Dual-signal reverse change ratio type upconversion nanoprobes for lead ion detection, and preparation method and application thereof

Abstract

The application relates to the technical field of analytical chemistry, and particularly discloses a dual-signal reverse change ratio type upconversion nanoprobe for lead ion detection as well as a preparation method and application thereof. The dual-signal reverse change ratio type upconversion nanoprobe for lead ion detection comprises polyacrylic acid coated upconversion nanoparticles and Cy3 modified DNA aptamer; wherein the Cy3 modified DNA aptamer serves as a specific recognition unit of Pb 2+ , Cy3 serves as an energy acceptor, and the polyacrylic acid coated upconversion nanoparticles serve as an energy donor. The dual-signal reverse change ratio type upconversion nanoprobe of the application realizes specific combination of the DNA aptamer and Pb 2+ , induces conformational change of the DNA aptamer, shortens the distance between the upconversion nanoparticles and the Cy3, constructs a ratio type detection signal through quenching of an emission peak of the upconversion nanoparticles at 540 nm and enhancement of an emission peak of the Cy3 at 565 nm, and finally realizes high-sensitivity and high-selectivity detection of trace Pb 2+ , and improves the detection sensitivity.

Description

Technical Field

[0001] This invention relates to the field of analytical chemistry, and in particular to a dual-signal inverse ratio upconversion nanoprobe for lead ion detection, its preparation method, and its application. Background Technology

[0002] Lead ions (Pb) 2+ Pb, a typical toxic heavy metal pollutant, is widely present in natural water bodies, soil, and the food chain related to human activities. It is difficult to biodegrade and easily accumulates in organisms, posing a serious and persistent threat to the ecological environment and human health. Studies have confirmed that even long-term exposure to low concentrations of Pb... 2+ In the environment, it may also cause irreversible damage to the human nervous system and kidney dysfunction; at the same time, Pb 2+ Its spread in the environment can also disrupt the ecological balance and harm the growth of plants and animals. Therefore, achieving trace Pb control is crucial. 2+ Rapid and accurate detection is of vital practical significance for safeguarding public health and safety, conducting environmental quality monitoring, and providing risk warnings.

[0003] Currently, it is used to detect Pb 2+ Traditional analytical methods for detecting Pb mainly include atomic absorption spectrometry, mass spectrometry, and electrochemical analysis. While these methods offer high detection sensitivity and reliable results, enabling the detection of trace and even ultra-trace Pb, they are not universally applicable. 2+ While quantitative analysis of Pb is possible, it generally suffers from significant drawbacks: firstly, it relies on expensive, precision instruments, resulting in high equipment maintenance costs; secondly, the operational procedures are complex (e.g., cumbersome sample pretreatment steps), requiring specially trained technicians, and the detection process is difficult to perform outside of a laboratory environment, failing to meet the practical needs of rapid on-site detection and real-time monitoring, thus greatly limiting its application in emergency detection, field environmental monitoring, and other scenarios. To overcome these shortcomings, fluorescence-based detection technology, with its advantages of high sensitivity, ease of operation, fast response speed, no need for complex pretreatment, and ease of on-site detection, is gradually becoming the preferred method for trace Pb analysis. 2+ A research hotspot in the field of detection. However, traditional fluorescent probes mostly use ultraviolet light as the excitation source. This excitation method easily excites the autofluorescence in the sample, resulting in significant background fluorescence interference, which in turn reduces the signal-to-noise ratio and accuracy of detection, making it difficult to meet the requirements for trace Pb in complex matrices. 2+ The need for ultra-sensitive detection.

[0004] Upconversion nanoparticles (UCNPs), as a novel type of fluorescent material, can break through the excitation-emission rules of traditional fluorescent materials, converting low-energy near-infrared light (e.g., 980 nm) excitation into high-energy visible light emission. Their unique optical properties give them unparalleled advantages in trace detection compared to traditional fluorescent materials: First, near-infrared excitation effectively avoids background autofluorescence interference from biomolecules or environmental components in the sample matrix, significantly improving detection specificity; second, near-infrared light has stronger tissue / matrix penetration and causes less photodamage to biological samples, making it suitable for detection in vivo or in complex environmental matrices; third, UCNPs have excellent photostability and are not prone to photobleaching, ensuring the stability and repeatability of detection results. Currently, when constructing traditional high-sensitivity sensing probes based on UCNPs, only a single energy donor emits light as the detection signal. However, fluorescence resonance energy transfer (FRET) is the core mechanism for achieving signal amplification and precise control. The efficiency of this mechanism is highly dependent on the distance between the energy donor (UCNPs) and the energy acceptor (typically less than 10 nm). However, the particle size of UCNPs, which act as energy donors, is usually ranging from tens to hundreds of nanometers. Therefore, the central luminescent region of UCNPs cannot be effectively quenched, resulting in low FRET efficiency and severely limiting its application in trace Pb. 2+ Sensitivity of the upconversion nanoprobe detected. Summary of the Invention

[0005] Based on this, the purpose of this invention is to provide a dual-signal inverse ratio-modulated upconversion nanoprobe for lead ion detection, its preparation method, and its application, enabling the detection of trace Pb in complex matrices such as agricultural products, soil, tap water, and serum. 2+ Accurate detection is needed to meet the requirements of environmental monitoring and public health protection for trace amounts of Pb. 2+ The purpose of testing requirements.

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

[0007] This invention first provides a dual-signal inverse ratio-modified upconversion nanoprobe for lead ion detection, comprising polyacrylic acid-coated upconversion nanoparticles and a Cy3-modified DNA aptamer; wherein the Cy3-modified DNA aptamer serves as the Pb... 2+ The specific recognition unit, Cy3 as the energy acceptor, and the polyacrylic acid-coated upconversion nanoparticles as the energy donor, the upconversion nanoparticles having a green emission peak at 540 nm; the dual-signal inverse ratio-modulated upconversion nanoprobe is constructed as follows: in the presence of Pb 2+ At that time, Pb 2+Binding to the DNA aptamer induces a conformational change in the DNA aptamer, shortening the distance between the upconversion nanoparticles and Cy3, thereby quenching the 540 nm green emission of the upconversion nanoparticles and enhancing the 565 nm fluorescence emission of Cy3. This is achieved by adjusting the luminescence intensity ratio at 540 nm to 565 nm. 540 / I 565 Implement Pb 2+ Ratio-based detection.

[0008] This invention provides a dual-signal inverse ratio-modified upconversion nanoprobe for lead ion detection, using a Cy3-modified DNA aptamer as a Pb ion. 2+ The system utilizes a specific recognition unit, with Cy3 as the energy acceptor and PAA-coated upconversion nanoparticles as the energy donor. The carboxyl groups of PAA undergo an amidation reaction with the amino groups of the aptamer, achieving stable modification of Cy3 on the surface of the upconversion nanoparticles (UCNPs). The absorption spectrum of Cy3 effectively overlaps with the 540 nm green emission spectrum of UCNPs; the green emission of UCNPs excites Cy3 to produce an emission peak at 565 nm. When Pb is present in the system... 2+ At that time, Pb 2+ Specific binding to DNA aptamers induces conformational changes in the DNA aptamers, shortening the distance between UCNPs (donors) and Cy3 (acceptors), significantly improving fluorescence resonance energy transfer (FRET) efficiency. This allows for the construction of a ratiometric detection signal through the quenching of the UCNP emission peak at 540 nm and the enhancement of the Cy3 emission peak at 565 nm, ultimately enabling the detection of trace Pb. 2+ This invention offers highly sensitive and selective detection. Compared to traditional single-emission nanoprobes, the dual-emission signal of this invention significantly improves signal contrast by constructing a ratiometric detection signal, thereby enhancing detection sensitivity.

[0009] As a further improvement to the above-described scheme of the present invention, the upconversion nanoparticles are NaYF4:Yb,Er@NaYF4 upconversion nanoparticles with a core-shell structure.

[0010] As a further improvement to the above-described scheme of the present invention, the upconversion nanoparticles are subjected to the following treatment: the upconversion nanoparticles are added to a hydrochloric acid solution, ultrasonically treated, centrifuged, and washed to obtain hydrophilic upconversion nanoparticles. This can improve the water solubility and biocompatibility of UCNPs. Acid treatment removes oleic acid from the surface of UCNPs, resulting in hydrophilic UCNPs.

[0011] As a further improvement to the above-described scheme of the present invention, in the dual-signal inverse ratio upconversion nanoprobe for lead ion detection, the concentration of Cy3 is 0.4-4.4 μM, preferably 4 μM.

[0012] This invention also provides a method for preparing a dual-signal inverse ratio-modulated upconversion nanoprobe for lead ion detection as described above, comprising the following steps:

[0013] Polyacrylic acid solution was added to upconversion nanoparticle solution and stirred to react. After post-treatment, polyacrylic acid coated upconversion nanoparticles were obtained.

[0014] The polyacrylic acid-coated upconversion nanoparticles were dispersed in HEPES buffer, and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS) were added. The mixture was reacted at room temperature. Then, a Cy3-modified DNA aptamer was added, and the mixture was reacted at room temperature. After post-processing, a dual-signal inverse ratio upconversion nanoprobe for lead ion detection was obtained.

[0015] The present invention also provides a detection device comprising a dual-signal inverse ratio upconversion nanoprobe for lead ion detection as described above.

[0016] This invention also provides a dual-signal inverse ratio-modulated upconversion nanoprobe for lead ion detection, as described above, for detecting Pb in agricultural products, soil, tap water, and human serum. 2+ Applications. For assessing Pb in humans and the environment. 2+ It provides a reliable detection method for exposing risks, carrying out public health protection, and early warning of health risks.

[0017] As a further improvement to the above-described solution of the present invention, the application involves fluorescence detection with an excitation wavelength of 980 nm and an emission wavelength of 500-700 nm, using I... 540 / I 565 This is a quantitative signal. The detection signal used in this invention is the ratio of the luminescence intensity at 540 nm and 565 nm. 540 / I 565 After the introduction of lead ions, the emission at 565 nm is enhanced and the emission at 540 nm is quenched through the fluorescence resonance energy transfer (FRET) process, forming a ratio signal.

[0018] As a further improvement to the above-described scheme of the present invention, the dual-signal inverse ratio-type upconversion nanoprobe for lead ion detection is used for Pb 2+ The detection limit is as low as 43 pM, and within the concentration range of 0-60 nM, Pb... 2+ Concentration and I 540 / I 565 The logarithm of is linearly related.

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] This invention provides a dual-signal inverse ratio-modified upconversion nanoprobe for lead ion detection, using a Cy3-modified DNA aptamer as a Pb ion. 2+ The system utilizes a specific recognition unit, with Cy3 as the energy acceptor and polyacrylic acid (PAA)-coated upconversion nanoparticles as the energy donor. Stable modification of the upconversion nanoparticles (UCNPs) surface is achieved through the amidation reaction between the carboxyl groups of PAA and the amino groups of the aptamer. The absorption spectrum of Cy3 effectively overlaps with the 540 nm green emission spectrum of UCNPs; the green emission of UCNPs excites Cy3, causing it to produce an emission peak at 565 nm. When Pb is present in the system... 2+ When it specifically binds to the aptamer, it can induce a conformational change in the aptamer, shortening the distance between UCNPs (donors) and Cy3 (acceptors), significantly improving FRET efficiency. Furthermore, by quenching the emission peak of UCNPs at 540 nm and enhancing the emission peak of Cy3 at 565 nm, a ratiometric detection signal is constructed, ultimately enabling the detection of trace Pb. 2+ This invention offers highly sensitive and selective detection of Pb. Compared to traditional single-emission nanoprobes, the dual-emission signal of this invention, by constructing a ratiometric detection signal, significantly improves signal contrast, thereby enhancing detection sensitivity. 2+ The detection limit is as low as 43 pM, and within the concentration range of 0-60 nM, Pb... 2+ Concentration and I 540 / I 565 The logarithm of is linearly related.

[0021] This invention combines near-infrared light (980 nm) excitation with a FRET-mediated ratiometric detection signal, effectively avoiding interference from the autofluorescence of biomolecules and environmental humic substances in the sample. This means that the dual-signal inverse-change ratiometric upconversion nanoprobe for lead ion detection in this invention can detect trace amounts of Pb. 2+ The high signal-to-noise ratio detection significantly improves detection accuracy, meeting the requirements for trace Pb in environmental and biological samples. 2+ The monitoring needs. Attached Figure Description

[0022] Figure 1 This is a schematic diagram illustrating the construction and sensing of the dual-signal inverse ratio-modulation upconversion nanoprobe for lead ion detection proposed in this invention; wherein... Figure 1 (A) is a schematic diagram of the construction of the nanoprobe; Figure 1 (B) Detection of Pb using nanoprobes 2+ A schematic diagram of the sensing mechanism.

[0023] Figure 2 Characterization diagram of UCNPs prepared in Example 1 of this invention: Figure 2(A) is a low-resolution TEM image of the initially synthesized nuclear structures UCNPs; Figure 2 (B) is a low-resolution TEM image of core-shell UCNPs; Figure 2 (C) is a particle size distribution diagram of nuclear UCNPs; Figure 2 (D) is a particle size distribution diagram of core-shell structured UCNPs; Figure 2 (E) is a high-resolution TEM image of core-shell UCNPs; Figure 2 (F) shows the XRD patterns of core and core-shell UCNPs; Figure 2 (G) is the upconversion emission spectrum of core-shell UCNPs. Figure 2 The inset in (G) shows images of core-shell UCNPs dispersed in water and excited by a 980 nm laser; Figure 2 (H) is a schematic diagram of energy transfer in upconversion luminescence.

[0024] Figure 3 Characterization diagram of the dual-signal inverse ratio upconversion nanoprobe for lead ion detection prepared in Example 1 of this invention: Figure 3 (A) shows the upconversion emission spectrum of UCNPs-PAA and the excitation and emission spectra of the aptamer-Cy3; Figure 3 (B) Absorbance spectra of PAA-modified PAA-coated UCNPs (UCNPs-PAA), aptamer-Cy3, and aptamer-Cy3-modified UCNPs (nanoparticle probes). Figure 3 (C) shows the FT-IR spectra of oleic acid-coated UCNPs (UCNPs-OA), UCNPs, UCNPs-PAA, and nanoprobes. Figure 3 (D) shows the zeta potential spectra of UCNPs-OA, UCNPs, UCNPs-PAA, and nanoprobes; Figure 3 (E) for the addition of Pb 2+ The emission spectra of the nanoprobes before and after; Figure 3 (F) is for the addition of Pb 2+ Time-resolved fluorescence spectra of the nanoprobes before and after; Figure 3 (G) 60 nM Pb was added as the Cy3 concentration increased. 2+ The emission spectrum of the post-nano probe; Figure 3 (H) is the addition of 60 nM Pb 2+ Afterwards, Cy3 concentration and I 540 / I 565 The concentration quenching curve.

[0025] Figure 4 The dual-signal inverse ratio-modulation upconversion nanoprobe for lead ion detection prepared in Example 1 is used for Pb. 2+Sensitivity analysis results and comparisons of the detection; Figure 4 (A) is a nanoprobe with added Pb 2+ Front and rear light emission diagrams; Figure 4 (B) represents different Pb 2+ UCL spectra of nanoprobes at concentrations (0, 1, 2, 4, 6, 8, 10, 20, 40, 60 nM); Figure 4 (C) represents the luminescence intensity at 540 nm and the Pb concentration. 2+ Linear fitting curve of concentration; Figure 4 (D) represents the luminescence intensity at 565 nm and the Pb concentration. 2+ Linear fitting curve of concentration; Figure 4 (E) for I 540 / I 565 Luminous intensity ratio and Pb 2+ The exponential fitting curve of concentration; Figure 4 (F) represents the logarithmized I 540 / I 565 Luminous intensity ratio and Pb 2+ Linear fitting curve of concentration; Figure 4 (G) is I 540 I 565 and I 540 / I 565 Detect the signal contrast; Figure 4 (H) is I 540 I 565 and I 540 / I 565 The detection limit of the detected signal.

[0026] Figure 5 The dual-signal inverse ratio-modulation upconversion nanoprobe for lead ion detection prepared in Example 1 is used for Pb. 2+ Results of the analysis of the selectivity and anti-interference ability of the detection; Figure 5 (A) Detection of Pb by nanoprobes in the presence of different interfering substances 2+ Selectivity and anti-interference experimental data graph; Figure 5 (B) is the selective emission spectrum of the nanoprobe in the presence of different interfering substances. Detailed Implementation

[0027] To facilitate understanding of the present invention, a more comprehensive description will be given below with reference to specific embodiments. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a more thorough and complete understanding of the disclosure of the present invention.

[0028] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used herein in the description of the invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

[0029] Example 1

[0030] This embodiment proposes a dual-signal inverse ratio-modified upconversion nanoprobe for lead ion detection, comprising polyacrylic acid-coated upconversion nanoparticles and a Cy3-modified DNA aptamer; wherein, the Cy3-modified DNA aptamer serves as the Pb... 2+ The specific recognition unit, with Cy3 as the energy acceptor and PAA-coated upconversion nanoparticles as the energy donor, is used to induce a ratiometric response of dual emission signals, thereby achieving the targeting of trace Pb. 2+ High selectivity and quantitative detection. The upconversion nanoparticles in this embodiment are NaYF4:Yb,Er@NaYF4 upconversion nanoparticles with a core-shell structure. Combined with... Figure 1 (A) The preparation method of the dual-signal inverse ratio-type upconversion nanoprobe for lead ion detection in this embodiment includes the following steps:

[0031] S1. Preparation of polyacrylic acid-coated upconversion nanoparticles

[0032] Preparation of NaYF4:Yb,Er core nanoparticles: 3.89 mmol of YCl3·6H2O, 0.1 mmol of ErCl3·6H2O, and 1 mmol of YbCl3·6H2O were added to 30 mL of oleic acid (OA) and 75 mL of 1-octadecene (ODE). The mixture was heated to 160 °C and held for 1 hour under an argon atmosphere until completely dissolved, then naturally cooled to room temperature to obtain a rare earth salt mixed solution. Simultaneously, 0.5 g of NaOH and 0.74 g of NH4F were dissolved in 50 mL of methanol, and the mixture was slowly added dropwise to the cooled rare earth salt mixed solution. The solution was then heated to 50 °C and held for 40 minutes; the solution was then heated to 100 °C and evacuated for 20 minutes; finally, it was heated to 300 °C and held for 1 hour under an argon atmosphere. After natural cooling, the solution was washed three times with cyclohexane and ethanol to obtain NaYF4:Yb,Er core nanoparticles, which were then stored in cyclohexane.

[0033] Preparation of core-shell structured NaYF4:Yb,Er@NaYF4 upconversion nanoparticles: 1 mmol of YCl3·6H2O was mixed into 6 mL of OA and 15 mL of ODE, heated to 160℃ and held for 1 hour to completely dissolve, then cooled to room temperature and added dropwise to the prepared NaYF4:Yb,Er core nanoparticle solution; 0.1 g of NaOH and 0.148 g of NH4F were dissolved in 15 mL of methanol and added dropwise to the solution, heated to 100℃ and held under vacuum for 20 minutes; finally, heated to 300℃ under argon atmosphere and held for 1 hour, then cooled to room temperature; washed three times with cyclohexane and ethanol to obtain core-shell structured NaYF4:Yb,Er@NaYF4 upconversion nanoparticles (denoted as UCNPs-OA) and stored in cyclohexane.

[0034] Preparation of hydrophilic UCNPs: Since the oleic acid ligands on the surface of the prepared UCNPs make them hydrophobic, acid treatment is used to remove the OA ligands to improve their hydrophilicity. Specifically, 200 mg of the prepared UCNPs (i.e., core-shell structured NaYF4:Yb,Er@NaYF4 upconversion nanoparticles) were added to 60 mL of 0.1 M hydrochloric acid solution and sonicated for 1 hour; then centrifuged at 10000 r / min and washed twice with deionized water; finally dispersed in 20 mL of water to form a 10 mg / mL hydrophilic UCNPs solution (denoted as UCNPs).

[0035] Preparation of polyacrylic acid (PAA) coated UCNPs: In order to add modification sites on the surface of UCNPs, polyacrylic acid (PAA) was used to coat UCNPs. Specifically, 5 mL of PAA solution (5 mg / mL) was added to a hydrophilic UCNPs solution (5 mL, 10 mg / mL), and stirred for 24 hours. Then, the UCNPs were washed three times with deionized water to obtain polyacrylic acid coated UCNPs (denoted as UCNPs-PAA), which were stored in 5 mL of HEPES buffer (10 mM).

[0036] Figure 2 Characterization diagram of the UCNPs prepared in this embodiment: Figure 2 (A) is a low-resolution TEM image of the initially synthesized NaYF4:Yb,Er core nanoparticles; Figure 2 (B) is a low-resolution TEM image of core-shell structured NaYF4:Yb,Er@NaYF4 upconversion nanoparticles; Figure 2 (C) is the particle size distribution diagram of NaYF4:Yb,Er core nanoparticles; Figure 2 (D) is the particle size distribution diagram of core-shell structured NaYF4:Yb,Er@NaYF4 upconversion nanoparticles; Figure 2(E) is a high-resolution TEM image of core-shell structured NaYF4:Yb,Er@NaYF4 upconversion nanoparticles; Figure 2 (F) shows the XRD patterns of NaYF4:Yb,Er core nanoparticles and core-shell structured NaYF4:Yb,Er@NaYF4 upconversion nanoparticles. Figure 2 (G) shows the upconversion luminescence spectrum of core-shell structured NaYF4:Yb,Er@NaYF4 upconversion nanoparticles. Figure 2 (G) The inset is an image of core-shell NaYF4:Yb,Er@NaYF4 upconversion nanoparticles dispersed in water and excited by a 980 nm laser; Figure 2 (H) is a schematic diagram of energy transfer in upconversion luminescence.

[0037] Depend on Figure 2 As can be seen from (A)-(D), the NaYF4:Yb / Er upconversion nanoparticles and NaYF4:Yb / Er@NaYF4 upconversion nanoparticles prepared in this embodiment both exhibit a uniform spherical structure, with the particle size increasing from 27.10 nm to 29.00 nm.

[0038] Depend on Figure 2 (E) It can be seen that the core-shell structured UCNPs prepared in this embodiment have clear lattice stripes and a crystal plane spacing of about 0.51 nm, corresponding to the (100) crystal plane of hexagonal phase NaYF4.

[0039] Figure 2 The XRD pattern of (F) corresponds highly to the standard card (PDF 16-0334) of hexagonal NaYF4, proving the successful synthesis of UCNPs in this embodiment.

[0040] S2. Preparation of a dual-signal inverse ratio-based upconversion nanoprobe for lead ion detection

[0041] 10 mg of polyacrylic acid-coated UCNPs were dispersed in 9 mL of 10 mM HEPES buffer, and 30 mg of EDC and 30 mg of NHS were added. The mixture was stirred at room temperature for 2 h to activate the carboxyl groups on the surface of the UCNPs. Then, 1000 μL of Cy3-modified DNA aptamer (Cy3 concentration of 40 μM) was added, and the mixture was stirred slowly overnight at room temperature. After the reaction was completed, the mixture was centrifuged and washed with ultrapure water to remove unbound aptamers. The centrifugation and washing process was repeated three times. Finally, a dual-signal inverse ratio-modified upconversion nanoprobe for lead ion detection (denoted as UCNPs-aptamer-Cy3) was obtained, dispersed in 10 mL of water, and stored at 4 °C for further application.

[0042] Example 2-11

[0043] The difference between Example 2-11 and Example 1 is that in step S2 of Example 2-11, the concentration of Cy3 in the Cy3-modified DNA aptamer is 4 μM, 8 μM, 12 μM, 16 μM, 20 μM, 24 μM, 28 μM, 32 μM, 36 μM, and 44 μM, respectively.

[0044] Test Example 1

[0045] 10 mg of the dual-signal inverse ratio change type upconversion nanoprobe for lead ion detection prepared in Example 1, the polyacrylic acid-coated UCNPs (UCNPs-PAA) prepared in Example 1, and the Cy3-modified DNA aptamer (denoted as aptamer-Cy3) were added to 10 mL of deionized water, respectively. Under excitation with 980 nm near-infrared light, the UCL spectra in the range of 500–700 nm were recorded using a Fluorolog-3 fluorescence spectrometer. Figure 3 A) Absorbance spectra in the range of 200–700 nm were recorded using a Shimadzu UV-2550 spectrophotometer, and the results were as follows: Figure 3 The UCL spectrum shown in A and as shown in Figure A Figure 3 The absorbance spectrum shown in B. Figure 3 A, Figure 3 Results B show that UCNPs-PAA, when excited by 980 nm near-infrared light, produces two emission peaks at 540 nm and 655 nm, and almost no light absorption in the 200-700 nm range; while aptamer-Cy3 produces a strong absorption peak at 550 nm, and an emission peak at 565 nm when excited at this wavelength; the dual-signal reverse-variation ratio upconversion nanoprobe for lead ion detection prepared in the example, after modifying the aptamer-Cy3 onto the surface of UCNPs-PAA, produces an absorption peak at 550 nm, thus constructing a dual-signal luminescent nanoprobe.

[0046] Test Example 2

[0047] The UCNPs-OA, UCNPs, UCNPs-PAA, and UCNPs-aptamer-Cy3 prepared in Example 1 were tested using an FT-IR spectrometer, and the results were as follows: Figure 3 The infrared spectrum shown in C and as shown in Figure 1 Figure 3 The Zeta potential spectrum is shown in Figure D. Figure 3 The results showed that UCNPs-OA was at 2926 cm⁻¹. -1 2853 cm -1 1558 cm -1 1460 cm -1The UCNPs exhibited strong absorption bands, corresponding to the stretching vibrations of the methylene (CH2) and carboxyl (COOH) groups, respectively. These peaks disappeared entirely after acid treatment, demonstrating the successful removal of the OA ligand. Upon the addition of PAA, these peaks reappeared, only to disappear again after modification with the aptamer-Cy3, confirming the successful modification of the UCNPs surface by PAA and the aptamer-Cy3. Zete potentials showed that before acid treatment, the OA-coated UCNPs were electronegative, after acid treatment they became electronegative, and after coating with PAA and the aptamer, they reverted to electronegativity, further confirming the successful modification by PAA and the aptamer.

[0048] Test Example 3

[0049] 60 nM of Pb was added to the dual-signal inverse ratio-modulation upconversion nanoprobe for lead ion detection prepared in Example 1. 2+ The UCL spectra in the 500–700 nm range were recorded using a Fluorolog-3 fluorescence spectrometer under 980 nm light excitation, yielding the following results: Figure 3 The UCL spectrum shown in E. Figure 3 The results showed that the addition of Pb 2+ Previously, the nanoprobe exhibited two emission peaks at 540 nm and 655 nm; with the addition of Pb... 2+ Subsequently, the emission peak at 540 nm decreased, and a new emission peak was generated at 565 nm. This indicates the presence of Pb. 2+ Subsequently, the UCL emission of UCNPs is absorbed and quenched by Cy3, after which Cy3 generates a new fluorescence emission peak. Figure 3 The time-resolved fluorescence spectrum of F showed that Pb was added. 2+ The luminescence lifetimes of the nanoprobes before and after addition of Pb were compared, and the results showed that the addition of Pb... 2 + The post-luminescence lifetime decreased from 264 μs to 178 μs, proving that the energy transfer mechanism was fluorescence resonance energy transfer (FRET).

[0050] Test Example 4

[0051] 10 mg of the nanoprobes prepared in Examples 1-11 and the UCNPs-PAA prepared in Example 1 were added to 10 mL of deionized water, respectively. Under excitation with 980 nm near-infrared light, the UCL spectra in the range of 500–700 nm were recorded using a Fluorolog-3 fluorescence spectrometer, yielding the following results: Figure 3 The UCL spectrum shown in G and as shown in the figure Figure 3 The luminous intensity shown by H is greater than that of I. 540 / I 565 Linear fitting curve with the concentration of Cy3 in the probe solution. Figure 3The H results showed that as the Cy3 concentration in the probe solution increased, I 540 / I 565 The concentration of Cy3 in the probe solution was continuously reduced until it reached 4 μM. 540 / I 565 To achieve stability, 4 μM was chosen as the optimal Cy3 concentration.

[0052] Test Example 5

[0053] Different concentrations of Pb were added to the nanoprobes prepared in Example 1. 2+ The emission spectra in the 500–700 nm range were recorded using a Fluorolog-3 fluorescence spectrometer, yielding the following results: Figure 4 The UCL spectrum shown in B. Figure 4 Results B showed that Pb... 2+ Concentrations are all related to I 540 ( Figure 4 C) and I 565 ( Figure 4 D) exhibits two different linear relationships; while the ratio of luminous intensity to I 540 / I 565 When detecting signals, Pb is in the range of 0-60 nM. 2+ Concentration and I 540 / I 565 It exhibits a strong exponential relationship (y=40.927*x-0.640, R0). 2 =0.996, Figure 4 E), the limit of detection (LOD) was 43 pM; further analysis of concentration and I 540 / I 565 Logarithmic calculations yielded a linear relationship between concentration and luminescence intensity (y = 3.609 - 0.57x, R0). 2 =0.984). After calculation, I 540 I 565 and I 540 / I 565 The signal strength ratios were 1.70, 5.61, and 10.33, respectively. Figure 4 G), the limits of detection (LOD) were 294 pM, 162 pM and 43 pM, respectively. Figure 4 H). The above results show that, compared with the single detection signal, the ratiometric emission signal not only improves the detection range, but also has a higher signal intensity ratio, lower LOD, and more sensitive detection.

[0054] Test Example 6

[0055] Pb was added to the nanoprobe prepared in Example 5 2+ and different interfering substances (As 3+Hg 3+ Cd 3+ Ni 3+ Co 3+ Fe 2+ Fe 3+ Ca 3+ Cr 3+ Al 3+ (albumin), research probes for Pb 2+ Selectivity and anti-interference ability, obtained Figure 5 The dual-signal inverse ratio-modulation upconversion nanoprobe for lead ion detection shown is for Pb. 2+ The results of the selectivity and anti-interference ability analysis of the detection are shown in the figure. When Pb is added alone... 2+ When there are interfering substances, only Pb 2+ Caused I 540 / I 565 The reduction demonstrates the effectiveness of the nanoprobe for Pb. 2+ It has specific detection; while Pb 2+ After being added to the nanoprobe along with the interfering agent, I 540 / I 565 The stability demonstrates that the nanoprobe has a certain degree of anti-interference capability. This strong selectivity and anti-interference capability will help the probe's sensing ability in complex mechanisms.

[0056] Application examples

[0057] To verify the effectiveness of dual-signal inverse ratio-based upconversion nanoprobes for lead ion detection in Pb 2+ To assess the applicability of the assay, cabbage, soil, tap water, and serum samples from healthy volunteers were used as real samples for spiked recovery experiments. Cabbage and soil samples were pulverized and soaked in deionized water for 12 hours, with the supernatant filtered through a 0.45 μm filter. Beverage and tap water samples were directly filtered through a 0.45 μm filter. Serum samples were diluted tenfold before use. Pb was added at concentrations of 10 nM, 30 nM, and 50 nM using the standard addition method. 2+ The standard solution was added to the dual-signal inverse ratio upconversion nanoprobe prepared in Example 1 for lead ion detection, and the results are shown in Table 1:

[0058] Table 1. Probe detection of Pb in real samples 2+ Spiked recovery data

[0059]

[0060] The results showed that the nanoprobe could accurately detect Pb in each sample. 2+The concentration, relative standard deviation (RSD) was less than 5.4%, and the recovery rate was between 98.80% and 107.50%. Furthermore, the detection results were verified using inductively coupled plasma mass spectrometry (ICP-MS), showing that the nanoprobe detection values ​​were almost identical to the ICP-MS detection values. This demonstrates that the detection device described in this invention can accurately detect Pb in complex biological matrices. 2+ It exhibits strong anti-interference capabilities, and its recovery rate and precision meet the requirements of clinical testing, providing a basis for the accurate measurement of trace Pb. 2+ The new method has been improved.

[0061] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0062] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. A dual-signal inverse ratio-modulated upconversion nanoprobe for lead ion detection, characterized in that, It comprises polyacrylic acid-coated upconversion nanoparticles and Cy3-modified DNA aptamers; wherein the Cy3-modified DNA aptamer serves as Pb 2+ The specific recognition unit, Cy3 as the energy acceptor, and the polyacrylic acid-coated upconversion nanoparticles as the energy donor; the upconversion nanoparticles have a green emission peak at 540 nm; the dual-signal inverse ratio-modulated upconversion nanoprobe is constructed as follows: in the presence of Pb 2+ At that time, Pb 2+ Binding to the DNA aptamer induces a conformational change in the DNA aptamer, shortening the distance between the upconversion nanoparticles and Cy3, thereby quenching the 540 nm green emission of the upconversion nanoparticles and enhancing the 565 nm fluorescence emission of Cy3. This is achieved by adjusting the luminescence intensity ratio at 540 nm to 565 nm. 540 / I 565 Implement Pb 2+ Ratio-based detection.

2. The dual-signal inverse ratio-modulated upconversion nanoprobe for lead ion detection according to claim 1, characterized in that, The upconversion nanoparticles are NaYF4:Yb,Er@NaYF4 upconversion nanoparticles with a core-shell structure.

3. The dual-signal inverse ratio-modulated upconversion nanoprobe for lead ion detection according to claim 2, characterized in that, The upconversion nanoparticles were processed as follows: the upconversion nanoparticles were added to a hydrochloric acid solution, sonicated, centrifuged, and washed to obtain hydrophilic upconversion nanoparticles.

4. The dual-signal inverse ratio-modulated upconversion nanoprobe for lead ion detection according to claim 1, characterized in that, In the dual-signal inverse ratio-modulation upconversion nanoprobe used for lead ion detection, the concentration of Cy3 is 0.4-4.4 μM.

5. A method for preparing a dual-signal inverse ratio-type upconversion nanoprobe for lead ion detection as described in any one of claims 1-4, characterized in that, It includes the following steps: The polyacrylic acid solution was added to the upconversion nanoparticle solution and stirred. Excess polyacrylic acid was removed by centrifugation to obtain polyacrylic acid-coated upconversion nanoparticles. The polyacrylic acid-coated upconversion nanoparticles were dispersed in HEPES buffer, and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide were added. The reaction was carried out at room temperature. Then, Cy3-modified DNA aptamers were added, and the reaction was carried out at room temperature. After post-processing, a dual-signal inverse ratio upconversion nanoprobe for lead ion detection was obtained.

6. A detection device, characterized in that, It includes the dual-signal inverse ratio upconversion nanoprobe for lead ion detection as described in any one of claims 1-4.

7. A dual-signal inverse ratio-modulated upconversion nanoprobe for lead ion detection as described in any one of claims 1-4, for detecting Pb in agricultural products, soil, tap water, and human serum. 2+ Applications.

8. The application according to claim 7, characterized in that, The application described involves fluorescence detection with an excitation wavelength of 980 nm and an emission wavelength in the range of 500-700 nm, using I... 540 / I 565 It is a quantitative signal.

9. The application according to claim 8, characterized in that, The dual-signal inverse ratio-modulation upconversion nanoprobe for lead ion detection targets Pb 2+ The detection limit is as low as 43 pM, and within the concentration range of 0-60 nM, Pb... 2+ Concentration and I 540 / I 565 The logarithm of is linearly related.

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