Method for fluorescence detection of perfluorooctanoic acid based on aptamer modified nanogold
By modifying the nano-gold fluorescent probe with aptamer and combining it with FAM fluorescent dye, the problem of lacking a rapid, convenient, and low-cost PFOA detection method in the existing technology is solved, and PFOA detection with high sensitivity and specificity is achieved.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- JIANGNAN UNIV
- Filing Date
- 2025-09-28
- Publication Date
- 2026-04-28
AI Technical Summary
Existing technologies lack rapid, convenient, and low-cost methods for detecting perfluorooctanoic acid (PFOA), making them particularly unsuitable for large-scale sample testing and on-site detection.
An aptamer-modified gold nanoparticle fluorescent probe is combined with FAM fluorescent dye. The aptamer binds to the gold nanoparticle and forms a probe with complementary strand cDNA, enabling rapid detection of PFOA using fluorescence properties.
It achieves highly sensitive PFOA detection with a detection limit of 1.3 nmol L⁻¹, exhibits good linearity and specificity, and is suitable for large-scale sample and rapid on-site detection.
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Figure CN121933719A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to a method for detecting perfluorooctanoic acid (PFOA) based on aptamer-modified gold nanoparticles using fluorescence, belonging to the fields of nanomaterials and molecular biology. Background Technology
[0002] Perfluorooctanoic acid (PFOA), also known as pentafluorooctanoic acid, is a perfluoroalkyl compound that is a white crystalline solid at room temperature. Its molecular formula is C8HF. 15 O2 has a molecular structure of an alkyl carboxylic acid containing eight carbon atoms, with all hydrogen atoms on the carbon atoms replaced by fluorine atoms. PFOA is a synthetic organic compound that is widely used in surfactants, food packaging materials, non-stick kitchenware, and fire-fighting foams due to its good stability, hydrophobic and oleophobic properties, and extremely low surface tension.
[0003] The main route of human exposure to PFOA is through drinking water and food intake, where it accumulates in human organs and blood. Long-term exposure to PFOA can lead to reproductive system abnormalities, affecting the development and health of offspring, and can also cause diabetes, uric acid, and high blood lipids. Animal experiments have found that PFOA can cause developmental cardiotoxicity in chicken embryos and chicks, and cause abnormal heart rate and affect the movement of zebrafish embryos, suggesting that PFOA may have cardiotoxic effects. GB5749-2022 specifies the limit for PFOA in drinking water as 80 ng / L; GB5009.253-2016 specifies the detection limit for PFOA in animal-derived foods as 2 ng / kg.
[0004] Currently, the main analytical methods for detecting PFOA are chromatographic techniques, including liquid chromatography-mass spectrometry (LC-MS), liquid chromatography-tandem mass spectrometry (LC-MS), and gas chromatography-mass spectrometry (GC-MS). While chromatographic methods offer high sensitivity, they suffer from drawbacks such as the need for expensive equipment, cumbersome sample pretreatment, and specialized personnel, making them unsuitable for large-scale sample collection and rapid on-site detection. Therefore, the development of rapid, convenient, and low-cost PFOA detection technologies is urgently needed. Summary of the Invention
[0005] To address the shortcomings of the existing technologies, this invention provides a highly sensitive method for rapid PFOA detection based on aptamer-modified gold nanoparticles forming a probe to capture perfluorooctanoic acid (PFOA) and combining the optical properties of FAM fluorescent dyes. The aim is to solve the technical problem of the current lack of rapid, convenient, and low-cost PFOA detection methods.
[0006] The first technical solution provided by the present invention is a method for preparing a perfluorooctanoic acid (PFOA) detection probe. The method involves linking the PFOA aptamer Apt to gold nanoparticles, modifying the 3' end of the cDNA of the aptamer's complementary strand with a FAM fluorescent group, and forming a probe by the complementary pairing of the aptamer with its complementary strand bases.
[0007] In some implementations, the following steps are included:
[0008] (1) The perfluorooctanoic acid aptamer was mixed with gold nanoparticles and incubated to obtain a mixture;
[0009] (2) Centrifuge and wash the mixture from step (1) to obtain nanoparticles;
[0010] (3) Add cDNA to the nanoparticles from step (2) to obtain a perfluorooctanoic acid detection probe.
[0011] In some embodiments, the molar mass ratio of the perfluorooctanoic acid aptamer to the gold nanoparticles is 200:1, and the reaction is carried out at -80°C for 18-20 min.
[0012] In some embodiments, the specific steps of centrifugation and washing are as follows: centrifuge at 4°C and 12,000 rpm for 30 min, aspirate the supernatant, add an equal volume of 10 mM phosphate (pH 7.4) solution and 0.1 M NaCl solution, shake and resuspend, and repeat the centrifugation and washing three times to remove Apt that is not bound to the gold nanoparticles.
[0013] In some embodiments, the obtained nanoparticles are dispersed in a PBS solution (0.3M NaCl, 10mM phosphate, pH 7.4).
[0014] In some embodiments, in step (3), the volume ratio of nanoparticles to cDNA is 3:10, and then sufficient Tris-HCl buffer is added to obtain the perfluorooctanoic acid detection probe.
[0015] In some implementations, the Apt sequence is 5'-aaaaaaaaaaaaaaaTCTCGGGACGACGGCGTGGGGTGGTAGGCTGTAAAGGGGGTCGTCG TCCC-3'.
[0016] In some embodiments, the cDNA sequence is 5'-GTCGTCCCGAGAG-3'.
[0017] In some embodiments, the preparation method of the gold nanoparticles includes the following steps:
[0018] A 1 / 10000 (w / w) chloroauric acid solution was heated to boiling while stirring. After 10 minutes, a 1% (w / w) sodium citrate solution was added. The color of the solution gradually changed from pale yellow to wine red. The solution was heated and stirred for another 10 minutes, and then allowed to cool to room temperature. The volume ratio of the chloroauric acid solution to the sodium citrate solution was 50:1.
[0019] Specifically, the preparation method of the gold nanoparticles includes the following steps:
[0020] The containers used were soaked in aqua regia overnight, then rinsed with ultrapure water and dried.
[0021] Add 100 mL of chloroauric acid solution (1 / 10000, w / w) to a dried container, heat to boiling while stirring in a heating mantle, and add 2 mL of 1% (w / w) sodium citrate (prepared fresh) after 10 min. The color of the solution gradually changes from pale yellow to wine red. Continue heating and stirring for 10 min, and then let it stand and cool to room temperature.
[0022] The prepared gold nanoparticle solution was diluted to 100 mL with pure water, filtered through a 0.45 μm filter membrane, and stored at 4 °C.
[0023] The resistivity of the pure water is greater than or equal to 18.2 MΩ·cm.
[0024] The second technical solution provided by the present invention is a perfluorooctanoic acid detection probe prepared by the method described in the first technical solution.
[0025] The third technical solution provided by the present invention is a method for detecting perfluorooctanoic acid (PFOA), wherein the method utilizes the PFOA detection probe described in the second technical solution to detect the PFOA content in the analyte.
[0026] In some embodiments, the method involves the binding of the target perfluorooctanoic acid (PFOA) in the analyte to the aptamer, causing the two complementary DNA strands to separate and fluorescence to recover (on state); as the concentration of PFOA increases, the fluorescence intensity gradually increases; 488 nm is the excitation wavelength, and the fluorescence spectrum in the range of 510-650 nm is collected to achieve quantitative detection of PFOA in the analyte.
[0027] In some embodiments, the volume ratio of the perfluorooctanoic acid detection probe to the analyte is 1:1, and a certain amount of Tris-HCl buffer is added after mixing.
[0028] In some embodiments, the Tris-HCl buffer is formulated as 20 mM Tris-base and 137 mM NaCl.
[0029] Detection Principle: Apt and cDNA have completely complementary base pairs. Apt can recognize and capture the target substance perfluorooctanoic acid (PFOA). The complementary strand of cDNA and PFOA compete for binding to the aptamer Apt. When the analyte contains PFOA, Apt binds to PFOA, and the cDNA is in a free state. The fluorescence of the FAM group attached to it recovers. The fluorescence intensity of FAM varies depending on the concentration of PFOA in the analyte. When the analyte does not contain PFOA, Apt and cDNA hybridize complementaryly, forming a stable network structure. A fluorescence resonance energy transfer (FRET) effect occurs, and the fluorescence is quenched. The visual detection of PFOA is achieved based on the change in fluorescence intensity of the solution system.
[0030] The fourth technical solution provided by the present invention is a reagent kit containing the perfluorooctanoic acid detection probe described in the second technical solution.
[0031] The fifth technical solution provided by the present invention is the application of the method described in the first technical solution, the perfluorooctanoic acid detection probe described in the second technical solution, or the kit described in the fourth technical solution in the detection of perfluorooctanoic acid.
[0032] Compared with the prior art, the beneficial effects of the present invention are as follows:
[0033] (1) The use of aptamers to specifically capture the substances being detected effectively improves the stability and accuracy of the detection.
[0034] (2) Compared with antibodies, aptamers have the advantages of being able to be artificially synthesized, not requiring animal or cell culture, being easy to chemically modify, having a short synthesis cycle, low cost, good stability, small batch-to-batch differences, and being able to be stored for a long time.
[0035] (3) The aptamer-complementary chain-gold nanoprobe is easy to prepare and store, reducing detection time and enabling rapid detection of perfluorooctanoic acid.
[0036] In summary, this invention utilizes the fluorescence quenching properties of gold nanoparticles and the specificity and high sensitivity of the aptamer to achieve a detection limit of 1.3 nmol L for perfluorooctanoic acid. -1 5 nmol L -1 Up to 200 nmol L -1 It exhibits a good linear relationship within the range (R) 2 =0.9970). Attached Figure Description
[0037] Figure 1 This is a method for detecting perfluorooctanoic acid (PFOA) using aptamer-modified gold nanoparticles.
[0038] Figure 2 This is a transmission electron microscope image of gold nanoparticles.
[0039] Figure 3 The images show the emission spectrum of FAM and the absorption spectrum of gold nanoparticles.
[0040] Figure 4 The images show the UV absorption spectra and physical images of AuNPs, AuNPs after freezing, and AuNPs-Apt after freezing.
[0041] Figure 5 UV absorption spectra and images of AuNPs, AuNPs after adding 300mM NaCl, and AuNPs-Apt after freezing and adding 300mM NaCl.
[0042] Figure 6 This is a hydration size distribution diagram of gold nanoparticles before and after DNA modification.
[0043] Figure 7 The quenching effect of different concentrations of AuNP-Apt on FAM-cDNA.
[0044] Figure 8 The fluorescence response curves are for different concentrations of PFOA.
[0045] Figure 9 This is a standard curve for PFOA detection.
[0046] Figure 10 Specific analysis for quantitative detection of PFOA.
[0047] Figure 11 The results show the reaction of different amounts of aptamers with gold nanoparticles. Detailed Implementation
[0048] The preferred embodiments of the present invention are described below. It should be understood that the embodiments are for better explanation of the present invention and are not intended to limit the present invention.
[0049] Raw materials used in the examples:
[0050] Anhydrous sodium dihydrogen phosphate (NaH2PO4), anhydrous disodium hydrogen phosphate (Na2HPO4), sodium citrate dihydrate (Shanghai Sangon Biotech Co., Ltd.); sodium tetrachloroaurate dihydrate (NaAuCl4·2H2O), perfluoroheptanoic acid (PFHA), 9H-perfluorononanoic acid, perfluoroundecanoic acid (PFU) n DA), perfluorododecanoic acid (Shanghai Aladdin Reagent Co., Ltd.); hydrochloric acid (HCl), nitric acid (HNO3), tris(hydroxymethyl)aminomethane (Tris-Base) (Sinopharm Chemical Reagent Co., Ltd.); perfluorooctane sulfonic acid (PFOS) (Beijing Bailingwei Technology Co., Ltd.); perfluorooctanoic acid (PFOA) (Shanghai Maclean Biochemical Technology Co., Ltd.). All reagents are analytical grade and require no further purification. Ultrapure water (Hangzhou Wahaha Group Co., Ltd.).
[0051] Example 1: Preparation of perfluorooctanoic acid detection probe
[0052] 1. Synthesize polyA-modified aptamer Apt and complementary strand cDNA that is complementary to the aptamer (purchased from Shanghai Sangon Biotech Co., Ltd.).
[0053] The polyA-modified aptamer Apt:
[0054] 5'-aaaaaaaaaaaaaaaTCTCGGGACGACGGCGTGGGGTGGTAGGCTGTAAAGGGGGTCGTCGTCCC-3';
[0055] Complementary cDNA strand: 5'-GTCGTCCCGAGAG-3';
[0056] 2. Preparation of gold nanoparticles (Reference) Figure 1 )
[0057] (1) The container used was soaked overnight in aqua regia (a mixture of concentrated hydrochloric acid and concentrated nitric acid in a volume ratio of 3:1), then rinsed with ultrapure water and dried.
[0058] (2) Add 100 mL of chloroauric acid solution (1 / 10000, w / w) to the container, stir well with a rotor, boil for 10 min, and add 2 mL of 1% (w / w) sodium citrate. The solution gradually turns wine red, and continue heating for 10 min.
[0059] (3) Stop heating, continue stirring, cool at room temperature, dilute with pure water to 100 mL, filter through a membrane, and obtain a wine-red gold nanoparticle solution.
[0060] The prepared gold nanoparticle solution was characterized by transmission electron microscopy and UV-Vis absorption spectroscopy. The characterization spectra are shown in the figure. Figure 2 and Figure 3 . Figure 2 The successful synthesis of gold nanoparticles with a particle size of approximately 14 nm was characterized. Figure 3 It is known that the characteristic absorption peak of gold nanoparticles is at approximately 518 nm, which highly overlaps with the emission peak of FAM (522 nm), resulting in a high energy transfer efficiency between the two.
[0061] 3. Preparation of detection probes
[0062] (1) Take 20 μL of 10 μmol / L aptamer Apt into a 1.5 mL centrifuge tube, add 400 μL of gold nanoparticle solution, shake, and incubate at -80℃ for 18 min.
[0063] (2) The solution after the reaction was centrifuged at 15,000 rpm for 30 min at 4℃, and the supernatant was precipitated. Then, an equal amount of 10 mM phosphate (pH 7.4) solution and 0.1 M NaCl solution were added and the mixture was shaken and resuspended. This process was repeated 3 times to remove aptamers that were not bound to the gold nanoparticles. The resulting AuNP-Apt nanoparticles were dispersed in PBS solution (0.3 M NaCl, 10 mM phosphate, pH 7.4) and stored at 4℃ in the dark for later use.
[0064] (3) Take 350 μL of Tris-HCl buffer into a 2 mL centrifuge tube, add 30 μL of 1 μmol / L complementary strand cDNA, and then add 120 μL of AuNP-Apt solution. The resulting probe is stored at 4 °C in the dark.
[0065] Depend on Figure 4 It is known that when naked AuNPs are frozen, the local salt ion concentration near the AuNPs increases, leading to irreversible aggregation and a blue solution. For probe sets containing Apt and AuNPs with polyA sequences, as the temperature decreases, water molecule crystallization repels AuNPs and Apt from the growing ice crystals, resulting in a high local concentration of these substances. This enhances the adhesion of Apt to the AuNP surface, causing a slight rightward shift of its maximum UV absorption peak, and the solution remains in a red, dispersed state. Because the aptamer DNA on the AuNPs-Apt surface is negatively charged, it increases the mutual repulsion between AuNPs, allowing them to remain dispersed even in the presence of 300 mM NaCl. In this state, AuNPs aggregate, such as... Figure 5 As shown. By Figure 6 It can be seen that after gold nanoparticles are modified with Apt, there is a significant red shift and the intensity of the characteristic absorption peak of DNA at 260 nm is significantly increased, and the hydrated particle size is increased, proving that Apt has been successfully modified onto the surface of gold nanoparticles.
[0066] Take 470, 450, 430, 410, 390, 370, and 350 μL of Tris-HCl buffer into 2 mL centrifuge tubes, add 30 μL of 1 μmol / L complementary strand cDNA, and then add 0, 20, 40, 60, 80, 100, and 120 μL of AuNP-Apt solution. Figure 7 It was observed that the luminescence intensity of FAM-cDNA gradually decreased while the quenching efficiency gradually increased with increasing AuNP-Apt concentrations (1, 2, 3, 4, 5, 6 nmol / L). Therefore, the concentration of 6 nmol / L, which showed the best quenching effect, was selected for subsequent experiments.
[0067] Example 2: Detection of perfluorooctanoic acid
[0068] Take 100 μL of the probe prepared in Example 1 (AuNP-Apt concentration of 6 nmol / L) and 100 μL of perfluorooctanoic acid solution and place them in a 2 mL centrifuge tube and mix well to make the target concentrations 150 pM, 300 pM, 500 pM, 5 nM, 35 nM, 100 nM, 150 nM, and 200 nM (diluted with Tris-HCl buffer). Incubate at room temperature for 30 min and measure the fluorescence value of each sample using a fluorescence spectrophotometer. The excitation wavelength is 488 nm and the scanning range is 510-650 nm.
[0069] Depend on Figure 8 , 9 It can be seen that when the target compound PFOA is present, the luminescence intensity of FAM-cDNA gradually recovers, and the luminescence intensity is related to the PFOA concentration within 5 nmol / L. -1 Up to 200 nmol L -1 It exhibits good linearity within the range, with a detection limit (3 times standard deviation) of 1.3 nmol L⁻¹. -1 The linear equation is y = 3.936x + 3127, R. 2 =0.9970.
[0070] Other perfluorinated compounds, including perfluorooctane sulfonic acid (PFOS), perfluoroheptanoic acid (PFHA), 9H-perfluorononanoic acid, and perfluoroundecanoic acid (PFU), were determined using the probe prepared in Example 1. n DA), perfluorododecanoic acid. From Figure 10 It can be seen that only PFOA significantly enhanced the luminescence intensity of the probe, while other perfluorinated compounds did not cause significant changes in luminescence intensity. This indicates that the method established in this study has good specificity for PFOA detection.
[0071] Comparative Example 1
[0072] 20 μL of 100 μmol / L aptamer Apt was added to a 1.5 mL centrifuge tube, followed by 400 μL of gold nanoparticle solution. The mixture was shaken and incubated at -80°C for 18 min. Excessive aptamer concentration caused the gold nanoparticles to aggregate. Figure 11 .
[0073] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.
Claims
1. A method for preparing a perfluorooctanoic acid (PFOA) detection probe, characterized in that, The method involves linking the perfluorooctanoic acid aptamer Apt to gold nanoparticles, modifying the 3' end of the complementary strand cDNA of the aptamer with a FAM fluorescent group, and forming a probe after the aptamer binds to the complementary strand bases of the aptamer through complementary pairing. The Apt sequence is 5'-aaaaaaaaaaaaaaaTCTCGGGACGACGGCGTGGGGTGGTAGGCTGTAAAGGGGGTCGTCG TCCC-3', and the cDNA sequence is 5'-GTCGTCCCGAGAG-3'.
2. The method according to claim 1, characterized in that, Includes the following steps: (1) The perfluorooctanoic acid aptamer was mixed with gold nanoparticles and incubated to obtain a mixture; (2) Centrifuge and wash the mixture from step (1) to obtain nanoparticles; (3) Add cDNA to the nanoparticles from step (2) to obtain a perfluorooctanoic acid detection probe.
3. The method according to claim 2, characterized in that, In step (1), the molar mass ratio of perfluorooctanoic acid aptamer to gold nanoparticles is 100:1-300:1, and the reaction is carried out at -80℃ for 18-20 min.
4. The method according to claim 2, characterized in that, In step (3), the volume ratio of nanoparticles to cDNA is 1:10-5:10, thus obtaining the perfluorooctanoic acid detection probe.
5. The method according to claim 1 or 2, characterized in that, The preparation method of the aforementioned gold nanoparticles involves the following steps: A 1 / 10000 (w / w) chloroauric acid solution was heated to boiling while stirring. After 10 minutes, a 1% (w / w) sodium citrate solution was added. The color of the solution gradually changed from pale yellow to wine red. The solution was heated and stirred for another 10 minutes, and then allowed to cool to room temperature. The volume ratio of the chloroauric acid solution to the sodium citrate solution was 50:
1.
6. A perfluorooctanoic acid detection probe prepared using the method described in any one of claims 1 to 5.
7. A method for detecting perfluorooctanoic acid (PFOA), characterized in that, The method involves using the perfluorooctanoic acid (PFOA) detection probe described in claim 6 to detect the PFOA content in the analyte.
8. The method according to claim 7, characterized in that, The method involves the binding of the target perfluorooctanoic acid (PFOA) in the analyte to the PFOA aptamer, causing the cDNA to separate from the two complementary DNA strands of the PFOA aptamer, resulting in fluorescence recovery. The excitation wavelength is 488 nm, and the fluorescence spectrum in the range of 510-650 nm is collected. The concentration of PFOA is characterized by the fluorescence intensity, thus enabling the quantitative detection of PFOA in the analyte.
9. A reagent kit, characterized in that, The kit contains the perfluorooctanoic acid detection probe as described in claim 6.
10. The application of the method according to any one of claims 1 to 5, the perfluorooctanoic acid detection probe according to claim 6, or the kit according to claim 9 in the detection of perfluorooctanoic acid.