 M; and the gold seed reaction stirring time is 20-40 min, preferably 30 min.

[0051] In some embodiments, in step (4), the CTAB concentration is 40-60 mM, preferably 50 mM; the NaI concentration is 0.1-0.15 M, preferably 0.1 M.

[0052] In some embodiments, in step (4), the HAuCl4 concentration is 20-25 mM, preferably 24 mM; the ascorbic acid concentration is 0.08-0.1 M, preferably 0.1 M; and the NaOH concentration is 0.1-0.15 M, preferably 0.1 M.

[0053] In some embodiments, in step (4), the aging reaction temperature is 25-30°C, preferably 30°C; the reaction time is 20-30h, preferably 24h.

[0054] In some embodiments, in step (5), the Au-MoO x The preparation method of the modified electrode comprises: preparing the Au-MoO x After mixing with 5 μL ultrapure water, add it dropwise onto the working electrode and dry it.

[0055] In some embodiments, in step (5), the Au-MoO x The amount added is 4-6 μL, preferably 5 μL.

[0056] In some embodiments, in step (5), the drying temperature is 20-30°C, preferably 30°C.

[0057] In some embodiments, the nucleotide sequences involved in each step are as follows: in step (2), the sequence of Apt is shown in SEQ ID NO: 1; the sequence of ssDNA is shown in SEQ ID NO: 2; in step (5), the Au-MoO x The sequence of the eDNA coupled to the modified electrode is shown in SEQ ID NO: 3; in step (6), the sequence of S1 is shown in SEQ ID NO: 4, the sequence of S2 is shown in SEQ ID NO: 5, the sequence of H1 is shown in SEQ ID NO: 6, and the sequence of H2 is shown in SEQ ID NO: 7.

[0058] The second aspect of the present invention provides a target cycle fluorescence-electrochemical dual-signal aptamer sensor for detecting lead ions, which is obtained by the preparation method of the target cycle fluorescence-electrochemical dual-signal aptamer sensor for detecting lead ions.

[0059] The third aspect of the present invention provides a method for detecting lead ions using the target cycle fluorescence-electrochemical dual signal aptamer sensor for detecting lead ions, comprising the following steps:

[0060] (i) The analyte is added to the fluorescence system for detection. Apt is separated from the double strand by specific capture, triggering the enzymatic hydrolysis process, resulting in the recycling of the analyte and the generation of fluorescence signals. The fluorescence signals are measured to detect Pb 2+ A one-time quantitative

[0061] (ii) After magnetic separation, the precipitate is detected in an electrochemical system. The ssDNA separated from the double-stranded DNA hybridizes with S1, and the released S2 acts as a secondary target to trigger a hybridization chain reaction. The electrochemical signal provided by the silver nanoclusters in the reaction is measured to achieve the detection of Pb 2+ Secondary quantification.

[0062] In some embodiments, in step (a), the Pb 2+ The detection method in the fluorescence system includes: adding Pb 2+ The standard solution and 10 μL RecJf were added. After the reaction, the supernatant was obtained by magnetic separation and its fluorescence spectrum was measured (excitation wavelength 360 nm, emission wavelength range 400 nm-800 nm).

[0063] In some embodiments, in step (i), the amount of the mixed solution of ssDNA / Au-MnFe2O4 and Apt / Au NPs@BSA@AuAg NCs added is 25-35 μL, preferably 30 μL.

[0064] In some embodiments, in step (a), the Pb 2+ The amount of standard solution added is 10-15 μL, preferably 10 μL.

[0065] In some embodiments, in step (i), the RecJf concentration is 0.3-0.5 U / μL, preferably 0.3 U / μL; the reaction temperature is 20-30°C, preferably 25°C; and the reaction time is 40-60 min, preferably 60 min.

[0066] In some embodiments, in step (ii), the Pb 2+The detection method in the electrochemical system includes: mixing the precipitate in the above magnetic separation process with 20μL S1-S2 crosslinked product, and then magnetically separating to obtain a supernatant. The supernatant is added dropwise to the eDNA modified electrode, reacted at 37°C for 30 minutes, and then the electrode is rinsed. Then 10μL H1-Ag NCs and 10μL H2-Ag NCs are added, and the HCR reaction is carried out at 37°C. After the reaction is completed, the electrode is rinsed. The DPV signal provided by AgNCs is measured in PBS (0.1M, pH 7.0) for Pb 2+ Carry out quantitative analysis.

[0067] In some embodiments, in step (ii), the concentration of the S1-S2 crosslinker is 20-30 μM, preferably 20 μM; the reaction temperature is 20-30°C, preferably 25°C; and the reaction time is 20-40 min, preferably 30 min.

[0068] In some embodiments, in step (ii), the concentration of H1-Ag NCs and H2-Ag NCs is 4-6 μM, preferably 5 μM.

[0069] In some embodiments, in step (ii), the HCR reaction time is 20-40 min, preferably 30 min.

[0070] In a fourth aspect of the present invention, a target cycle fluorescence-electrochemical dual signal aptamer sensor for detecting lead ions is provided for detecting heavy metal ions Pb in food. 2+ Application in.

[0071] In some embodiments, the heavy metal ion Pb in tea and vegetables is detected 2+ The method is realized by the target cycle fluorescence-electrochemical dual signal aptamer sensor for detecting lead ions, comprising the following steps:

[0072] (a) Preparation of different concentration gradients of Pb 2+ The standard solution is dripped onto the target cycle fluorescence-electrochemical dual-signal aptamer sensor for detecting lead ions, and its fluorescence signal intensity and electrochemical characteristic peak signal intensity are measured to fit the standard curve;

[0073] (b) The tea leaves and vegetables were pre-treated and then added to the target cycle fluorescence-electrochemical dual signal aptamer sensor for detecting lead ions, and the Pb was determined according to the fluorescence and electrochemical detection linear models respectively. 2+ concentration.

[0074] In some embodiments, in step (a), Pb 2+ The standard curve fitting method: Pb 2+The standard was diluted with ultrapure water to different concentrations, and the solutions of different concentrations were dripped onto the target cycle fluorescence-electrochemical dual-signal aptamer sensor for detecting lead ions, and the fluorescence signal and the electrochemical DPV signal of the silver nanoclusters were detected, and the standard curve was fitted according to the signal peak intensity values.

[0075] In some embodiments, in step (a), Pb 2+ The different concentrations of the solution were 0 ng / mL, 0.001 ng / mL, 0.01 ng / mL, 0.1 ng / mL, 0.2 ng / mL, 0.5 ng / mL, 1 ng / mL, 2 ng / mL, 5 ng / mL, 10 ng / mL, 50 ng / mL, 100 ng / mL, and 200 ng / mL;

[0076] In some embodiments, in step (b), nitric acid and hydrogen peroxide are added to the crushed tea powder and vegetables, and then digested in a graphite digestion furnace; after the digestion is completed, the solution is diluted with water, filtered and impurities are removed, and then the solution required for detection is added dropwise to the target cycle fluorescence-electrochemical dual signal aptamer sensor for detecting lead ions, and the electrochemical signal is detected, and the Pb in tea and vegetables is calculated according to the standard curve. 2+ The content.

[0077] Due to the adoption of the above scheme, the beneficial effects of the present invention are:

[0078] 1. The enzyme-assisted target cyclic metal cluster fluorescence-electrochemical dual-signal aptamer sensor constructed by the present invention degrades the aptamer nucleotide by RecJf exonuclease, releases the target cycle into the fluorescence system, and realizes the primary signal amplification; the S2 obtained by magnetic separation enters the electrochemical detection system as a secondary target substitute to trigger the hybridization chain reaction process, and the DNA-stabilized silver nanoclusters can serve as cross-linking sequences and provide electrochemical signals, realizing secondary signal amplification and improving the sensitivity of the detection system.

[0079] 2. Composite nanomaterial Au-MoO2 of gold nanoparticles and molybdenum oxide nanosheets prepared by the present invention x It has excellent conductivity and can effectively improve the conductivity and specific surface area of ​​screen-printed electrodes. The constructed sensor uses fluorescence and electrochemical dual signal responses as analysis objects. 2+ The detection has high accuracy and reliability. In addition, the specific recognition function of the aptamer effectively avoids potential interference from other heavy metal ions, and has the advantages of strong specificity and high accuracy.

[0080] 3. The present invention provides a new method for preparing a multi-signal biosensor for detecting heavy metal ions with high sensitivity and high specificity. The preparation method is novel and the preparation process is simple. It can be applied to the food testing industry. For example, it can quickly detect the heavy metal Pb in tea and vegetable samples. 2+Content, has important practical application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0081] Figure 1 The target cycle combined with hybridization chain reaction signal amplification fluorescence-electrochemical sensing system of the present invention detects Pb 2+ Schematic diagram.

[0082] Figure 2 This is the fluorescence lifetime and quantum yield diagram of Au NPs@BSA@AuAg NCs in Example 1.

[0083] Figure 3 This is the hysteresis loop diagram of MnFe2O4 and Au-MnFe2O4 in Example 1.

[0084] Figure 4 The Au-MoO x Transmission electron microscopy image of .

[0085] Figure 5 This is the cyclic voltammetry curve of the gradually modified electrode in Example 1.

[0086] Figure 6 For the fluorescence system detection of Pb in Example 1 2+ Fluorescence spectrum of .

[0087] Figure 7 For the fluorescence system detection of Pb in Example 1 2+ The standard curve fitting diagram.

[0088] Figure 8 For the electrochemical system detection of Pb in Example 1 2+ DPV diagram of .

[0089] Fig. 9 Detection of Pb in the chemical system in Example 1 2+ The standard curve fitting diagram.

[0090] Fig.10 Detection of Pb for Examples 1-4 2+ The reliability verification results of . DETAILED DESCRIPTION

[0091] The present invention will be further described below with reference to the embodiments.

[0092] The nucleotide sequences involved in each embodiment are as follows:

[0093] The sequence of Apt (SEQ ID NO: 1) is: 5′-GGTTGGTGTGGTTGGAAAAAA-NH2-3′;

[0094] The sequence of the ssDNA (SEQ ID NO: 2) is: 5′-HS-SH-CCAACCACACCAACC-3′;

[0095] The sequence of the cDNA (SEQ ID NO: 3) is: 5′-HS-SH-AAAAAATGTGGTTGG-3′;

[0096] The sequence of S1 (SEQ ID NO: 4) is: 5′-GGTTGGTGTGGTTGG-3′;

[0097] The sequence of S2 (SEQ ID NO: 5) is: 5′-GAGTAAGCAGATTCCCAACCACA-3′;

[0098] The sequence of H1 (SEQ ID NO: 6) is:

[0099] 5′-GAATCTGCTTACTCTTGCCAGTGAGTAAGCAAAAACCCCCCTTAATCCCCCC-3′;

[0100] The sequence of H2 (SEQ ID NO: 7) is:

[0101] 5′-CCCCCCTAATTCCCCCCAAAAGAGTAAGCAGATTCTGCTTACTCACTGGCAA-3′.

[0102] like Figure 1 As shown, the preparation and detection process of the target cycle fluorescence-electrochemical dual signal aptamer sensor for detecting lead ions in the present invention is as follows:

[0103] (1) Preparation of fluorescent nanomaterials Au NPs@BSA@AuAg NCs and magnetic nanomaterials Au-MnFe2O4;

[0104] (2) Apt and ssDNA were coupled with the fluorescent nanomaterial Au NPs@BSA@AuAg NCs and the magnetic nanomaterial Au-MnFe2O4, respectively, and complemented to form double-stranded DNA to construct a fluorescence detection system;

[0105] (3) During the electrochemical detection process, the biochemical reaction is carried out on a screen-printed electrode, the electrode is based on polyethylene terephthalate, and a working electrode, a counter electrode and a reference electrode are fixed on the substrate by screen printing, wherein the working electrode and the counter electrode are composed of graphite and a polyacrylic acid derivative, and the reference electrode is composed of silver / silver chloride paste;

[0106] (4) In situ generation of gold nanoparticles on the surface of molybdenum oxide nanosheets to obtain the composite Au-MoO x ;

[0107] (5) Preparation of Au-MoO x Modification of screen-printed electrodes to improve conductivity and specific surface area to obtain AuNPs-MoO x Modified electrodes;

[0108] (6) The analyte is added to the fluorescence detection system, and Apt is separated from the double strand by specific capture, triggering the enzymatic hydrolysis process, resulting in the recycling of the analyte and the generation of fluorescence signals. The fluorescence signals are measured to detect Pb 2+ Quantification of

[0109] (7) After magnetic separation, the ssDNA separated from the double-stranded DNA hybridizes with S1, and the released S2 enters the electrochemical detection system as a secondary target to trigger a hybridization chain reaction, and the electrochemical signal provided by the silver nanoclusters is measured to achieve the detection of Pb 2+ Secondary quantification.

[0110] Embodiment 1:

[0111] This example uses a target cycle fluorescence-electrochemical dual signal aptamer sensor to detect Pb in tea. 2+ The process includes the following steps:

[0112] (1) Preparation of fluorescent nanomaterials Au NPs@BSA@AuAg NCs:

[0113] 2.5 mL of Au NPs was mixed with 2.5 mL of BSA solution (50 mg / mL), and the mixture was stirred at 37 °C for 5 h to obtain AuNPs@BSA. 4 mL of Au NPs@BSA was mixed with 1 mL of a mixed solution of HAuCl4 (10 mM) and AgNO3 (10 mM) (the molar ratio of Au to Ag was 18:2), and then an appropriate amount of NaOH (1 M) was added to adjust the pH of the solution system to 12, and the mixture was stirred at 37 °C for 5 h to obtain Au NPs@BSA@AuAg NCs.

[0114] (2) Preparation of magnetic nanomaterials Au-MnFe2O4:

[0115] 360mg FeCl3·6H2O, 132mg MnCl2·4H2O and 2g PVP were dissolved in 20mL ethylene glycol / diethylene glycol (V1:V2=1:1), stirred at 120℃ for 10min until the solution was transparent, and 1.5g sodium acetate was added and stirred for 30min. Then the above solution was transferred to a reactor and reacted at 200℃ for 10h. After the reaction, magnetic separation and washing were performed, and MnFe2O4 powder was obtained by vacuum drying at 60℃ overnight. 10mL HAuCl4 (5mM), 10mL trisodium citrate (5mM) and 5mL NaBH4 (0.1M) were added to 180mL ultrapure water in turn, stirred until the solution color turned orange, and continued to stir and react at room temperature for 4h to obtain gold seeds (particle size of 4nm). 100mg MnFe2O4 powder was added to 50mL PEI solution (10mg / mL), and PEI-MnFe2O4 was obtained after ultrasonic reaction for 20min and magnetic separation and washing. The above solid was dispersed in 200mL gold seed solution and ultrasonic reaction for 1h. After magnetic separation and washing, it was dispersed in 100mL ultrapure water to obtain Au NPs-MnFe2O4. 50mL Au NPs-MnFe2O4 was dispersed in 150mL ultrapure water, and 2.5g PVP was added. After complete dissolution, 2mL hydroxylamine hydrochloride (50mg / mL) was added. Finally, 1.5mL HAuCl4 (24mM) was added, and Au-MnFe2O4 was obtained after ultrasonic reaction for 5min and magnetic separation and washing.

[0116] (3) Preparation of conjugates of Apt and Au NPs@BSA@AuAg NCs:

[0117] 40 μL Au NPs@BSA@AuAg NCs were dispersed in 160 μL PBS (10 mM, pH 7.0), and 4.48 mg EDC and 2.5 mg NHS were added in sequence, and the reaction was continued at 37°C for 30 min. 10 μL amino-modified Apt (50 μM) was added to the above system, and the reaction was carried out at 37°C for 4 h to obtain Apt / Au NPs@BSA@AuAg NCs.

[0118] (4) Preparation of conjugates of ssDNA and magnetic nanomaterials Au-MnFe2O4:

[0119] 50 μL of thiol-modified ssDNA (50 μM) was reduced with TCEP, mixed with 5 mg of Au-MnFe2O4, and stirred overnight at 25° C. After the reaction, it was washed by magnetic separation and redissolved in 500 μL of ultrapure water to obtain ssDNA / Au-MnFe2O4.

[0120] (5) Preparation of Au-MoO modified electrode x Composite Materials:

[0121] Add 1.96g of molybdenum powder to 8mL of ultrapure water, ultrasonicate for 60min at room temperature until uniformly dispersed, slowly add 12mL of H2O2, dilute with water to a total volume of 20mL after complete dissolution, then add 10g of polyethylene glycol 2000, continuously and rapidly stir magnetically to form a transparent yellow solution, and transfer it to a 50mL reactor and react at 80℃ for 12h. After the reaction is completed, centrifuge and wash to collect the blue MoO x The nanosheet precipitate was redissolved in 20 mL of water. 50 μL HAuCl4 (24 mM) and 100 μL trisodium citrate (10 mM) were added to 3.5 mL of ultrapure water in sequence, and 100 μL NaBH4 (0.1 M) was added while stirring. The gold seeds were obtained by vigorous stirring for 30 min. 110 mL CTAB (50 mM) and 100 μL NaI (0.1 M) were fully mixed and divided into three portions of different volumes: A (10 mL), B (10 mL), and C (90 mL). 1.2 mL HAuCl4 (24 mM), 0.5 mL ascorbic acid (0.1 M), and 0.5 mL NaOH (0.1 M) were fully mixed and added to systems A and B respectively, and 1.2 mL HAuCl4 was added to system C. Then, 1 mL of the gold seed solution was added to system A and mixed for 10 seconds. Then, 1 mL of the mixed solution was added to system B and mixed for 10 seconds. Finally, all the solutions in system B were mixed with 500 μL of the MoO prepared above. x The solution was added to system C and stirred until the solution turned blue-purple. The above solution was placed in a dark environment at 30°C for 24 hours. After the reaction was completed, it was centrifuged and washed, and finally dispersed in 10 mL of ultrapure water to obtain Au-MoO x .

[0122] (6) Preparation of Au-MoO x Modified electrode:

[0123] 5 μL of the Au-MoO prepared above was added x After mixing with 5 μL ultrapure water, add it dropwise onto the working electrode and dry it at 30°C.

[0124] (7) Fitting Pb 2+ Standard curve:

[0125] 10 μL of Pb was added to the mixed solution of 30 μL ssDNA / Au-MnFe2O4 and Apt / Au NPs@BSA@AuAg NCs (V1:V2=20:10) 2+The standard solution and 10 μL RecJf (0.3U / μL) were reacted at 25°C for 60 min, and the supernatant was obtained by magnetic separation. The fluorescence spectrum (excitation wavelength 360nm, emission wavelength range 400nm-800nm) was measured, and the fluorescence standard curve was fitted by the fluorescence emission peak intensity value. The precipitate in the above magnetic separation process was mixed with 20 μL S1-S2 crosslinker (20 μM), reacted at 25°C for 30 min, and magnetic separation was performed to obtain the supernatant. The supernatant was added dropwise to the eDNA modified electrode, reacted at 37°C for 30 min, and then the electrode was rinsed. Then 10 μL H1-Ag NCs (5 μM) and 10 μL H2-Ag NCs (5 μM) were added, and the HCR reaction was carried out at 37°C (30 min). The electrode was rinsed after the reaction. The DPV signal provided by Ag NCs was measured in PBS (0.1 M, pH 7.0), and the electrochemical standard curve was fitted based on the DPV peak intensity value.

[0126] (8) Detection of Pb in tea 2+ Content:

[0127] 3 mL of nitric acid and 1 mL of hydrogen peroxide (30%) were added to 0.5 g of crushed green tea powder, and then heated to 120 ° C in a graphite digestion furnace for 90 min. After digestion, the volume was adjusted to 25 mL with water, filtered and impurities were removed as the solution required for detection, and then dripped onto the constructed dual-signal aptamer sensor. The fluorescence and electrochemical signals were reacted and detected according to the above steps, and the Pb content in green tea was calculated according to the standard curve. 2+ The content.

[0128] from Figure 2 It can be seen that the quantum yield QY of Au NPs@BSA@AuAg NCs in this embodiment is 15.25%, which is the result of the fluorescence enhancement effect of Au NPs surface plasmon coupling and the restriction of the BSA layer on the non-radiative relaxation process of AuAg NCs. The average fluorescence lifetime of Au NPs@BSA@AuAg NCs is 3.876μs.

[0129] from Figure 3 It can be seen that no hysteresis loop is observed in MnFe2O4 and Au-MnFe2O4, indicating that both have superparamagnetism, and the magnetic saturation value of MnFe2O4 is about 81emu / g. When a gold shell is formed on its surface, the magnetic saturation value decreases to 57emu / g, which is due to the fact that the surface of MnFe2O4 is covered by non-magnetic materials such as PEI and Au NPs.

[0130] from Figure 4 It can be seen that Au-MoO was prepared by gold seed-mediated growth method. xThe particle size of Au NPs is about 30 nm and is evenly distributed in MoO x The surface of the nanosheets shows that Au-MoO x Successful synthesis of nanocomposites.

[0131] from Figure 5 It can be seen that the unmodified screen-printed electrode has a pair of weak reversible redox peaks. x After modification, the redox peaks are enhanced, proving that Au-MoO x The nanocomposite material has a strong electron transfer ability. After the eDNA is fixed on the electrode surface and the nonspecific binding sites are blocked by MCH, the electron movement is hindered and the I pa S2 can be fixed on the electrode surface through complementary pairing with eDNA, which further increases the amount of nucleic acid, thus leading to I pa When S2 is present, adding H1 and H2 to the system can trigger the HCR process, which significantly increases the negative charge and steric hindrance on the electrode surface and reduces the redox peak current.

[0132] from Figure 6 and 7 It can be seen that in Pb 2+ When the concentration increased from 0 ng / mL to 200 ng / mL, the characteristic peak of fluorescence emission changed from weak to strong. The linear range obtained by fitting was 0.1-200 ng / mL, and the detection limit was 0.028 ng / mL.

[0133] from Figure 8 and 9 It can be seen that in Pb 2+ When the concentration increased from 0 ng / mL to 200 ng / mL, the DPV characteristic peak of silver nanoclusters changed from weak to strong. The linear range obtained by fitting was 0.001-100 ng / mL, and the detection limit was 0.788 pg / mL.

[0134] Embodiment 2:

[0135] This example uses a target cycle fluorescence-electrochemical dual signal aptamer sensor to detect Pb in tea. 2+ The process includes the following steps:

[0136] (1) Preparation of fluorescent nanomaterials Au NPs@BSA@AuAg NCs:

[0137] 2 mL of Au NPs was mixed with 2 mL of BSA solution (60 mg / mL), and the mixture was stirred at 30 °C for 6 h to obtain Au NPs@BSA. 4.5 mL of Au NPs@BSA was mixed with 1.2 mL of a mixed solution of HAuCl4 (10 mM) and AgNO3 (10 mM) (the molar ratio of Au to Ag was 19:1), and then an appropriate amount of NaOH (0.8 M) was added to adjust the pH of the solution system to 13, and the mixture was stirred at 30 °C for 6 h to obtain Au NPs@BSA@AuAg NCs.

[0138] (2) Preparation of magnetic nanomaterials Au-MnFe2O4:

[0139] 350mg FeCl3·6H2O, 130mg MnCl2·4H2O and 1.5g PVP were dissolved in 19mL ethylene glycol / diethylene glycol (V1:V2=1:1), stirred at 110℃ for 12min until the solution was transparent, and 1.2g sodium acetate was added and stirred for 35min. Then the above solution was transferred to a reactor and reacted at 190℃ for 9h. After the reaction, magnetic separation and washing were performed, and MnFe2O4 powder was obtained by vacuum drying at 55℃ overnight. 8mL HAuCl4 (4mM), 8mL trisodium citrate (4mM) and 6mL NaBH4 (0.1M) were added to 184mL ultrapure water in sequence, stirred until the solution color turned orange, and continued to stir and react at room temperature for 5h to obtain gold seeds (particle size of 3nm). 80mg MnFe2O4 powder was added to 45mL PEI solution (9mg / mL), and PEI-MnFe2O4 was obtained after ultrasonic reaction for 25min and magnetic separation and washing. The above solid was dispersed in 180mL gold seed solution and ultrasonic reaction was carried out for 1.2h. After magnetic separation and washing, it was dispersed in 100mL ultrapure water to obtain Au NPs-MnFe2O4. 45mL Au NPs-MnFe2O4 was dispersed in 150mL ultrapure water, 2.6g PVP was added, and 2.5mL hydroxylamine hydrochloride (45mg / mL) was added after complete dissolution. Finally, 1.2mL HAuCl4 (22mM) was added, and Au-MnFe2O4 was obtained after ultrasonic reaction for 6min and magnetic separation and washing.

[0140] (3) Preparation of conjugates of Apt and Au NPs@BSA@AuAg NCs:

[0141] 45 μL Au NPs@BSA@AuAg NCs were dispersed in 155 μL PBS (10 mM, pH 7.0), and 4.5 mg EDC and 2.5 mg NHS were added in sequence, and the reaction was continued at 37°C for 20 min. 12 μL amino-modified Apt (45 μM) was added to the above system, and the reaction was carried out at 35°C for 4.5 h to obtain Apt / Au NPs@BSA@AuAg NCs.

[0142] (4) Preparation of conjugates of ssDNA and magnetic nanomaterials Au-MnFe2O4:

[0143] 45 μL of thiol-modified ssDNA (60 μM) was reduced with TCEP, mixed with 6 mg of Au-MnFe2O4, and stirred overnight at 26° C. After the reaction, it was washed by magnetic separation and redissolved in 500 μL of ultrapure water to obtain ssDNA / Au-MnFe2O4.

[0144] (5) Preparation of Au-MoO modified electrode x Composite Materials:

[0145] Add 1.8g molybdenum powder to 8mL ultrapure water, ultrasonicate for 50min at room temperature until evenly dispersed, slowly add 12mL H2O2, dilute with water to a total volume of 20mL after complete dissolution, then add 8g polyethylene glycol 2000, continuously and rapidly stir magnetically to form a transparent yellow solution, and transfer it to a 50mL reactor and react at 75℃ for 11h. After the reaction is completed, centrifuge and wash to collect the blue MoO x The nanosheet precipitate was redissolved in 20 mL of water. 50 μL HAuCl4 (20 mM) and 100 μL trisodium citrate (8 mM) were added to 3.5 mL of ultrapure water in sequence, and 100 μL NaBH4 (0.12 M) was added while stirring. The gold seeds were obtained by vigorous stirring for 25 min. 110 mL CTAB (40 mM) and 100 μL NaI (0.12 M) were fully mixed and divided into three portions of different volumes: A (10 mL), B (10 mL), and C (90 mL). 1.2 mL HAuCl4 (20 mM), 0.5 mL ascorbic acid (0.08 M), and 0.5 mL NaOH (0.12 M) were fully mixed and added to systems A and B respectively, and 1.2 mL HAuCl4 was added to system C. Then, 1 mL of the gold seed solution was added to system A and mixed for 10 seconds. Then, 1 mL of the mixed solution was added to system B and mixed for 10 seconds. Finally, all the solutions in system B were mixed with 500 μL of the MoO prepared above. x The solution was added to system C and stirred until the solution turned blue-purple. The above solution was placed in a dark environment at 25°C for 20 hours. After the reaction was completed, it was centrifuged and washed, and finally dispersed in 10 mL of ultrapure water to obtain Au-MoOx .

[0146] (6) Preparation of Au-MoO x Modified electrode:

[0147] 4 μL of the Au-MoO prepared above was added x After mixing with 5 μL ultrapure water, add it dropwise onto the working electrode and dry it at 25°C.

[0148] (7) Fitting Pb 2+ Standard curve:

[0149] 12 μL Pb was added to the mixed solution of 25 μL ssDNA / Au-MnFe2O4 and Apt / Au NPs@BSA@AuAg NCs (V1:V2=20:10) 2+ The standard solution and 10 μL RecJf (0.5U / μL) were reacted at 30°C for 40 minutes, and the supernatant was obtained by magnetic separation. The fluorescence spectrum (excitation wavelength 360nm, emission wavelength range 400nm-800nm) was measured, and the fluorescence standard curve was fitted by the fluorescence emission peak intensity value. The precipitate in the above magnetic separation process was mixed with 20 μL S1-S2 crosslinker (30 μM), reacted at 30°C for 20 minutes, and magnetic separation was obtained to obtain the supernatant. The supernatant was added dropwise to the eDNA modified electrode, reacted at 37°C for 30 minutes, and then the electrode was rinsed. Then 10 μL H1-Ag NCs (4 μM) and 10 μL H2-Ag NCs (4 μM) were added, and the HCR reaction was carried out at 37°C (25 minutes). The electrode was rinsed after the reaction. The DPV signal provided by Ag NCs was measured in PBS (0.1 M, pH 7.0), and the electrochemical standard curve was fitted based on the DPV peak intensity value.

[0150] (8) Detection of Pb in tea 2+ Content:

[0151] 3 mL of nitric acid and 1 mL of hydrogen peroxide (30%) were added to 0.5 g of crushed black tea powder, and then heated to 120 ° C in a graphite digestion furnace for 90 min. After digestion, the volume was adjusted to 25 mL with water, filtered and impurities were removed, and the solution required for detection was added dropwise to the constructed electrochemical aptamer sensor. The electrochemical signal was detected according to the above steps, and the Pb content in black tea was calculated according to the standard curve. 2+ The content.

[0152] Embodiment 3:

[0153] This example uses a target cycle fluorescence-electrochemical dual signal aptamer sensor to detect Pb in tea. 2+ The process includes the following steps:

[0154] (1) Preparation of fluorescent nanomaterials Au NPs@BSA@AuAg NCs:

[0155] 3 mL of Au NPs was mixed with 3 mL of BSA solution (50 mg / mL), and the mixture was stirred at 35 °C for 5 h to obtain Au NPs@BSA. 5 mL of Au NPs@BSA was mixed with 1.5 mL of a mixed solution of HAuCl4 (8 mM) and AgNO3 (8 mM) (the molar ratio of Au to Ag was 17:3), and then an appropriate amount of NaOH (0.5 M) was added to adjust the pH of the solution system to 12, and the mixture was stirred at 35 °C for 5 h to obtain Au NPs@BSA@AuAg NCs.

[0156] (2) Preparation of magnetic nanomaterials Au-MnFe2O4:

[0157] 380mg FeCl3·6H2O, 135mg MnCl2·4H2O and 1.8g PVP were dissolved in 18mL ethylene glycol / diethylene glycol (V1:V2=1:1), stirred at 115℃ for 15min until the solution was transparent, and 1.3g sodium acetate was added and stirred for 25min. Then the above solution was transferred to a reactor and reacted at 200℃ for 10h. After the reaction, magnetic separation and washing were performed, and MnFe2O4 powder was obtained by vacuum drying at 60℃ overnight. 9mL HAuCl4 (6mM), 9mL trisodium citrate (6mM) and 4mL NaBH4 (0.15M) were added to 182mL ultrapure water in turn, stirred until the solution color changed to orange, and continued to stir and react at room temperature for 4h to obtain gold seeds (particle size of 5nm). 90mg MnFe2O4 powder was added to 40mL PEI solution (9mg / mL), and PEI-MnFe2O4 was obtained after ultrasonic reaction for 30min and magnetic separation and washing. The above solid was dispersed in 190mL gold seed solution and ultrasonic reaction for 1h, and dispersed in 100mL ultrapure water after magnetic separation and washing to obtain Au NPs-MnFe2O4. 48mL Au NPs-MnFe2O4 was dispersed in 150mL ultrapure water, and 2.8g PVP was added. After complete dissolution, 2.4mL hydroxylamine hydrochloride (48mg / mL) was added. Finally, 1.3mL HAuCl4 (20mM) was added, and Au-MnFe2O4 was obtained after ultrasonic reaction for 8min and magnetic separation and washing.

[0158] (3) Preparation of conjugates of Apt and Au NPs@BSA@AuAg NCs:

[0159] 50 μL Au NPs@BSA@AuAg NCs were dispersed in 150 μL PBS (10 mM, pH 7.0), and 4.2 mg EDC and 2.0 mg NHS were added in sequence, and the reaction was continued at 37°C for 25 min. 15 μL amino-modified Apt (40 μM) was added to the above system, and the reaction was carried out at 40°C for 5 h to obtain Apt / Au NPs@BSA@AuAg NCs.

[0160] (4) Preparation of conjugates of ssDNA and magnetic nanomaterials Au-MnFe2O4:

[0161] 48 μL of thiol-modified ssDNA (80 μM) was reduced with TCEP, mixed with 4 mg of Au-MnFe2O4, and stirred overnight at 30° C. After the reaction, it was washed by magnetic separation and redissolved in 500 μL of ultrapure water to obtain ssDNA / Au-MnFe2O4.

[0162] (5) Preparation of Au-MoO modified electrode x Composite Materials:

[0163] Add 1.9g molybdenum powder to 8mL ultrapure water, ultrasonicate for 40min at room temperature until uniformly dispersed, slowly add 12mL H2O2, dilute with water to a total volume of 20mL after complete dissolution, then add 9g polyethylene glycol 2000, continuously and rapidly stir magnetically to form a transparent yellow solution, and transfer it to a 50mL reactor and react at 85℃ for 10h. After the reaction is completed, centrifuge and wash to collect the blue MoO x The nanosheet precipitate was redissolved in 20 mL of water. 50 μL HAuCl4 (24 mM) and 100 μL trisodium citrate (9 mM) were added to 3.5 mL of ultrapure water in sequence, and 100 μL NaBH4 (0.15 M) was added while stirring. The gold seeds were obtained by vigorous stirring for 20 min. 110 mL CTAB (60 mM) and 100 μL NaI (0.15 M) were fully mixed and divided into three portions of different volumes: A (10 mL), B (10 mL), and C (90 mL). 1.2 mL HAuCl4 (24 mM), 0.5 mL ascorbic acid (0.09 M), and 0.5 mL NaOH (0.15 M) were fully mixed and added to systems A and B respectively, and 1.2 mL HAuCl4 was added to system C. Then, 1 mL of the gold seed solution was added to system A and mixed for 10 seconds. Then, 1 mL of the mixed solution was added to system B and mixed for 10 seconds. Finally, all the solutions in system B were mixed with 500 μL of the MoO prepared above. x The solution was added to system C and stirred until the solution turned blue-purple. The above solution was placed in a dark environment at 26°C for 30 hours. After the reaction was completed, it was centrifuged and washed, and finally dispersed in 10 mL of ultrapure water to obtain Au-MoOx .

[0164] (6) Preparation of Au-MoO x Modified electrode:

[0165] 6 μL of the Au-MoO prepared above was added x After mixing with 5 μL ultrapure water, add it dropwise onto the working electrode and dry it at 26°C.

[0166] (7) Fitting Pb 2+ Standard curve:

[0167] 10 μL of Pb was added to the mixed solution of 35 μL ssDNA / Au-MnFe2O4 and Apt / Au NPs@BSA@AuAg NCs (V1:V2=20:10) 2+ The standard solution and 10 μL RecJf (0.4 U / μL) were reacted at 20°C for 50 min, and the supernatant was obtained by magnetic separation. The fluorescence spectrum (excitation wavelength 360 nm, emission wavelength range 400 nm-800 nm) was measured, and the fluorescence standard curve was fitted by the fluorescence emission peak intensity value. The precipitate in the above magnetic separation process was mixed with 20 μL S1-S2 crosslinker (25 μM), reacted at 20°C for 25 min, and magnetic separation was obtained to obtain the supernatant. The supernatant was added dropwise to the eDNA modified electrode, reacted at 37°C for 30 min, and then the electrode was rinsed. Then 10 μL H1-Ag NCs (5 μM) and 10 μL H2-Ag NCs (5 μM) were added, and the HCR reaction was carried out at 37°C (20 min). The electrode was rinsed after the reaction. The DPV signal provided by Ag NCs was measured in PBS (0.1 M, pH 7.0), and the electrochemical standard curve was fitted based on the DPV peak intensity value.

[0168] (8) Detection of Pb in tea 2+ Content:

[0169] 3 mL of nitric acid and 1 mL of hydrogen peroxide (30%) were added to 0.5 g of crushed dark tea powder, and then heated to 120 ° C in a graphite digestion furnace for 90 min. After digestion, the volume was adjusted to 25 mL with water, filtered and impurities were removed, and the solution required for detection was added dropwise to the constructed electrochemical aptamer sensor. The electrochemical signal was detected according to the above steps, and the Pb content in dark tea was calculated according to the standard curve. 2+ The content.

[0170] Embodiment 4:

[0171] This example uses a target cycle fluorescence-electrochemical dual signal aptamer sensor to detect Pb in vegetables. 2+ The process includes the following steps:

[0172] (1) Preparation of fluorescent nanomaterials Au NPs@BSA@AuAg NCs:

[0173] 2.5 mL of Au NPs was mixed with 2.5 mL of BSA solution (55 mg / mL), and the mixture was stirred at 40 °C for 4 h to obtain AuNPs@BSA. 5 mL of Au NPs@BSA was mixed with 1.2 mL of a mixed solution of HAuCl4 (8 mM) and AgNO3 (8 mM) (the molar ratio of Au to Ag was 18:2), and then an appropriate amount of NaOH (1 M) was added to adjust the pH of the solution system to 12, and the mixture was stirred at 40 °C for 4 h to obtain Au NPs@BSA@AuAg NCs.

[0174] (2) Preparation of magnetic nanomaterials Au-MnFe2O4:

[0175] 400mg FeCl3·6H2O, 135mg MnCl2·4H2O and 1.6g PVP were dissolved in 20mL ethylene glycol / diethylene glycol (V1:V2=1:1), stirred at 120℃ for 8min until the solution was transparent, and 1.4g sodium acetate was added and stirred for 20min. Then the above solution was transferred to a reactor and reacted at 210℃ for 10h. After the reaction, magnetic separation and washing were performed, and MnFe2O4 powder was obtained by vacuum drying at 50℃ overnight. 10mL HAuCl4 (5mM), 10mL trisodium citrate (5mM) and 5mL NaBH4 (0.12M) were added to 180mL ultrapure water in sequence, stirred until the solution color turned orange, and continued to stir and react at room temperature for 6h to obtain gold seeds (particle size of 4nm). 90mg MnFe2O4 powder was added to 50mL PEI solution (9.5mg / mL), and PEI-MnFe2O4 was obtained by magnetic separation and washing after ultrasonic reaction for 30min. The above solid was dispersed in 200mL gold seed solution and ultrasonic reaction was carried out for 1.5h. After magnetic separation and washing, it was dispersed in 100mL ultrapure water to obtain Au NPs-MnFe2O4. 50mL Au NPs-MnFe2O4 was dispersed in 150mL ultrapure water, 3g PVP was added, and 2mL hydroxylamine hydrochloride (50mg / mL) was added after complete dissolution. Finally, 1.4mL HAuCl4 (25mM) was added, and Au-MnFe2O4 was obtained by magnetic separation and washing after ultrasonic reaction for 6min.

[0176] (3) Preparation of conjugates of Apt and Au NPs@BSA@AuAg NCs:

[0177] 50 μL Au NPs@BSA@AuAg NCs were dispersed in 160 μL PBS (10 mM, pH 7.0), and 4 mg EDC and 2.2 mg NHS were added in sequence, and the reaction was continued at 37°C for 35 min. 10 μL amino-modified Apt (50 μM) was added to the above system, and the reaction was carried out at 36°C for 5 h to obtain Apt / Au NPs@BSA@AuAg NCs.

[0178] (4) Preparation of conjugates of ssDNA and magnetic nanomaterials Au-MnFe2O4:

[0179] 50 μL of thiol-modified ssDNA (70 μM) was reduced with TCEP, mixed with 5 mg of Au-MnFe2O4, and stirred overnight at 28° C. After the reaction, it was washed by magnetic separation and redissolved in 500 μL of ultrapure water to obtain ssDNA / Au-MnFe2O4.

[0180] (5) Preparation of Au-MoO modified electrode x Composite Materials:

[0181] Add 2g of molybdenum powder to 8mL of ultrapure water, ultrasonicate at room temperature for 45min until uniformly dispersed, slowly add 12mL of H2O2, dilute with water to a total volume of 20mL after complete dissolution, then add 8g of polyethylene glycol 2000, continuously and rapidly stir magnetically to form a transparent yellow solution, and transfer it to a 50mL reactor and react at 80℃ for 12h. After the reaction is completed, centrifuge and wash to collect the blue MoO x The nanosheet precipitate was redissolved in 20 mL of water. 50 μL HAuCl4 (25 mM) and 100 μL trisodium citrate (8 mM) were added to 3.5 mL of ultrapure water in sequence, and 100 μL NaBH4 (0.1 M) was added while stirring. The gold seed was obtained by vigorous stirring for 35 min. 110 mL CTAB (45 mM) and 100 μL NaI (0.1 M) were fully mixed and divided into three parts with different volumes: A (10 mL), B (10 mL), and C (90 mL). 1.2 mL HAuCl4 (25 mM), 0.5 mL ascorbic acid (0.1 M), and 0.5 mL NaOH (0.1 M) were fully mixed and added to systems A and B respectively, and 1.2 mL HAuCl4 was added to system C. Then, 1 mL of the gold seed solution was added to system A, mixed for 10 s, and 1 mL of the mixed solution was added to system B and mixed for 10 s. Finally, all the solutions in system B were mixed with 500 μL of MoO prepared above. x The solution was added to system C and stirred until the solution turned blue-purple. The above solution was placed in a dark environment at 28°C for 25 hours. After the reaction was completed, it was centrifuged and washed, and finally dispersed in 10 mL of ultrapure water to obtain Au-MoO x .

[0182] (6) Preparation of Au-MoO x Modified electrode:

[0183] 5 μL of the Au-MoO prepared above was added x After mixing with 5 μL ultrapure water, add it dropwise onto the working electrode and dry it at 28°C.

[0184] (7) Fitting Pb 2+ Standard curve:

[0185] 15 μL Pb was added to the mixed solution of 30 μL ssDNA / Au-MnFe2O4 and Apt / Au NPs@BSA@AuAg NCs (V1:V2=20:10) 2+ The standard solution and 10 μL RecJf (0.4 U / μL) were reacted at 28°C for 45 min, and the supernatant was obtained by magnetic separation. The fluorescence spectrum (excitation wavelength 360 nm, emission wavelength range 400 nm-800 nm) was measured, and the fluorescence standard curve was fitted by the fluorescence emission peak intensity value. The precipitate in the above magnetic separation process was mixed with 20 μL S1-S2 crosslinker (25 μM), reacted at 28°C for 30 min, and magnetic separation was performed to obtain the supernatant. The supernatant was added dropwise to the eDNA modified electrode, reacted at 37°C for 30 min, and then the electrode was rinsed. Then 10 μL H1-Ag NCs (4.5 μM) and 10 μL H2-Ag NCs (4.5 μM) were added, and the HCR reaction was carried out at 37°C (40 min). The electrode was rinsed after the reaction. The DPV signal provided by AgNCs was measured in PBS (0.1 M, pH 7.0), and the electrochemical standard curve was fitted based on the DPV peak intensity value.

[0186] (8) Detection of Pb in vegetables 2+ Content:

[0187] 3 mL of nitric acid and 1 mL of hydrogen peroxide (30%) were added to 0.5 g of crushed Shanghai Qing, and then heated to 120 ° C in a graphite digestion furnace for 90 min. After digestion, the volume was adjusted to 25 mL with water, filtered and impurities were removed, and the solution required for detection was added dropwise to the constructed electrochemical aptamer sensor. The reaction and electrochemical signals were detected according to the above steps, and the Pb content in Shanghai Qing was calculated according to the standard curve. 2+ The content.

[0188] Embodiment 5:

[0189] This example uses a target cycle fluorescence-electrochemical dual signal aptamer sensor to detect Pb in vegetables. 2+ The process includes the following steps:

[0190] (1) Preparation of fluorescent nanomaterials Au NPs@BSA@AuAg NCs:

[0191] 3 mL of Au NPs was mixed with 3 mL of BSA solution (50 mg / mL), and the mixture was stirred at 40 °C for 4 h to obtain Au NPs@BSA. 4 mL of Au NPs@BSA was mixed with 1 mL of a mixed solution of HAuCl4 (12 mM) and AgNO3 (12 mM) (the molar ratio of Au to Ag was 19:1), and then an appropriate amount of NaOH (0.6 M) was added to adjust the pH of the solution system to 13, and the mixture was stirred at 40 °C for 4 h to obtain Au NPs@BSA@AuAg NCs.

[0192] (2) Preparation of magnetic nanomaterials Au-MnFe2O4:

[0193] 360mg FeCl3·6H2O, 130mg MnCl2·4H2O and 2g PVP were dissolved in 18mL ethylene glycol / diethylene glycol (V1:V2=1:1), stirred at 100℃ for 15min until the solution was transparent, and 1.2g sodium acetate was added and stirred for 40min. Then the above solution was transferred to a reactor and reacted at 205℃ for 9h. After the reaction, magnetic separation and washing were performed, and MnFe2O4 powder was obtained by vacuum drying at 50℃ overnight. 8mL HAuCl4 (6mM), 8mL trisodium citrate (6mM) and 6mL NaBH4 (0.14M) were added to 190mL ultrapure water in turn, stirred until the solution color turned orange, and continued to stir and react at room temperature for 4h to obtain gold seeds (particle size of 5nm). 80mg MnFe2O4 powder was added to 45mL PEI solution (10mg / mL), and PEI-MnFe2O4 was obtained after ultrasonic reaction for 25min and magnetic separation and washing. The above solid was dispersed in 200mL gold seed solution and ultrasonic reaction was carried out for 1.5h. After magnetic separation and washing, it was dispersed in 100mL ultrapure water to obtain Au NPs-MnFe2O4. 50mL Au NPs-MnFe2O4 was dispersed in 150mL ultrapure water, 3g PVP was added, and 2.2mL hydroxylamine hydrochloride (45mg / mL) was added after complete dissolution. Finally, 1.5mL HAuCl4 (24mM) was added, and Au-MnFe2O4 was obtained after ultrasonic reaction for 6min and magnetic separation and washing.

[0194] (3) Preparation of conjugates of Apt and Au NPs@BSA@AuAg NCs:

[0195] 40 μL Au NPs@BSA@AuAg NCs were dispersed in 150 μL PBS (10 mM, pH 7.0), and 4.1 mg EDC and 2.4 mg NHS were added in sequence, and the reaction was continued at 37°C for 40 min. 12 μL amino-modified Apt (45 μM) was added to the above system, and the reaction was carried out at 37°C for 4 h to obtain Apt / Au NPs@BSA@AuAg NCs.

[0196] (4) Preparation of conjugates of ssDNA and magnetic nanomaterials Au-MnFe2O4:

[0197] 50 μL of thiol-modified ssDNA (65 μM) was reduced with TCEP, mixed with 6 mg of Au-MnFe2O4, and stirred overnight at 25° C. After the reaction, it was washed by magnetic separation and redissolved in 500 μL of ultrapure water to obtain ssDNA / Au-MnFe2O4.

[0198] (5) Preparation of Au-MoO modified electrode x Composite Materials:

[0199] Add 1.9g molybdenum powder to 8mL ultrapure water, ultrasonicate for 50min at room temperature until evenly dispersed, slowly add 12mL H2O2, dilute with water to a total volume of 20mL after complete dissolution, then add 10g polyethylene glycol 2000, continuously and rapidly stir magnetically to form a transparent yellow solution, and transfer it to a 50mL reactor and react at 75℃ for 10h. After the reaction is completed, centrifuge and wash to collect the blue MoO x The nanosheet precipitate was redissolved in 20 mL of water. 50 μL HAuCl4 (22 mM) and 100 μL trisodium citrate (10 mM) were added to 3.5 mL of ultrapure water in sequence, and 100 μL NaBH4 (0.12 M) was added while stirring. The gold seeds were obtained by vigorous stirring for 40 min. 110 mL CTAB (60 mM) and 100 μL NaI (0.12 M) were fully mixed and divided into three portions of different volumes: A (10 mL), B (10 mL), and C (90 mL). 1.2 mL HAuCl4 (22 mM), 0.5 mL ascorbic acid (0.1 M), and 0.5 mL NaOH (0.12 M) were fully mixed and added to systems A and B respectively, and 1.2 mL HAuCl4 was added to system C. Then, 1 mL of the gold seed solution was added to system A and mixed for 10 seconds. Then, 1 mL of the mixed solution was added to system B and mixed for 10 seconds. Finally, all the solutions in system B were mixed with 500 μL of the MoO prepared above. x The solution was added to system C and stirred until the solution turned blue-purple. The above solution was placed in a dark environment at 30°C for 26 hours. After the reaction was completed, it was centrifuged and washed, and finally dispersed in 10 mL of ultrapure water to obtain Au-MoOx .

[0200] (6) Preparation of Au-MoO x Modified electrode:

[0201] 4 μL of the Au-MoO prepared above was added x After mixing with 5 μL ultrapure water, add it dropwise onto the working electrode and dry it at 30°C.

[0202] (7) Fitting Pb 2+ Standard curve:

[0203] 15 μL of Pb was added to the mixed solution of 25 μL ssDNA / Au-MnFe2O4 and Apt / Au NPs@BSA@AuAg NCs (V1:V2=20:10) 2+ The standard solution and 10 μL RecJf (0.3U / μL) were reacted at 30°C for 60 min, and the supernatant was obtained by magnetic separation. The fluorescence spectrum (excitation wavelength 360nm, emission wavelength range 400nm-800nm) was measured, and the fluorescence standard curve was fitted by the fluorescence emission peak intensity value. The precipitate in the above magnetic separation process was mixed with 20 μL S1-S2 crosslinker (20 μM), reacted at 30°C for 40 min, and magnetic separation was performed to obtain the supernatant. The supernatant was added dropwise to the eDNA modified electrode, reacted at 37°C for 30 min, and then the electrode was rinsed. Then 10 μL H1-Ag NCs (6 μM) and 10 μL H2-Ag NCs (6 μM) were added, and the HCR reaction was carried out at 37°C (35 min). The electrode was rinsed after the reaction. The DPV signal provided by Ag NCs was measured in PBS (0.1 M, pH 7.0), and the electrochemical standard curve was fitted based on the DPV peak intensity value.

[0204] (8) Detection of Pb in vegetables 2+ Content:

[0205] 3 mL of nitric acid and 1 mL of hydrogen peroxide (30%) were added to 0.5 g of crushed Shanghai Qing, and then heated to 120 ° C in a graphite digestion furnace for 90 min. After digestion, the volume was adjusted to 25 mL with water, filtered and impurities were removed, and the solution required for detection was added dropwise to the constructed electrochemical aptamer sensor. The reaction and electrochemical signals were detected according to the above steps, and the Pb content in Shanghai Qing was calculated according to the standard curve. 2+ The content.

[0206] According to the method of Examples 1-4, different samples (tea and vegetables) were subjected to Pb 2+ The content of the test, the spike recovery results are as follows Fig.10As shown in the figure, it can be seen that the FL-EC dual-signal aptasensor constructed in the present invention has a good 2+ It has high recovery rate, good accuracy and strong reliability.

[0207] The above description of the embodiments is to facilitate the understanding and use of the present invention by those skilled in the art. It is obvious that those skilled in the art can easily make various modifications to these embodiments and apply the general principles described herein to other embodiments without creative work. Therefore, the present invention is not limited to the above embodiments. Improvements and modifications made by those skilled in the art based on the principles of the present invention without departing from the scope of the present invention should be within the protection scope of the present invention.

Claims

1. A method for preparing a target cycle fluorescence-electrochemical dual-signal aptamer sensor for detecting lead ions, characterized in that: The steps include: (1) Preparation of fluorescent nanomaterials Au NPs@BSA@AuAg NCs and magnetic nanomaterials Au-MnFe2O4; (2) Apt and ssDNA were coupled with the fluorescent nanomaterial Au NPs@BSA@AuAg NCs and the magnetic nanomaterial Au-MnFe2O4, respectively, and complemented to form double-stranded DNA to construct a fluorescence detection system; (3) During the electrochemical detection process, the biochemical reaction is carried out on a screen-printed electrode, the electrode is based on polyethylene terephthalate, and a working electrode, a counter electrode and a reference electrode are fixed on the substrate by screen printing, wherein the working electrode and the counter electrode are composed of graphite and a polyacrylic acid derivative, and the reference electrode is composed of silver / silver chloride paste; (4) In situ generation of gold nanoparticles on the surface of molybdenum oxide nanosheets to obtain the composite Au-MoO x ; (5) Au-MoO prepared in step (4) x Modification of screen-printed electrodes to improve conductivity and specific surface area to obtain AuNPs-MoO x modifying an electrode, and coupling the modified electrode to eDNA; (6) Prepare the S1-S2 crosslinked product, H1-Ag NCs and H2-Ag NCs required for the HCR reaction, and construct an electrochemical detection system with the screen-printed electrode treated in step (5).

2. The method for preparing the target cycle fluorescence-electrochemical dual signal aptasensor for detecting lead ions according to claim 1, characterized in that: In step (1), the preparation method of the fluorescent nanomaterial Au NPs@BSA@AuAg NCs comprises: mixing Au NPs with a BSA solution, stirring and reacting to obtain Au NPs@BSA; mixing Au NPs@BSA with a mixed solution of HAuCl4 and AgNO3, adding NaOH to adjust the pH of the solution system, and continuing to stir and react to obtain Au NPs@BSA@AuAg NCs; wherein: The amount of Au NPs added is 2-3 mL; and / or the amount of the BSA solution added is 2-3 mL, and the concentration is 50-60 mg / mL; And / or the preparation reaction temperature of Au NPs@BSA is 30-40°C and the reaction time is 4-6h; and / or the amount of Au NPs@BSA added to the mixed solution of HAuCl4 and AgNO3 is 4-5 mL; and / or the amount of the mixed solution of HAuCl4 and AgNO3 added is 1-1.5 mL, wherein the concentration of HAuCl4 is 8-12 mM, the concentration of AgNO3 is 8-12 mM, and the molar ratio of Au to Ag is 19:1-17:3; And / or the concentration of NaOH is 0.5-1M, and the pH range of the system is adjusted to 12-13; And / or the Au NPs@BSA@AuAg NCs preparation reaction temperature is 30-40°C and the reaction time is 4-6h.

3. The method for preparing the target cycle fluorescence-electrochemical dual signal aptasensor for detecting lead ions according to claim 1, characterized in that: In step (1), the preparation method of the magnetic nanomaterial Au-MnFe2O4 includes: FeCl3·6H2O, MnCl2·4H2O and PVP are dissolved in ethylene glycol / diethylene glycol with a volume ratio of 1:1, stirred to react until the solution is transparent, and sodium acetate is added and stirred; then the above solution is transferred to a reactor for reaction; after the reaction is completed, magnetic separation and washing are performed, and vacuum drying is performed overnight to obtain MnFe2O4 powder; HAuCl4, trisodium citrate and NaBH4 are added to ultrapure water in sequence, stirred until the solution color changes to orange, and continued to stir and react at room temperature to obtain gold seeds; MnFe2O4 powder is added to PEI solution, ultrasonically reacted and then magnetically separated and washed to obtain PEI-MnFe2O4; the above solid is dispersed in the gold seed solution for ultrasonic reaction, magnetically separated and washed, and then dispersed in 100 mL of ultrapure water to obtain Au NPs-MnFe2O4; Au NPs-MnFe2O4 was dispersed in 150 mL ultrapure water, and PVP was added. After PVP was completely dissolved, hydroxylamine hydrochloride was added. Finally, HAuCl4 was added, and Au-MnFe2O4 was obtained by magnetic separation and washing after ultrasonic reaction. The FeCl3·6H2O addition amount is 350-400 mg, the MnCl2·4H2O addition amount is 130-135 mg, and the PVP addition amount is 1.5-2 g; and / or the volume of the ethylene glycol / diethylene glycol solution is 18-20 mL; and / or the stirring reaction temperature of the solution containing FeCl3·6H2O, MnCl2·4H2O and PVP is 100-120°C and the time is 8-15min; And / or the amount of sodium acetate added is 1.2-1.5 g, and the stirring time is 20-40 min; and / or the reaction temperature in the reactor is 190-210° C. and the reaction time is 9-10 h; And / or the vacuum drying temperature is 50-60°C; and / or the HAuCl4 is added in an amount of 8-10 mL, with a concentration of 4-6 mM, trisodium citrate is added in an amount of 8-10 mL, with a concentration of 4-6 mM, NaBH4 is added in an amount of 4-6 mL, with a concentration of 0.1-0.15 M, and the volume of ultrapure water is 180-190 mL; and / or the gold seed preparation reaction time is 4-6h, and the particle size is 3-5nm; And / or the MnFe2O4 powder is added in an amount of 80-100 mg, the PEI solution is added in an amount of 45-50 mL, the concentration is 9-10 mg / mL, and the ultrasonic reaction time is 20-30 min; and / or the volume of the gold seed solution used for dispersing PEI-MnFe2O4 is 180-200 mL, and the ultrasonic reaction time is 1-1.5 h; and / or the amount of Au NPs-MnFe2O4 added is 45-50 mL, the amount of PVP added is 2.5-3 g, the amount of hydroxylamine hydrochloride added is 2-2.5 mL, and the concentration is 45-50 mg / mL; And / or the amount of HAuCl4 added is 1.2-1.5 mL, the concentration is 20-25 mM, and the ultrasonic reaction time is 5-8 min.

4. The method for preparing the target cycle fluorescence-electrochemical dual signal aptasensor for detecting lead ions according to claim 1, characterized in that: In step (2), the preparation method of coupling Apt with fluorescent nanomaterial Au NPs@BSA@AuAg NCs includes: dispersing Au NPs@BSA@AuAg NCs in PBS (10mM, pH 7.0), adding EDC and NHS in sequence, and continuing the reaction at 37°C; adding amino-modified Apt to the above system to react to obtain Apt / Au NPs@BSA@AuAg NCs; wherein: The amount of Au NPs@BSA@AuAg NCs added is 40-50 μL; and / or the volume of the PBS solution is 150-160 μL; and / or the amount of EDC added is 4-4.5 mg, the amount of NHS added is 2-2.5 mg, and the reaction time is 20-40 min; And / or the amount of the amino-modified Apt added is 10-15 μL, the concentration is 40-50 μM, the reaction temperature is 35-40° C., and the reaction time is 4-5 h.

5. The method for preparing the target cycle fluorescence-electrochemical dual signal aptasensor for detecting lead ions according to claim 1, characterized in that: In step (2), the preparation method of coupling ssDNA with magnetic nanomaterial Au-MnFe2O4 includes: reducing ssDNA modified with thiol by TCEP, mixing with Au-MnFe2O4 and stirring overnight. After the reaction is completed, washing by magnetic separation, and redissolving in 500 μL ultrapure water to obtain ssDNA / Au-MnFe2O4; wherein: The amount of thiol-modified ssDNA added is 45-50 μL, and the concentration is 50-80 μM; And / or the amount of Au-MnFe2O4 added is 4-6 mg, and the reaction temperature is 25-30°C.

6. The method for preparing the target cycle fluorescence-electrochemical dual signal aptasensor for detecting lead ions according to claim 1, characterized in that: In step (3), the working electrode diameter of the screen-printed electrode is 3.5 mm.

7. The method for preparing the target cycle fluorescence-electrochemical dual signal aptasensor for detecting lead ions according to claim 1, characterized in that: In step (4), the Au-MoO x The preparation method of the composite material includes: adding molybdenum powder to 8 mL ultrapure water, ultrasonicating at room temperature until uniformly dispersed, slowly adding 12 mL H2O2, diluting with water to a total volume of 20 mL after complete dissolution, and then adding polyethylene glycol 2000, continuously and rapidly stirring with a magnetic stirrer to form a transparent yellow solution, and transferring the solution to a 50 mL reactor for reaction; after the reaction is completed, centrifuging and washing to collect the blue MoO x The nanosheet precipitate was redissolved in 20 mL of water; 50 μL of HAuCl4 and 100 μL of trisodium citrate were added to 3.5 mL of ultrapure water in sequence, and 100 μL of NaBH4 was added while stirring, and the gold seed was obtained by vigorous stirring; 110 mL of CTAB and 100 μL of NaI were fully mixed and divided into three parts of different volumes: A 10 mL, B 10 mL, and C 90 mL; 1.2 mL of HAuCl4, 0.5 mL of ascorbic acid, and 0.5 mL of NaOH were fully mixed and added to systems A and B respectively, and 1.2 mL of HAuCl4 was added to system C; then, 1 mL of the gold seed solution was added to system A, mixed for 10 seconds, and 1 mL of the mixed solution was added to system B and mixed for 10 seconds; finally, all the solutions in system B were mixed with 500 μL of the MoO prepared above. x The solution was added to system C and stirred until the solution turned blue-purple. The solution was placed in a light-proof environment for aging. After the reaction was completed, it was centrifuged and washed, and finally dispersed in 10 mL of ultrapure water to obtain Au-MoO x ;in: The molybdenum powder addition amount is 1.8-2g, the ultrasonic time is 40-60min, the polyethylene glycol 2000 addition amount is 8-10g, the reaction temperature in the reactor is 75-85°C, and the reaction time is 10-12h; and / or the HAuCl4 concentration is 20-25 mM, the trisodium citrate concentration is 8-10 mM, the NaBH4 concentration is 0.1-0.15 M, and the gold seed reaction stirring time is 20-40 min; and / or the CTAB concentration is 40-60 mM, and the NaI concentration is 0.1-0.15 M; and / or the HAuCl4 concentration is 20-25 mM, the ascorbic acid concentration is 0.08-0.1 M, and the NaOH concentration is 0.1-0.15 M; And / or the aging reaction temperature is 25-30°C, and the reaction time is 20-30h.

8. The method for preparing the target cycle fluorescence-electrochemical dual signal aptasensor for detecting lead ions according to claim 1, characterized in that: In step (5), the Au-MoO x The preparation method of the modified electrode comprises: preparing the Au-MoO x After mixing with 5 μL ultrapure water, drip it onto the working electrode and dry it; where: The Au-MoO x The amount added is 4-6 μL; And / or the drying temperature is 20-30°C.

9. The method for preparing the target cycle fluorescence-electrochemical dual signal aptasensor for detecting lead ions according to claim 1, characterized in that: In step (2), the sequence of Apt is shown in SEQ ID NO: 1; the sequence of ssDNA is shown in SEQ ID NO: 2; in step (5), the Au-MoO x The sequence of the eDNA coupled to the modified electrode is shown in SEQ ID NO: 3; in step (6), the sequence of S1 is shown in SEQ ID NO: 4, the sequence of S2 is shown in SEQ ID NO: 5, the sequence of H1 is shown in SEQ ID NO: 6, and the sequence of H2 is shown in SEQ ID NO:

7.

10. A target cycle fluorescence-electrochemical dual signal aptamer sensor for detecting lead ions, characterized in that: The method is obtained by the preparation method of the target cycle fluorescence-electrochemical dual-signal aptamer sensor for detecting lead ions according to any one of claims 1 to 9.

11. A method for detecting lead ions using the target cycle fluorescence-electrochemical dual signal aptamer sensor according to claim 10, characterized in that: The following steps are involved: (i) The analyte is added to the fluorescence system for detection. Apt is separated from the double strand by specific capture, triggering the enzymatic hydrolysis process, resulting in the recycling of the analyte and the generation of fluorescence signals. The fluorescence signals are measured to detect Pb 2+ A one-time quantitative (ii) After magnetic separation, the precipitate is detected in an electrochemical system. The ssDNA separated from the double-stranded DNA hybridizes with S1, and the released S2 acts as a secondary target to trigger a hybridization chain reaction. The electrochemical signal provided by the silver nanoclusters in the reaction is measured to achieve the detection of Pb 2+ Secondary quantification.

12. The method for detecting lead ions using a target cycle fluorescence-electrochemical dual signal aptamer sensor according to claim 11, characterized in that: In step (a), Pb 2+ The detection method in the fluorescence system includes: adding Pb 2+ The standard solution and 10 μL RecJf were added. After the reaction, the supernatant was obtained by magnetic separation and its fluorescence spectrum was measured. The fluorescence spectrum measurement conditions were an excitation wavelength of 360 nm and an emission wavelength range of 400 nm-800 nm; wherein: The amount of the mixed solution of ssDNA / Au-MnFe2O4 and Apt / Au NPs@BSA@AuAg NCs added is 25-35 μL; and / or the Pb 2+ The amount of standard solution added is 10-15 μL; And / or the RecJf concentration is 0.3-0.5U / μL, the reaction temperature is 20-30°C, and the reaction time is 40-60min.

13. The method for detecting lead ions using a target cycle fluorescence-electrochemical dual signal aptamer sensor according to claim 12, characterized in that: In step (ii), Pb 2+ The detection method in the electrochemical system includes: mixing the precipitate in the magnetic separation process with 20 μL of S1-S2 crosslinked material, and then magnetically separating to obtain a supernatant; dropping the supernatant onto the eDNA modified electrode, reacting at 37°C for 30 minutes, and then rinsing the electrode; then adding 10 μL of H1-Ag NCs and 10 μL of H2-Ag NCs, and performing HCR reaction at 37°C, and then rinsing the electrode after the reaction; and measuring the DPV signal provided by Ag NCs for Pb in PBS with a concentration of 0.1 M and a pH of 7.

0. 2+ To quantify; where: The concentration of the S1-S2 crosslinker is 20-30 μM, the reaction temperature is 20-30°C, and the reaction time is 20-40 min; and / or the concentration of the H1-Ag NCs and H2-Ag NCs is 4-6 μM; And / or the HCR reaction time is 20-40 min.

14. The target cycle fluorescence-electrochemical dual signal aptamer sensor for detecting lead ions according to claim 10 is used to detect heavy metal ions Pb in food 2+ Application in.

15. The use according to claim 14, characterized in that: Detection of heavy metal ions Pb in tea and vegetables 2+ The method is realized by the target cycle fluorescence-electrochemical dual signal aptamer sensor for detecting lead ions, comprising the following steps: (a) Preparation of different concentration gradients of Pb 2+ The standard solution is dripped onto the target cycle fluorescence-electrochemical dual-signal aptamer sensor for detecting lead ions, and its fluorescence signal intensity and electrochemical characteristic peak signal intensity are measured to fit the standard curve; (b) The tea leaves and vegetables were pre-treated and then added to the target cycle fluorescence-electrochemical dual signal aptamer sensor for detecting lead ions, and the Pb was determined according to the fluorescence and electrochemical detection linear models respectively. 2+ concentration.

16. The use according to claim 15, characterized in that In step (a), Pb 2+ The standard curve fitting method: Pb 2+ The standard is diluted with ultrapure water to different concentrations, and the solutions of different concentrations are dripped onto the target cycle fluorescence-electrochemical dual signal aptamer sensor for detecting lead ions, and the fluorescence signal and the electrochemical DPV signal of the silver nanoclusters are detected, and the standard curve is fitted by the signal peak intensity value respectively; wherein: Pb 2+ The different concentrations of the solution were 0 ng / mL, 0.001 ng / mL, 0.01 ng / mL, 0.1 ng / mL, 0.2 ng / mL, 0.5 ng / mL, 1 ng / mL, 2 ng / mL, 5 ng / mL, 10 ng / mL, 50 ng / mL, 100 ng / mL, and 200 ng / mL; And / or in step (b), nitric acid and hydrogen peroxide are added to the crushed tea powder and vegetables, and then digested in a graphite digestion furnace; after the digestion is completed, the volume is fixed with water, and after filtering and removing impurities, the solution required for detection is added dropwise to the target cycle fluorescence-electrochemical dual signal aptamer sensor for detecting lead ions, the electrochemical signal is detected, and the Pb in tea and vegetables is calculated according to the standard curve 2+ The content.

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