A fluorescent biosensor for detecting lead ions and a preparation method and application thereof

Abstract

The application belongs to the technical field of detection, and more particularly relates to a fluorescent biosensor for lead ion detection and a preparation method and application thereof. The application uses thio GR-5S and thio Poly-T as reactants after thiol activation treatment by TCEP and gold nanoparticles as reactants, constructs a primary gold label probe through a halide assisted modification strategy, uses the primary gold label probe and biotin labeled GR-5E as reactants, and obtains a bifunctional gold label probe through hybridization and annealing treatment. The bifunctional gold label probe and streptavidin magnetic beads are used as reactants, and the AuNP@MB probe is obtained through a biotin-streptavidin binding reaction. The probe is used for lead ion detection, shows a linear detection range of 1-500 nM, a detection limit of 0.34 nM, and excellent performance in actual sample analysis, with a recovery rate of 91.6-117%.

Description

Technical Field

[0001] This invention belongs to the field of detection technology, and more specifically relates to a fluorescent biosensor for lead ion detection, its preparation method, and its application. Background Technology

[0002] Lead, as a toxic heavy metal, seriously endangers food and environmental safety. Traditional detection methods include inductively coupled plasma mass spectrometry (ICP-MS), atomic absorption spectrometry (AAS), atomic fluorescence spectrometry (AFS), and electrochemical methods. Although these methods have high accuracy and sensitivity, they are limited by equipment cost, operational complexity, and on-site applicability / portability, making them unsuitable for on-site and rapid detection. Therefore, there is an urgent need to develop new detection methods that are economical, rapid, highly sensitive, and highly specific.

[0003] GR-5 DNAzyme is a type of Pb 2+ Pb is a functional double-stranded nucleic acid (composed of a base strand and an enzyme strand) with enzymatic catalytic activity. 2+ It can specifically cleave the rA site in its base strand. GR-5 DNAzyme has gained favor among researchers due to its high specificity and sequence programmability. In recent years, the introduction of nanomaterials has provided broad prospects for the development of GR-5 DNAzyme biosensors. Robust, reliable, low-cost, and easily prepared nanomaterials are crucial for the widespread application of GR-5 DNAzyme.

[0004] Among numerous nanomaterials, gold nanoparticles (AuNPs) are widely used in the construction of biosensors due to their excellent optoelectronic properties and the versatility and ease of preparation and surface functionalization. Especially in fluorescence-based biosensors, gold nanoparticles, as strong fluorescence acceptors, can efficiently quench fluorescent group signals through the fluorescence energy resonance transfer (FRET) effect.

[0005] Terminal deoxyribonuclease (TdT) is a template-independent DNA polymerase that catalyzes the addition of dNTPs to the 3'-OH ends of oligonucleotides, single-stranded DNA, or double-stranded DNA with sticky ends. The minimum length of the oligonucleotide it catalyzes is three nucleotides. This enzyme is an isothermal amplification polymerase, requiring simple amplification conditions and eliminating the need for large-scale equipment, thus possessing great application potential.

[0006] Traditional FRET mainly involves modifying the GR-5 DNAzyme onto the surface of gold nanoparticles, Pb 2+ The base strand is cleaved at rA, releasing a fluorescent group, which is then collected to capture the fluorescence signal. For example, Kim et al. modified the GR-5 DNAzyme with a fluorescent group, and then collected the signal using Pb. 2+Enzymatic cleavage releases a fluorescent group, causing the fluorescence signal to change from "quenching" to "on". However, this process is overly dependent on Pb. 2+ Cutting efficiency.

[0007] Therefore, it is necessary to develop a novel FRET biosensor for Pb in matrices such as food and the environment. 2+ Rapid, low-cost, and instant detection is of great significance. Summary of the Invention

[0008] The purpose of this invention is to provide a fluorescent biosensor for lead ion detection, its preparation method, and its application. More specifically, it provides a TdT-induced fluorescence resonance energy transfer (FRET) signal amplification biosensor for Pb detection. 2+ The detection method utilizes TdT to immobilize Cy3 fluorescently modified dUTP onto the DNA strand on the surface of gold nanoparticles, thereby realizing a simple and efficient novel FRET detection strategy. A magnetic particle-gold nanoparticle probe (AuNP@MB probe) was constructed by binding a bifunctional gold-labeled probe (Poly-T / GR-5DNAzyme@AuNP) with SA@MB, achieving Pb detection during the detection process. 2+ Highly efficient enzymatic digestion and magnetic separation.

[0009] To achieve the above objectives, the present invention provides the following solution:

[0010] One of the technical solutions of this invention is to provide a method for preparing an AuNP@MB probe, comprising the following steps:

[0011] Thio-GR-5S and thio-Poly-T were activated with thiol groups using TCEP and then reacted with gold nanoparticles (AuNPs) to construct a primary gold-labeled probe (Poly-T / GR-5S@AuNP) through a halide ion-assisted modification strategy.

[0012] Using the primary gold-labeled probe and biotin-labeled GR-5E as reactants, a bifunctional gold-labeled probe (Poly-T / GR-5 DNAzyme@AuNP) was obtained by hybridization annealing.

[0013] Using the bifunctional gold-labeled probe and streptavidin magnetic beads (SA@MB) as reactants, the AuNP@MB probe (Poly-T / GR-5 DNAzyme@AuNP@MB) was obtained through a biotin-streptavidin binding reaction.

[0014] Furthermore, the molar ratio of the thiolated GR-5S to the gold nanoparticles is 15-40:1, preferably 20:1.

[0015] Furthermore, the molar ratio of the thiopoly-T to the gold nanoparticles is 150-250:1, preferably 180:1.

[0016] Furthermore, the step of the thiol activation treatment includes: mixing the thio-GR-5S solution and the thio-Poly-T solution with the TCEP solution respectively, wherein the concentration of TCEP in the mixed system is 3 mM, and the treatment time is 2 h.

[0017] Furthermore, the steps of the halide ion-assisted modification strategy include: mixing thio-activated GR-5S and thio-Poly-T with gold nanoparticles, then first adding NaF solution to make the final concentration of NaF 15-25 mM (preferably 20 mM), then adding NaBr solution to make the final concentration of NaBr 260-300 mM (preferably 280 mM), reacting for 1.5-2.5 h (preferably 2 h), centrifuging, and resuspending to obtain the primary gold-labeled probe.

[0018] Optionally, the concentration of the gold nanoparticles in the system mixed with thio-activated GR-5S and thio-Poly-T is 15-25 nM (preferably 20 nM).

[0019] Furthermore, the molar ratio of the thio-GR-5S to the biotin-labeled GR-5E is 1:1.2.

[0020] Furthermore, the hybridization annealing process includes: mixing biotin-labeled GR-5E with a primary gold-labeled probe, reacting at 60 °C for 10 min, cooling and continuing the reaction for 3 h, centrifuging and resuspending to obtain the bifunctional gold-labeled probe.

[0021] Furthermore, the ratio of the bifunctional gold-labeled probe to streptavidin magnetic beads is 20-30 pmol: 1 mg.

[0022] Furthermore, the biotin-streptavidin binding reaction includes the following steps:

[0023] The streptavidin magnetic bead solution (10 mg / mL) was magnetically separated to remove the supernatant, washed, resuspended, and then mixed with 1 wt% BSA solution and reacted for 1 h. After washing and resuspending, the solution was brought to a final volume of 5 mg / mL (this operation is to block unmodified sites on the SA@MB surface, thereby reducing nonspecific adsorption). The solution was then mixed with the bifunctional gold-labeled probe and reacted for 30 min. The free bifunctional gold-labeled probe was removed by magnetic separation to obtain the AuNP@MB probe.

[0024] The second technical solution of the present invention is to provide an AuNP@MB probe, which is prepared by the above-described preparation method.

[0025] The third technical solution of the present invention provides an application of the above-mentioned AuNP@MB probe in a fluorescent biosensor for lead ion detection.

[0026] The fourth technical solution of the present invention provides a fluorescent biosensor for lead ion detection, wherein the fluorescent biosensor for lead ion detection uses the above-mentioned AuNP@MB probe as a biorecognition element.

[0027] Fifth technical solution of the present invention: to provide an application of the above-mentioned fluorescent biosensor in the detection of lead ions in food and environmental samples.

[0028] In this invention, GR-5 DNA enzyme is used as Pb 2+ This invention provides a novel TdT-induced fluorescence resonance energy transfer (FRET) signal amplification biosensor for detecting Pb in food and environmental samples. 2+ The core of this FRET-based biosensor lies in its ultra-sensitive detection.

[0029] (1) A bifunctional DNA-gold nanoparticle probe (Poly-T / GR-5 DNAzyme@AuNP) was constructed by halide ion-assisted modification, which can efficiently bind streptavidin magnetic beads (SA@MB) to achieve rapid magnetic separation;

[0030] (2) The introduction of TdT overcomes the limitations of the traditional FRET method on Pb. 2+ Dependence on DNase cleavage efficiency. TdT utilizes Cy3-dUTP as a substrate to catalyze the cleavage of a bifunctional DNA-AuNP probe (made from Pb). 2+ The strategy of efficiently extending the Poly-T sequence on the cleaved GR-5 DNAzyme (released from the DNA) is based on the strong FRET effect of AuNPs, which amplifies the Pb-related gene. 2+ The detection signal.

[0031] Under optimized conditions, the proposed Pb 2+ The biosensor exhibits a linear detection range of 1–500 nM and a limit of detection (LOD) of 0.34 nM. It demonstrates excellent performance in real-world sample analysis (preserved eggs, tea, lake water, and tap water), with recoveries ranging from 91.6% to 117%. Furthermore, this method eliminates the need for complex separation and purification steps, highlighting its strong potential for in-situ detection.

[0032] The sixth technical solution of the present invention provides a method for quantitative detection of lead ions. The method uses the above-mentioned AuNP@MB probe as the core identification and signal activation element of the lead ion detection system, and realizes quantitative detection of lead ions through cascade amplification effect.

[0033] Furthermore, the step of detecting lead ions through cascade amplification includes:

[0034] The AuNP@MB probe was brought to a final volume of 2 mg / mL to obtain the AuNP@MB probe solution.

[0035] Take 45 μL of the AuNP@MB probe solution and add 5 μL of Pb-containing solutions of different concentrations. 2+ In the solution, the reaction was carried out for 45 min. Unreacted MB@AuNPs probes were removed by magnetic separation. The supernatant after magnetic separation was collected, and 2.7 μL of extension reaction reagent (20 U / μL), 3 μL of Cy3-dUTP (100 μM), and 12.5 μL of extension reaction buffer were added. The amplification reaction was carried out at 37 °C for 90 min, and then the temperature was raised to 70 °C and held for 10 min to terminate the reaction. Simultaneously, a Pb-free setting was used. 2+ For the blank control experiment, 60 μL of the reaction solution was taken and its fluorescence intensity was measured by an enzyme-linked immunosorbent assay (ELISA) reader, with the excitation wavelength set to 532 nm and the emission wavelength set to 570 nm, to obtain the standard curve.

[0036] The AuNP@MB probe solution is then added to the test solution, and the fluorescence intensity is obtained using the same steps as in preparing the standard curve. The fluorescence intensity is then compared with the standard curve to calculate the lead ion concentration of the test solution.

[0037] Optionally, the extension reaction reagent is terminal deoxynucleotidyl transferase (TdT); the extension reaction buffer is a solution composed of 1 M potassium cacodylate, 0.05% (v / v) polyethylene glycol octylphenyl ether (Triton X-100) and 5 mM CoCl2.

[0038] In lead ion detection, when Pb 2+ In its presence, the base chain is cleaved at rA, resulting in the release and separation of AuNPs carrying the Poly-T sequence. Unreacted AuNP@MB is then removed via magnetic separation. The introduction of extension reagents such as TdT adds Cy3-dUTP to the 3'-OH end of the Poly-T on the AuNP, forming a long Poly-U chain on the AuNP surface. This enables highly efficient FRET quenching of Cy3-dUTP by AuNPs, and the fluorescence signal is used to detect Pb. 2+ Quantitative detection was performed.

[0039] The present invention discloses the following technical effects:

[0040] This invention uses the AuNP@MB probe for lead ion detection. Compared with traditional detection methods, it does not require complex professional instruments and can meet the needs of on-site detection.

[0041] This invention provides a novel detection mechanism for TdT-induced FRET signal amplification, which, compared to traditional FRET detection methods, reduces the impact on Pb. 2+ Dependence on enzyme digestion efficiency.

[0042] The preparation and detection methods of this invention do not require complex separation and purification steps, and have the advantages of being simple and rapid. Attached Figure Description

[0043] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an undue limitation of the invention. In the drawings:

[0044] Figure 1 For the detection of Pb using AuNP@MB probe 2+ A schematic diagram illustrating the principle.

[0045] Figure 2 Characterization diagrams of AuNPs, BSA / SA@MB, Poly-T / GR-5S@AuNP, Poly-T / GR-5 DNAzyme@AuNP, and BSA / SA@MB+GR-5 DNAzyme@AuNP@MB (AuNP@MB probes). A represents UV-vis characterization. Figure I From left to right: electrophoretic characterization of AuNP, Poly-T / GR-5S@AuNP, and Poly-T / GR-5 DNAzyme@AuNP. Figure II In the figures, a and e are images of AuNPs, BSA / SA@MB, Poly-T / GR-5S@AuNP, Poly-T / GR-5 DNAzyme@AuNP, and BSA / SA@MB+GR-5 DNAzyme@AuNP@MB, respectively; B is the dynamic light scattering (DLS) distribution curve of AuNPs, Poly-T / GR-5S@AuNP, and Poly-T / GR-5 DNAzyme@AuNP; C is a transmission electron microscope image of AuNP; D is a transmission electron microscope image of Poly-T / GR-5S@AuNP; E and F are transmission electron microscope images of the AuNP@MB probe.

[0046] Figure 3 In the diagram, A represents the Pb of TdT enzyme-induced FRET. 2+ Feasibility of the test, based on FRET Pb 2+ Detection diagram; B represents Pb 2+Specific gel electrophoresis results of GR-5 DNAzyme lysis; C is the transmission electron microscope image of the product after TdT enzyme amplification; D shows the UV-vis and electrophoretic characterization of Poly-T / GR-5 DNAzyme@AuNP under different conditions; E shows the effect of different Poly-T / GR-5 DNAzyme@AuNP concentrations and different TdT concentrations on fluorescence intensity, with the blue histogram representing Pb. 2+ The fluorescence intensity value (ΔF) of FRET induced by TdT enzyme after the enzyme digestion reaction.

[0047] Figure 4 The results show the optimization of the AuNP@MB probe preparation scheme. Among them, A and B are the optimization of the molar ratio of thio-GR-5S to gold nanoparticles, C is the optimization of AuNP@MB probe concentration, D is the optimization of the type of lysis buffer, E is the optimization of the NaCl concentration of the lysis buffer, F is the optimization of the lysis time, G is the optimization of the TdT concentration, and H is the optimization of the amplification time.

[0048] Figure 5 In the middle, A is Pb 2+ The concentration detection curve, where B represents the ΔF / F0 value versus Pb. 2+ The standard curve for concentration is shown. C represents the selectivity of the AuNP@MB probe for different ions, D represents the stability of the AuNP@MB probe, and E represents the comparison between the AuNP@MB probe measurement results and the ICP-MS method for Pb in actual samples. 2+ The detection results are compared, with F representing the Pb content in samples of preserved eggs, tea, lake water, and tap water obtained using the AuNP@MB probe. 2+ The detection discrimination. Detailed Implementation

[0049] 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.

[0050] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0051] 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.

[0052] 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.

[0053] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0054] Unless otherwise specified, all raw materials and reagents involved in the specific embodiments of this invention are commercially available products.

[0055] Unless otherwise specified, room temperature and ambient temperature in the specific embodiments of this invention refer to 20-37℃.

[0056] It should be noted that any aspects not described in detail in this invention are conventional practices in the field and are not the focus of this invention.

[0057] The preparation steps of the gold nanoparticles (AuNPs) used in the specific embodiments of the present invention are as follows:

[0058] 30 mL of ultrapure water was injected into a round-bottom flask equipped with a reflux condenser and heated to boiling under reflux. Then, 300 μL of HAuCl4 aqueous solution (1%) was added to the flask. After boiling, 900 μL of trisodium citrate solution (1%) was added to the flask. The mixture was heated under reflux with strong magnetic stirring. The color of the solution changed from colorless to purplish-red and then gradually turned red. The mixture was heated for another 15 min and then cooled to room temperature to obtain gold nanoparticles with a particle size of approximately 13 nm. These nanoparticles were stored in a refrigerator at 4 °C for later use.

[0059] The nucleotide sequences of SH-GR-5S, SH-Poly-T, and Bio-GR-5E involved in the specific embodiments of the present invention are shown in Table 1.

[0060] Table 1

[0061]

[0062] Note: In SH-GR-5S, rA is the cleavage site after the DNAzyme structure is formed. "rA" is adenine ribonucleic acid, which is embedded in the DNA sequence as a cleavage site.

[0063] Example 1

[0064] The preparation steps of the AuNP@MB probe include:

[0065] S1. Thio-GR-5S and thio-Poly-T were treated with TCEP (final concentration 3mM) for 2 hours at room temperature to remove disulfide bonds, resulting in SH-GR-5S and SH-Poly-T.

[0066] S2. Mix SH-GR-5S, SH-Poly-T and the above gold nanoparticles to obtain a mixed solution with a gold nanoparticle concentration of 20 nM.

[0067] The molar ratio of SH-GR-5S to gold nanoparticles is 20:1, and the molar ratio of SH-Poly-T to gold nanoparticles is 180:1.

[0068] S3. First, add NaF (500 mM) solution to the above mixed solution to make the final concentration 20 mM, then add NaBr (500 mM) solution to make the final concentration 280 mM. Place the mixture in a constant temperature (24℃) track shaker for 2 hours to complete the modification of AuNPs. Centrifuge at 12000 rpm for 5 min, repeat 3 times, and resuspend in Tris-HCl buffer (10 mM Tris-HCl, 150 mM NaCl, 0.05% Tween-20, pH 7.4) to obtain Poly-T / GR-5S@AuNP;

[0069] S4. Mix the biotin-labeled GR-5E (Bio-GR-5E) with the Poly-T / GR-5S@AuNP obtained in step S3. After mixing, place the mixture in a 60 ℃ water bath for 10 min, slowly cool to room temperature and let it stand for 3 h. After that, remove the mixture and centrifuge at 10500 rpm. Repeat twice to remove the free biotin-labeled GR-5E. Resuspend the mixture in Tris-HCl buffer (10 mM Tris-HCl, 150 mM NaCl, pH 7.4) to obtain Poly-T / GR-5 DNAzyme@AuNP.

[0070] In step S2, the molar ratio of SH-GR-5S to biotin-labeled GR-5E is 1:1.2.

[0071] S5. The SA@MB sample solution (10 mg / mL) was magnetically separated to remove the supernatant. An appropriate amount of Tris-HCl buffer (10 mM Tris-HCl, 150 mM NaCl, pH 7.4) was added, and the mixture was washed three times and resuspended. SA@MB was then mixed with 1% BSA solution (dissolved in Tris-HCl buffer, 10 mM Tris-HCl, 150 mM NaCl, pH 7.4) and reacted on a rotary mixer for 1 h. The mixture was washed three times with Tris-HCl buffer (10 mM Tris-HCl, 150 mM NaCl, pH 7.4), resuspended, and brought to a final volume of 5 mg / mL. This operation blocks unmodified sites on the SA@MB surface, thereby reducing non-specific adsorption, resulting in a pretreated SA@MB solution, denoted as BSA / SA@MB.

[0072] S6. Mix Poly-T / GR-5 DNAzyme@AuNP and pretreated SA@MB solution at a ratio of 30 pmol: 1 mg, and react in a rotary vortex mixer for 30 min. Remove free Poly-T / GR-5 DNAzyme@AuNP by magnetic separation. Wash with Tris-HCl buffer (10 mM Tris-HCl, 150 mM NaCl, pH 7.4) three times to obtain the AuNP@MB probe (denoted as Poly-T / GR-5 DNAzyme@AuNP@MB or BSA / SA@MB+GR-5 DNAzyme@AuNP@MB).

[0073] Test case

[0074] The AuNP@MB probe prepared in Example 1 was brought to a concentration of 2 mg / mL for later use.

[0075] 5 μL of Pb at different concentrations 2+ The standard solution was mixed with 45 μL of MB@AuNPs probes and reacted for 45 min. Unreacted MB@AuNPs probes were removed by magnetic separation. The supernatant was collected, and 2.7 μL of TdT (20 U / μL), 3 μL of Cy3-dUTP (100 μM), and 12.5 μL of extension reaction buffer (1 M potassium cacodylate, 0.05% (v / v) Triton X-100, 5 mM CoCl2) were added. The mixture was incubated at 37 °C for 90 min, and then the temperature was raised to 70 °C and held for 10 min to terminate the reaction. A Pb-free setting was also included. 2+In the blank control experiment, 60 μL of the reaction solution was taken from each well and placed in a microplate. The fluorescence intensity was measured using a microplate reader, with the excitation wavelength set to 532 nm and the emission wavelength set to 570 nm. A standard curve was obtained, and the detection principle is as follows: Figure 1 As shown.

[0076] The fluorescence intensity in the actual sample was then detected using the method for preparing the standard curve, and the lead ion concentration in the test solution was calculated by comparing it with the standard curve.

[0077] Figure 2 Characterization diagrams of AuNPs, BSA / SA@MB, Poly-T / GR-5S@AuNP, Poly-T / GR-5 DNAzyme@AuNP, and BSA / SA@MB+GR-5 DNAzyme@AuNP@MB (AuNP@MB probes). A represents UV-vis characterization. Figure I Electrophoretic characterization of AuNP, Poly-T / GR-5S@AuNP, and Poly-T / GR-5 DNAzyme@AuNP, insert Figure II In the figures, a and e are images of AuNPs, BSA / SA@MB, Poly-T / GR-5S@AuNP, Poly-T / GR-5 DNAzyme@AuNP, and BSA / SA@MB+GR-5 DNAzyme@AuNP@MB, respectively; B is the dynamic light scattering (DLS) distribution curve of AuNPs, Poly-T / GR-5S@AuNP, and Poly-T / GR-5 DNAzyme@AuNP; C is a transmission electron microscope image of AuNP; D is a transmission electron microscope image of Poly-T / GR-5S@AuNP; E and F are transmission electron microscope images of the AuNP@MB probe. As shown in the figure, in A, the SPR absorption wavelength gradually red-shifts with the increase of the molecular weight of the DNA modified on the AuNP surface; in B, the hydrated particle size of Poly-T / GR-5 DNAzyme@AuNP is the largest; in E and F, the prepared Poly-T / GR-5 DNAzyme@AuNP successfully binds to SA@MB (the small particles in the figure are Poly-T / GR-5 DNAzyme@AuNP). These characterization results demonstrate that AuNPs and their probes were successfully prepared.

[0078] Figure 3 In the diagram, A represents the Pb of TdT enzyme-induced FRET. 2+ Feasibility of the test, based on FRET Pb 2+ Detection diagram; B represents Pb 2+Specific gel electrophoresis results of GR-5 DNAzyme lysis; C is the transmission electron microscope image of the product after TdT enzyme amplification; D shows the UV-vis and electrophoretic characterization of Poly-T / GR-5 DNAzyme@AuNP under different conditions; E shows the effect of different Poly-T / GR-5 DNAzyme@AuNP concentrations and different TdT concentrations on fluorescence intensity, with the blue histogram representing Pb. 2+ Following the enzyme digestion reaction, the fluorescence intensity value (ΔF) of FRET induced by TdT enzyme was observed. As shown in the figure, B represents Pb. 2+ Specific gel electrophoresis of lysed GR-5 DNAzyme, where lane 1: GR-5S; lane 2: GR-5E; lane 3: GR-5 DNAzyme; lane 4: GR-5 DNAzyme + Pb. 2+ Agarose gel concentration 3.5%, voltage 120 V, time 30 min; results showed that GR-5 DNAzyme was precipitated by Pb. 2+ Successful enzyme digestion; Figure C shows that the amplified gold-labeled probe is well dispersed and does not aggregate; the parameters for D are: Pb 2+ 3 μM, agarose gel concentration 1%, voltage 100 V, time 12 min, UV-vis characterization results showed that Pb 2+ The cleaved probe exhibited a blue shift in SPR absorption wavelength compared to the uncleaved probe; absorption peaks appeared near 523 nm and 547 nm in the amplified product; the inset in D shows that the probe migration speed was slower after amplification; E shows that the fluorescence signal after TdT amplification was significantly reduced (quenched by AuNP), and this was unaffected by the concentration of the unamplified gold-labeled probe or TdT. These results indicate that the TdT-induced FRET protocol is feasible.

[0079] Optimization of AuNP@MB probe preparation scheme:

[0080] AuNP@MB probe concentration optimization ( Figure 4 (C) Take an appropriate amount of AuNP@MB probe, wash it, and prepare 50 μL solutions with concentrations of 0.5, 1, 2, 3, and 4 mg / mL, respectively. Remove 5 μL of supernatant by magnetic separation. Add 5 μL of 2 μM Pb to each solution. 2+ A standard solution was prepared to a concentration of 200 nM, and then the mixture was placed in a test tube and allowed to react for 30 min using a rotary mixer. At this point, Pb... 2+The GR-5 DNAzyme was lysed to detach AuNPs. After the reaction, the supernatant was magnetically separated into test tubes, and 12.5 μL of extension buffer, 2 μL of TdT, and 3 μL of Cy3-dUTP were added. The tubes were then placed in a 37°C water bath for 60 min. After the reaction, the temperature was increased to 70°C and held for 10 min to inactivate TdT. 60 μL of each solution was then transferred to black microplates, and the fluorescence intensity was measured using a microplate reader. Three parallel experiments were performed for each concentration.

[0081] Optimization of lysis buffer types ( Figure 4 D): Prepare HEPES (10 mM HEPES, 150 mM NaCl, 0.05% Tween-20) buffer, Tris-HCl (10 mM Tris-HCl, 150 mM NaCl, 0.05% Tween-20, pH 7.4) buffer, 4×SSC (containing 0.05% Tween-20) buffer, and PBS (containing 0.05% Tween-20) buffer, respectively. Take an appropriate amount of AuNP@MB probe, wash it, and then adjust the volume to 2 mg / mL with the above buffers, 50 μL of each. Remove 5 μL of supernatant by magnetic separation. Add 5 μL of 2 μM Pb to each. 2+ A standard solution was prepared to a concentration of 200 nM and then reacted on a rotary mixer for 30 min. After the reaction, the supernatant was magnetically separated into test tubes, and 12.5 μL of extension reaction buffer, 2 μL of TdT, and 3 μL of Cy3-dUTP were added. The mixture was then placed in a water bath at 37°C for 60 min. After the reaction, the temperature was increased to 70°C and maintained for 10 min to inactivate TdT. 60 μL of each solution was then transferred to a black ELISA plate, and the fluorescence intensity was measured using an ELISA reader. Three parallel experiments were performed for each buffer.

[0082] Optimization of NaCl concentration in lysis buffer ( Figure 4 (E): Prepare Tris-HCl buffer solutions with concentrations of 100, 150, 300, 400, and 500 mM, respectively. Take an appropriate amount of AuNP@MB probe, wash it, and then adjust the volume to 2 mg / mL with the buffer solutions of the above different salt concentrations, 50 μL for each. Remove 5 μL of supernatant by magnetic separation. Add 5 μL of 2 μM Pb to each solution. 2+A standard solution was prepared to a concentration of 200 nM and then reacted on a rotary mixer for 30 min. After the reaction, the supernatant was magnetically separated into test tubes, and 12.5 μL of extension reaction buffer, 2 μL of TdT, and 3 μL of Cy3-dUTP were added. The tubes were then placed in a 37°C water bath for 60 min. After the reaction, the temperature was increased to 70°C and maintained for 10 min to inactivate TdT. 60 μL of each solution was then transferred to a black ELISA plate, and the fluorescence intensity was measured using an ELISA reader. Three parallel experiments were performed for each salt concentration.

[0083] Optimization of pyrolysis time ( Figure 4 (F in the original text) Take an appropriate amount of AuNP@MB probe, adjust the volume to 50 μL with Tris-HCl buffer, and remove 5 μL of supernatant by magnetic separation. Add 5 μL of 2 μM Pb solution. 2+ A standard solution was prepared to a concentration of 200 nM in the solution, and then the mixture was reacted in a test tube using a rotary mixer for 15 min, 30 min, 45 min, 60 min, and 90 min, respectively. After the reaction, the supernatant was magnetically separated into test tubes, and 12.5 μL of extension reaction buffer, 2 μL of TdT, and 3 μL of Cy3-dUTP were added. The mixture was then placed in a water bath at 37°C for 60 min. After the reaction, the temperature was increased to 70°C and maintained for 10 min to inactivate TdT. Then, 60 μL of each solution was transferred to a black ELISA plate, and the fluorescence intensity was measured using an ELISA reader. Three parallel experiments were performed for each lysis time.

[0084] TdT concentration optimization ( Figure 4 (G in the original text) Take an appropriate amount of AuNP@MB probe, adjust the volume to 50 μL with Tris-HCl buffer, and remove 5 μL of supernatant by magnetic separation. Add 5 μL of 2 μM Pb solution. 2+ A standard solution was prepared to a concentration of 200 nM in the solution. The solution was then placed in a rotary vortex mixer and reacted for 45 min. After the reaction, the supernatant was magnetically separated and placed in a test tube. 12.5 μL of extension reaction buffer was added, followed by 0.6, 1.4, 2, 2.7, and 3.5 μL of TdT (concentration 20 U / μL) and 3 μL of Ly3-dUTP, respectively. The mixture was then placed in a water bath at 37°C and reacted for 60 min. After the reaction, the temperature was raised to 70°C and held for 10 min to inactivate TdT. 60 μL of each solution was then transferred to a black ELISA plate, and the fluorescence intensity was measured using an ELISA reader. Three parallel experiments were performed for each TdT concentration.

[0085] Amplification time optimization ( Figure 4(H in the text) Take an appropriate amount of AuNP@MB probe, adjust the volume to 50 μL with Tris-HCl buffer, and remove 5 μL of supernatant by magnetic separation. Add 5 μL of 2 μM Pb solution. 2+ The standard solution was prepared to a concentration of 200 nM and then reacted on a rotary mixer for 45 min. After the reaction, the supernatant was magnetically separated into test tubes, and 12.5 μL of extension reaction buffer, 2.7 μL of TdT (concentration 20 U / μL), and 3 μL of Cy3-dUTP were added. The tubes were then placed in a water bath at 37℃ for 20, 40, 60, 90, and 120 min, respectively. After the reaction was completed, the temperature was raised to 70℃ and held for 10 min to inactivate TdT. 60 μL of each solution was then transferred to a black ELISA plate, and the fluorescence intensity was measured using an ELISA reader. Three parallel experiments were performed for each amplification reaction time.

[0086] Figure 4 The results show the optimization of the AuNP@MB probe preparation scheme. Among them, A and B are the optimization of the molar ratio of thio-GR-5S to gold nanoparticles, C is the optimization of AuNP@MB probe concentration, D is the optimization of the type of lysis buffer, E is the optimization of the NaCl concentration of the lysis buffer, F is the optimization of the lysis time, G is the optimization of the TdT concentration, and H is the optimization of the amplification time.

[0087] Pb solutions of 1 nM, 10 nM, 1 μM, 2 μM, 5 μM, 10 μM, 20 μM, and 30 μM were prepared respectively. 2+ For the standard solution, take an appropriate amount of AuNP@MB probe and dilute to 50 μL with Tris-HCl buffer. Remove 5 μL of supernatant using magnetic separation. Add 5 μL of Pb at different concentrations respectively. 2+ The standard solution was prepared and then reacted on a rotary mixer for 45 min. After the reaction, the supernatant was magnetically separated into test tubes, and 12.5 μL of extension reaction buffer, 2.7 μL of TdT (concentration 20 U / μL), and 3 μL of Ly3-dUTP were added. The mixture was then placed in a 37℃ water bath for 90 min. After the reaction, the temperature was increased to 70℃ and held for 10 min to inactivate TdT. 60 μL of each solution was then transferred to a black ELISA plate, and the fluorescence intensity was measured using an ELISA reader. The fluorescence intensity was measured for each Pb. 2+ Three parallel experiments were conducted to determine the concentration. The obtained points were used to obtain a curve through linear fitting, as shown below. Figure 5 A and B in the example.

[0088] Pb used for detection 2+ The initial concentration was 50 nM, and the concentrations of other metal ions were 5 μM. Subsequent steps were performed as described above. Three parallel experiments were conducted for each metal ion. Results are shown below. Figure 5 C in the middle.

[0089] The AuNP@MB probe was stored at 4°C for different periods (2, 4, 6, 8, 10, 12, and 14 days). After being removed and washed, it was used for detection. The Pb content of the detection solution was measured. 2+ The concentration was 200 nM (stock solution 2 μM), and subsequent steps were as described above. Three parallel experiments were performed for each storage time. Results are shown below. Figure 5 D in the middle.

[0090] Comparison of ICP-MS detection and this method ( Figure 5 E in the middle):

[0091] ICP-MS detection: Take 0.0588 g of preserved egg sample and 0.2012 g of tea sample respectively, add 3 mL of nitric acid and 1 mL of hydrogen peroxide, heat at 180℃ on a hot plate for about 30 min to digest, remove acid until the digestion solution is ≤1 mL, cool, and then make up to 10 mL with 2% dilute nitric acid before testing. Lake water and tap water samples were filtered through a microporous membrane and then directly tested.

[0092] For detection using this method: the solid sample mass is consistent with the ICP-MS sample volume. After digestion, the volume is adjusted to 1 mL with dilute nitric acid, and then 5 μL is added to the detection system for analysis. Lake water and tap water samples were filtered through a microporous membrane and used directly to prepare Tris-HCl lysis buffer, which was then added to the washed AuNP@MB probe for reaction. Subsequent steps are as described above.

[0093] Different Pb samples 2+ Significant differences in concentration: 1 μM and 2 μM Pb were added to different samples respectively. 2+ The mother liquor (for preserved egg and tea samples, it needs to be added to the digestion solution) and subsequent methods and procedures are as described above. Six parallel experiments were performed for each concentration. Results are shown below. Figure 5 F in the middle.

[0094] Figure 5 In the middle, A is Pb 2+ The concentration detection curve, where B represents the ΔF / F0 value versus Pb. 2+ The concentration standard curve, where C represents the selectivity of the AuNP@MB probe for different ions (Pb). 2+ Concentration: 50 nM, other metal ion concentration: 5 μM), D represents the stability of the AuNP@MB probe, and E represents the difference between the AuNP@MB probe measurement results and the ICP-MS method for Pb in the actual sample. 2+The detection results are compared (AuNP@MB probe concentration: 2 mg / mL, lysis time: 45 min, TdT concentration: 0.8 U / μL, error bars obtained from three independent parallel experiments), F represents the detection results of Pb in preserved egg, tea, lake water and tap water samples using the AuNP@MB probe. 2+ The detection discrimination (asterisk indicates statistically significant difference (****p<0.0001), error bars were obtained from six independent parallel experiments). As shown in Figure A, the biosensor prepared with the AuNP@MB probe has a detection range of 1 nM to 3 μM; Figure B shows that Pb... 2+ It exhibits a good linear relationship with ΔF / F0 value in the concentration range of 1~500 nM (R 2 =0.9925), detection limit is 0.34 nM (3δ / slope); C shows only the target analyte Pb. 2+ (or Mix) showed a significant response (ΔF / F0) to the FRET biosensor, and Pb 2+ The response signals showed extremely significant differences from those of other metal ions (p < 0.0001); D showed that the FRET biosensor had good stability over 14 days, and the ΔF / F0 value of the sensor decreased slightly over time; E and F showed that the sensor had good accuracy in the detection of actual samples such as preserved eggs, tea, lake water, and tap water.

[0095] The results of the spiked recovery experiment are shown in Table 2. The steps are as follows:

[0096] The digestion solutions of preserved eggs and tea leaves, lake water, and tap water were diluted 20 times, and Pb was added at concentrations of 100 nM, 500 nM, and 1 μM, respectively. 2+ For the mother liquor, the subsequent methods and procedures are as described above. Substitute the measured fluorescence value into the standard curve to calculate Pb. 2+ Concentration value, compared with the actual Pb added 2+ The recovery rate is calculated from the concentration.

[0097] Table 2

[0098]

[0099] As shown in Table 2, the spiked recovery experiment showed that Pb 2+ The spiked recoveries ranged from 91.6% to 117%, all of which demonstrate that this method is reliable and robust for Pb in real-world samples. 2+ Its detection performance makes it applicable to Pb in the environment and food. 2+ Monitoring and analysis.

[0100] The various embodiments in this specification are described in a progressive manner, with each embodiment focusing on the differences from other embodiments. The same or similar parts between the various embodiments can be referred to each other.

[0101] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for preparing an AuNP@MB probe, characterized in that the steps include... include: After thiolated GR-5S and thiolated Poly-T were activated with thiol groups using TCEP, they were reacted with gold nanoparticles to construct a primary gold-labeled probe via a halide-assisted modification strategy. The GR-5S after thiol activation treatment is 5'-SH-TTTTTTGGTCTCACTAT / rA / GGAAGAGATGGCGTC-3'; The thiol-activated Poly-T is 5'-SH-TTTTTTTTTTTTTTTTTTTT-3'; Using the primary gold-labeled probe and biotin-labeled GR-5E as reactants, a bifunctional gold-labeled probe was obtained through hybridization annealing. The biotin-labeled GR-5E is 5'-Bio-TTTTTTCATCTCTGAAGTAGCGCCGCCGTATAGTGAG-3'; The AuNP@MB probe was obtained by reacting the bifunctional gold-labeled probe with streptavidin magnetic beads via a biotin-streptavidin binding reaction. The molar ratio of the thio-GR-5S to the gold nanoparticles is 15-40:1; The molar ratio of the thiopoly-T to the gold nanoparticles is 150-250:1; The steps of the halide ion-assisted modification strategy include: mixing thio-activated GR-5S and thio-Poly-T with gold nanoparticles, then first adding NaF solution to make the final concentration of NaF 15-25 mM, then adding NaBr solution to make the final concentration of NaBr 260-300 mM, reacting for 1.5-2.5 h, centrifuging and resuspending to obtain the primary gold-labeled probe; The steps of the biotin-streptavidin binding reaction include: magnetically separating a 10 mg / mL streptavidin magnetic bead solution to remove the supernatant, washing, resuspending, mixing with 1% BSA solution and reacting for 1 h, washing, resuspending, then adjusting the volume to 5 mg / mL, then mixing with the bifunctional gold-labeled probe, reacting for 30 min, magnetically separating to remove the free bifunctional gold-labeled probe, and obtaining the AuNP@MB probe.

2. The preparation method according to claim 1, characterized in that, The steps of the thiol activation treatment include: mixing thio-GR-5S solution and thio-Poly-T solution with TCEP solution respectively, wherein the concentration of TCEP in the mixed system is 3 mM, and the treatment time is 2 h.

3. The preparation method according to claim 1, characterized in that, The concentration of the gold nanoparticles in the system mixed with thio-activated GR-5S and thio-Poly-T was 15-25 nM.

4. The preparation method according to claim 1, characterized in that, The molar ratio of the thio-GR-5S to the biotin-labeled GR-5E is 1:1.2; The hybridization annealing process includes: mixing biotin-labeled GR-5E with a primary gold-labeled probe, reacting at 60°C for 10 min, cooling, and continuing the reaction for 3 h to obtain the bifunctional gold-labeled probe.

5. The preparation method according to claim 1, characterized in that, The ratio of the bifunctional gold-labeled probe to streptavidin magnetic beads is 20-30 pmol: 1 mg.

6. An AuNP@MB probe, characterized in that, The AuNP@MB probe is prepared by the preparation method according to any one of claims 1-5.

7. The application of the AuNP@MB probe of claim 6 in the preparation of a fluorescent biosensor for lead ion detection.

8. A fluorescent biosensor for lead ion detection, characterized in that, The fluorescent biosensor for lead ion detection uses the AuNP@MB probe of claim 6 as a biorecognition element.

9. The application of the fluorescent biosensor of claim 8 in the detection of lead ions in food and environmental samples.

10. A method for quantitative detection of lead ions, characterized in that, The method uses the AuNP@MB probe described in claim 6 as the core identification and signal activation element of the lead ion detection system, and achieves quantitative detection of lead ions through cascade amplification effect. The method is used for the detection of lead ions in food and environmental matrices.

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