Method for detecting perfluorooctanoic acid based on aptamer-triggered nucleic acid amplification strategy and electrogenerated chemiluminescence

Abstract

The invention discloses a method for detecting perfluorooctanoic acid based on an aptamer-triggered nucleic acid amplification strategy and electrogenerated chemiluminescence, and relates to the technical field of perfluorooctanoic acid detection. The method comprises the following steps: synthesizing PFN NPs, using a step pulse method as an electrochemical excitation method, enabling the PFN NPs to induce a strong ECL signal only by using a + 0.80 V pulse voltage under the condition that an additional co-reaction reagent is not needed, and adopting a PFN NPs coupled rolling circle amplification to assist a PAM sequence-free CRISPR / Cas12a system to construct an ultra-sensitive ECL aptamer sensor, so that the detection limit is as low as 1.97 * 10 <-15 > M. The method provided by the invention is low in cost, higher in detection sensitivity and low in ECL trigger potential, and effectively solves the problems that the ECL trigger potential required by the existing detection method is too high, an effective signal amplification strategy is lacked, the detection sensitivity is limited, and the requirement of ultra-trace detection cannot be met.

Description

Technical Field

[0001] The present invention relates to the technical field of perfluorooctanoic acid detection, and in particular to a nucleic acid amplification strategy based on aptamer triggering and a method for detecting perfluorooctanoic acid by electrochemiluminescence. Background Art

[0002] Per- and poly-fluoroalkyl substances (PFASs), as man-made chemicals, have unique chemical stability, antifouling and water-repellent properties and are widely used in industrial production and daily life. However, PFASs are highly toxic and have significant impacts on environmental pollution and human health. As an important representative of PFASs, perfluorooctanoic acid (PFOA) is an environmental pollutant widely present in water systems around the world, which has adverse effects on human reproduction, development, nervous system, immune system, etc. Due to the stable chemical structure of PFOA, it is not easy to degrade and easily accumulates in the body. The bioaccumulation of PFOA means that even if the concentration of PFOA in the environmental medium is far below the limit, it will pose a significant environmental and health risk. Therefore, strengthening the high-sensitivity monitoring of PFAS is one of the main needs in the environmental field. As a standard method for detecting PFOA, high-performance liquid chromatography-mass spectrometry (HPLC-MS) can reach a detection limit of ng / L, but it cannot meet the detection of ultra-low concentrations of PFOA. In addition, this method has the disadvantages of expensive equipment and long time consumption. Although the fluorescence method and electrochemical method have the advantages of low cost, simple operation and fast speed, their detection limits for PFOA are generally higher than the ng / L level, which is difficult to meet the actual demand for ultra-sensitive detection of PFOA with extremely low abundance (such as pg / L). Therefore, it is particularly important to develop a simple, low-cost, ultra-sensitive new method for monitoring PFOA.

[0003] Aptamer-triggered nucleic acid amplification technology can convert non-nucleic acid targets into nucleic acid molecules, and then significantly amplify the detection signal with the help of various nucleic acid amplification technologies, creating extremely favorable conditions for ultra-sensitive analysis of non-nucleic acid targets. This amplification strategy combines the high specificity and affinity of aptamers with the high efficiency of nucleic acid signal amplification, and has high sensitivity, selectivity and stability. Therefore, it is very effective in the detection of pesticides such as organophosphates, acetamiprid, and heavy metal ions such as Hg 2+ PFOA has attracted wide attention in the detection of biomacromolecules such as proteins and even living cells. The current expert team has successfully screened out high-affinity aptamers for PFOA, opening up a new way to establish PFOA aptamer detection with higher sensitivity than existing detection methods.

[0004] As a technology that combines chemiluminescence with electrochemistry, electrochemiluminescence technology has the advantages of high sensitivity, simple equipment, low background signal, fast analysis speed, and wide dynamic response range. ECL sensing has been widely used in clinical diagnosis, drug analysis, food testing, environmental monitoring and other fields. This will certainly provide strong technical support for the high-sensitivity detection of PFOA, but there are few related studies at present. Although ECL sensors based on molecular imprinting (MIP) probes have been reported, S2O8 2- As a coreaction reagent, PFOA detection was achieved under the condition of a trigger potential of -1.30V, but the toxicity and stability of exogenous coreaction reagents and the electrochemical interference of undesirable oxidation / reduction reactions when the trigger potential exceeds ±1.00V cannot be ignored. In addition, the sensor also has the defect of insufficient sensitivity. Therefore, it is of great value to develop an ECL method for detecting PFOA with low trigger potential, high sensitivity and no exogenous coreaction reagents.

[0005] Various excellent ECL luminophores, including metal coordination compounds (such as ruthenium bipyridine), metal nanoclusters (such as gold nanoclusters), polymer dots (such as polyfluorene nanoparticles), etc., have been developed to achieve high-sensitivity ECL detection, but their trigger potentials are usually above ±1.00V. Electrochemical interference and electrode damage caused by high trigger potentials are inevitable. Selecting or designing suitable co-reactants, introducing functionalized electroactive agents or groups, and applying ECL resonance energy transfer (RET) are common strategies to reduce the ECL trigger potential. For example, a team of experts introduced stronger electron-donating and catalytic groups into the co-reactant triethylamine, successfully reducing the ECL trigger potential of gold nanoclusters to +0.75V. Photonic crystal nanofilms and deposited gold nanoparticles were also used to successfully reduce the ECL trigger potential of the ruthenium bipyridine / tripropylamine system to +0.95V. The instability and toxicity of exogenous coreactants, the complex process of introducing functionalized electroactive agents or groups, and the distance dependence of ECL-RET limit the application of these strategies. Currently, polyfluorene and its derivatives, as typical conjugated polymers with low toxicity and excellent luminescence properties, have been widely used in ECL analysis. In addition, we have discovered a series of polyfluorene-based luminophores, such as poly[9,9-bis(3'-(N,N-dimethylamino)propyl)-2,7-fluorene]-alt-2,7-(9,9-dioctylfluorene)] nanoparticles (PFN NPs), which exhibit ECL emission without exogenous coreactants at +1.20 V. Obviously, polyfluorene-based materials provide new hope for ECL luminophores without exogenous coreactants with low trigger potentials. Compared with the commonly used cyclic voltammetry (CV) as an electrochemical triggering method for ECL, the step pulse method (SP), as a transient electrochemical method, can generate more photons in the same time. More importantly, the reaction time of SP is much shorter than that of CV, which can reduce the decay of free radicals before the reaction and obtain stronger ECL emission. Therefore, by simply replacing CV with SP as the electrochemical triggering method, it is expected to reduce the ECL triggering potential of polyfluorene-based luminophores, but there is no relevant report yet. Summary of the invention

[0006] In view of the above-mentioned deficiencies in the prior art, the present invention provides a nucleic acid amplification strategy based on aptamer triggering and a method for detecting perfluorooctanoic acid by electrochemiluminescence. The method has low cost, higher detection sensitivity and low ECL triggering potential, and effectively solves the problems that the current detection method requires an ECL triggering potential that is too high, lacks an effective signal amplification strategy, and has limited detection sensitivity and cannot meet the requirements of ultra-trace detection.

[0007] To achieve the above-mentioned purpose, the technical solution adopted by the present invention to solve the technical problem is: to provide a nucleic acid amplification strategy based on aptamer triggering and a method for detecting perfluorooctanoic acid by electrochemiluminescence, comprising the following steps:

[0008] S1. Add poly[9,9-bis(3′-(N,N-dimethylamino)propyl)-2,7-fluorene]-alkyl-2,7-(9,9-dioctylfluorene)] to tetrahydrofuran and perform ultrasonication to obtain a mixed solution 1, then add poly(styrene-comaleic anhydride) to tetrahydrofuran and perform ultrasonication to obtain a mixed solution 2, mix the mixed solution 1 and the mixed solution 2 uniformly, add ultrapure water and perform ultrasonication, stir at high temperature until the tetrahydrofuran evaporates, and then filter to obtain a nanoparticle dispersion (PFN NPs dispersion);

[0009] S2, incubate the corresponding linker chain (L) and the corresponding aptamer chain (A) to form a duplex, then add the sample to be tested for reaction, then add the padlock probe and T4 DNA ligase to react to form a circular template, heat to inactivate the T4 DNA ligase, then add dNTPs, restriction endonuclease, DNA polymerase and polymerase buffer to perform rolling circle amplification (RCA), and finally heat to obtain a rolling circle amplification product;

[0010] S3, incubate the corresponding single strand 1 (S1) and the corresponding single strand 2 (S2) to form a duplex (S1 / S2), then react with the rolling circle amplification product obtained in step S2, and then add N-ethylmaleimide buffer, crRNA and Cas12a to activate the CRISPR / Cas12a system;

[0011] S4, using a coupling agent to activate the carboxyl group of the DNA hairpin structure H1, adding dopamine and stirring to obtain dopamine-labeled H1, then polishing the glassy carbon electrode with alumina powder and ultrasonically cleaning it, adding the nanoparticle dispersion obtained in step S1 and drying it to obtain a modified glassy carbon electrode, activating it with a coupling agent and incubating it with dopamine-labeled H1, then adding hexylene glycol, and finally placing the activated CRISPR / Cas12a system obtained in step S3 on the glassy carbon electrode for incubation, and then washing away the excess solution with phosphate buffer;

[0012] S5. The step pulse method is used for measurement, with the modified glassy carbon electrode obtained in step S4 as the working electrode, the Ag / AgCl electrode as the reference electrode, and the Pt electrode as the counter electrode, to measure the content of perfluorooctanoic acid.

[0013] Furthermore, in step S1, the concentration of poly[9,9-di(3′-(N,N-dimethylamino)propyl)-2,7-fluorene]-alkyl-2,7-(9,9-dioctylfluorene)] in the mixed solution 1 is 0.5-1.5 mg / mL.

[0014] Furthermore, in step S1, the concentration of poly[9,9-di(3′-(N,N-dimethylamino)propyl)-2,7-fluorene]-alkyl-2,7-(9,9-dioctylfluorene)] in the mixed solution 1 is 1 mg / mL.

[0015] Furthermore, in step S1, the concentration of poly(styrene-co-maleic anhydride) in the second mixed solution is 0.5-1.5 mg / mL.

[0016] Further, in step S1, the concentration of poly(styrene-co-maleic anhydride) in the second mixed solution is 1 mg / mL.

[0017] Furthermore, in step S1, the ultrasonic time of mixed solution 1 and mixed solution 2 is 4-6 hours.

[0018] Furthermore, in step S1, the ultrasonic time of mixed solution 1 and mixed solution 2 is 5 hours.

[0019] Further, in step S1, the mixed solution 1 and the mixed solution 2 are mixed for 1.5-2.5 hours.

[0020] Further, in step S1, the mixed solution 1 and the mixed solution 2 are mixed for 2 hours.

[0021] Furthermore, in step S1, the volume ratio of the mixed solution 1 to the mixed solution 2 is 4.5-5.5:0.5-1.5.

[0022] Furthermore, in step S1, the volume ratio of the mixed solution 1 to the mixed solution 2 is 5:1.

[0023] Furthermore, in step S1, the volume ratio of ultrapure water to mixed solution 1 is 9-11:4-6.

[0024] Furthermore, in step S1, the volume ratio of ultrapure water to mixed solution 1 is 10:5.

[0025] Furthermore, in step S1, ultrapure water is added and ultrasonicated for 4-6 minutes.

[0026] Furthermore, in step S1, ultrapure water is added and ultrasonicated for 5 minutes.

[0027] Furthermore, in step S1, stirring is performed at 75-85°C.

[0028] Furthermore, in step S1, stirring is performed at 80°C.

[0029] Furthermore, in step S1, filtration is performed using a 0.44 μm membrane filter.

[0030] Further, in step S2, the nucleotide sequence of the corresponding linker chain is shown as SEQ ID NO: 1, the nucleotide sequence of the corresponding aptamer chain is shown as SEQ ID NO: 2, and the nucleotide sequence of the padlock probe is shown as SEQ ID NO: 3.

[0031] Further, in step S2, the concentration of the corresponding linker chain is 1-3 μM.

[0032] Further, in step S2, the concentration of the corresponding linker chain is 2 μM.

[0033] Furthermore, in step S2, the concentration of the corresponding aptamer chain is 1-3 μM.

[0034] Furthermore, in step S2, the concentration of the corresponding aptamer chain is 2 μM.

[0035] Further, in step S2, the volume ratio of the corresponding linker chain to the corresponding aptamer chain is 9-11:9-11.

[0036] Further, in step S2, the mixture is incubated at 36-38°C for 0.5-1.5h.

[0037] Furthermore, in step S2, the mixture was incubated at 37°C for 1 hour.

[0038] Furthermore, in step S2, the volume ratio of the sample to be tested to the duplex is 9-11:9-11.

[0039] Furthermore, in step S2, the volume ratio of the sample to be tested to the duplex is 10:10.

[0040] Further, in step S2, after adding the sample to be tested, the mixture is reacted at 36-38° C. for 0.5-1.5 h.

[0041] Furthermore, in step S2, the sample to be tested is added and reacted at 37° C. for 1 hour.

[0042] The beneficial effect of adopting the above further scheme is that the corresponding aptamer chain in the duplex is made to specifically bind to perfluorooctanoic acid, thereby releasing the corresponding linking chain.

[0043] Further, in step S2, the concentration of the padlock probe is 1-3 μM.

[0044] Further, in step S2, the concentration of the padlock probe is 2 μM.

[0045] Furthermore, in step S2, the volume ratio of the padlock probe to the duplex is 4-6:9-11.

[0046] Furthermore, in step S2, the volume ratio of the padlock probe to the duplex is 5:10.

[0047] Furthermore, in step S2, the enzyme activity unit of T4 DNA ligase is 9-11U.

[0048] Furthermore, in step S2, the enzyme activity unit of T4 DNA ligase is 10U.

[0049] Furthermore, in step S2, the padlock probe and T4 DNA ligase are added and reacted at 36-38° C. for 0.5-1.5 h.

[0050] Furthermore, in step S2, the padlock probe and T4 DNA ligase were added and reacted at 37° C. for 1 h.

[0051] Further, in step S2, heating is performed at 60-70° C. for 10-20 min.

[0052] Furthermore, in step S2, heating is performed at 65°C for 15 minutes.

[0053] The beneficial effect of adopting the above further scheme is that T4 DNA ligase is inactivated.

[0054] Furthermore, in step S2, the restriction endonuclease is NB.BbvCI.

[0055] Furthermore, in step S2, the DNA polymerase is Phi29 DNA polymerase.

[0056] Further, in step S2, the polymerase buffer is Phi29 polymerase buffer.

[0057] Furthermore, in step S2, the concentration of dNTPs is 9-11 mM.

[0058] Furthermore, in step S2, the concentration of dNTPs is 10 mM.

[0059] Furthermore, the enzyme activity unit of the restriction endonuclease is 7-9U.

[0060] Furthermore, the enzyme activity unit of the restriction endonuclease is 8U.

[0061] Furthermore, the enzyme activity unit of DNA polymerase is 4-6U.

[0062] Furthermore, the enzyme activity unit of DNA polymerase is 5U.

[0063] Furthermore, the concentration of Phi29 polymerase buffer was 1×.

[0064] Furthermore, in step S2, the volume ratio of dNTPs to duplex is 1-1.5:10-11.

[0065] Furthermore, in step S2, the volume ratio of dNTPs to duplex is 1:10.

[0066] Furthermore, in step S2, a rolling circle amplification reaction is performed at 36-38°C for 0.5-1.5h.

[0067] Furthermore, in step S2, a rolling circle amplification reaction was performed at 37° C. for 1 h.

[0068] Further, in step S2, heating is performed at 60-70° C. for 10-20 min.

[0069] Further, in step S2, heating is performed at 65°C for 15 minutes.

[0070] The beneficial effect of adopting the above further scheme is: inactivating the enzyme.

[0071] Furthermore, in step S3, the nucleotide sequence corresponding to single strand 1 is shown as SEQ ID NO: 4, and the nucleotide sequence corresponding to single strand 2 is shown as SEQ ID NO: 5.

[0072] Furthermore, in step S3, the concentration of the corresponding single-stranded protein 1 is 1-3 μM.

[0073] Furthermore, in step S3, the concentration of the corresponding single-stranded protein 1 is 2 μM.

[0074] Furthermore, in step S3, the concentration of the corresponding single-stranded protein 2 is 1-3 μM.

[0075] Furthermore, in step S3, the concentration of the corresponding single-stranded protein 2 is 2 μM.

[0076] Further, in step S3, the volume ratio of the corresponding single strand 1 and the corresponding single strand 2 is 9-11:9-11.

[0077] Furthermore, in step S3, the volume ratio of the corresponding single strand 1 to the corresponding single strand 2 is 10:10.

[0078] Further, in step S3, the mixture is incubated at 36-38° C. for 0.5-1.5 h.

[0079] Further, in step S3, the mixture was incubated at 37° C. for 1 h.

[0080] Furthermore, in step S3, the volume ratio of the single strand 1 to the rolling circle amplification product obtained in step S2 is 9-11:9-11.

[0081] Further, in step S3, the volume ratio of the single strand 1 to the rolling circle amplification product obtained in step S2 is 10:10.

[0082] Further, in step S3, the reaction is carried out at 36-38° C. for 0.5-1.5 h.

[0083] Furthermore, in step S3, the reaction was carried out at 37° C. for 1 h.

[0084] The beneficial effect of adopting the above further scheme is: releasing the specific fragment corresponding to the single strand 2 through branch migration.

[0085] Furthermore, the concentration of the N-ethylmaleimide buffer was 1×.

[0086] Further, the concentration of crRNA was 45-55nM.

[0087] Furthermore, the concentration of crRNA was 50 nM.

[0088] Further, the concentration of Cas12a is 45-55nM.

[0089] Further, the concentration of Cas12a is 50nM.

[0090] Further, in step S3, the volume ratio of single-stranded 1, crRNA and Cas12a is 9-11:1-2:0.5-1.5.

[0091] Further, in step S3, the volume ratio of single-stranded 1, crRNA and Cas12a is 10:1.5:1.

[0092] The beneficial effect of adopting the above further scheme is: crRNA is bound to the corresponding single strand 2 through chain displacement mediated by a specific fragment, thereby activating the CRISPR / Cas12a system.

[0093] Furthermore, in step S4, the nucleotide sequence of the DNA hairpin structure H1 is shown in SEQ ID NO:6.

[0094] Further, in step S4, the coupling agent includes 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide solution and N-hydroxysuccinimide solution.

[0095] Furthermore, the concentration of the 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide solution is 0.01-0.03 g / mL.

[0096] Furthermore, the concentration of the 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide solution was 0.02 g / mL.

[0097] Furthermore, the concentration of the N-hydroxysuccinimide solution is 0.004-0.006 g / mL.

[0098] Furthermore, the concentration of the N-hydroxysuccinimide solution was 0.005 g / mL.

[0099] Furthermore, the volume ratio of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide to N-hydroxysuccinimide is 9-11:9-11.

[0100] Furthermore, the volume ratio of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide to N-hydroxysuccinimide is 10:10.

[0101] Further, in step S4, activation is performed at 3-5° C. for 1-3 h.

[0102] Furthermore, in step S4, activation is performed at 4° C. for 2 h.

[0103] Furthermore, in step S4, the concentration of the DNA hairpin structure H1 is 1-3 μM.

[0104] Furthermore, in step S4, the concentration of the DNA hairpin structure H1 is 2 μM.

[0105] Furthermore, in step S4, the concentration of dopamine is 9-11 mM.

[0106] Furthermore, in step S4, the concentration of dopamine is 10 mM.

[0107] Furthermore, in step S4, the volume ratio of the coupling agent, the DNA hairpin structure H1 and dopamine is 9-11:9-11:2-3.

[0108] Furthermore, in step S4, the volume ratio of the coupling agent, the DNA hairpin structure H1 and dopamine is 10:10:2.5.

[0109] Further, in step S4, stirring is performed at 3-5°C for 1-3h.

[0110] Further, in step S4, stirring is performed at 4° C. for 2 h.

[0111] Further, in step S4, ultrasonic cleaning is performed with water, anhydrous ethanol and water in sequence, each ultrasonic cleaning lasting 4-6 minutes.

[0112] Further, in step S4, ultrasonic cleaning is performed with water, anhydrous ethanol and water in sequence, each time for 5 minutes.

[0113] Further, in step S4, the amount of the nanoparticle dispersion obtained in step S1 added is 9-11 μL.

[0114] Further, in step S4, the amount of the nanoparticle dispersion obtained in step S1 added is 10 μL.

[0115] Further, in step S4, the nanoparticle dispersion is dropped onto the glassy carbon electrode.

[0116] Further, in step S4, natural drying is performed.

[0117] Further, in step S4, the coupling agent is used for activation for 15-25 minutes.

[0118] Further, in step S4, the coupling agent is used for activation for 20 minutes.

[0119] Further, in step S4, the mixture is incubated at 3-5°C overnight.

[0120] Further, in step S4, the mixture is incubated at 4°C overnight.

[0121] Further, in step S4, the volume ratio of the nanoparticle dispersion obtained in step S1, dopamine-labeled H1 and hexylene glycol is 9-11:9-11:9-11.

[0122] Furthermore, in step S4, the volume ratio of the nanoparticle dispersion obtained in step S1, dopamine-labeled H1 and hexylene glycol is 10:10:10.

[0123] Further, in step S4, the concentration of hexylene glycol is 0.5-1.5 mM.

[0124] Furthermore, in step S4, the concentration of hexylene glycol is 1 mM.

[0125] Furthermore, in step S4, hexylene glycol is added and the mixture is treated at 3-5° C. overnight.

[0126] Furthermore, in step S4, hexylene glycol is added and the mixture is treated at 4° C. overnight.

[0127] The beneficial effect of adopting the above further solution is: blocking non-specific binding sites on the electrode.

[0128] Further, in step S4, the activated CRISPR / Cas12a system obtained in step S3 is placed on a glassy carbon electrode and incubated at 36-38° C. for 1 h.

[0129] Further, in step S4, the activated CRISPR / Cas12a system obtained in step S3 is placed on a glassy carbon electrode and incubated at 37° C. for 0.5-1.5 h.

[0130] The beneficial effect of adopting the above further scheme is: cutting dopamine-labeled H1 to restore the signal.

[0131] Further, in step S5, the pulse voltage is +0.8-+0.9V, and the pulse time is 1-2s.

[0132] Further, in step S5, the pulse voltage is +0.8V and the pulse time is 1s.

[0133] Furthermore, in step S5, the photomultiplier tube is 500-700V, and the amplification level is 2-4.

[0134] Furthermore, in step S5, the photomultiplier tube is 600V and the amplification level is 3.

[0135] Further, in step S5, the PFN NPs on the modified glassy carbon electrode emit a strong electrochemiluminescence (ECL) signal at a low trigger potential of +0.80 V, and the relationship between the ECL intensity and the perfluorooctanoic acid concentration is: I=9665.38+495.20lg c, where I is the ECL intensity and c is the perfluorooctanoic acid concentration.

[0136] The present invention has the following beneficial effects:

[0137] 1. The present invention combines the advantages of aptamer-mediated nucleic acid amplification strategy with the advantages of efficient electrochemiluminescence method to construct a new highly sensitive aptamer sensor. Compared with the commonly used method for detecting perfluorooctanoic acid: high performance liquid chromatography-mass spectrometry, the biosensor constructed by the present invention not only avoids the problem of expensive equipment, but also has higher detection sensitivity, with a detection limit as low as 1.97×10 -15 M, provides a new idea for the detection of trace amounts of perfluorooctanoic acid.

[0138] 2. The present invention uses aptamer-mediated rolling circle amplification (RCA) to assist the PAM-free regularly interspaced short palindromic repeats (CRISPR) / Cas12a nucleic acid amplification system, develops an ultrasensitive ECL aptamer sensor for PFOA analysis, creatively develops SP to replace CV as an electrochemical triggering method, and adjusts the ECL triggering potential of PFNNPs to +0.80V in the absence of exogenous co-reacting reagents, achieving a strong electrochemiluminescence (ECL) signal induced by a low triggering potential. And the PFN NPs are prepared by a typical nanoprecipitation method, and the target conversion triggers the RCA reaction and activates the CRISPR / Cas12a system. PFOA binds to the aptamer, releasing single-stranded L, which binds to the padlock probe, triggering the RCA reaction and producing a large amount of single-stranded S0. S0 further binds to the DNA double-stranded S1 / S2, and releases a specific fragment of S2 through a branch migration reaction. Subsequently, the PAM-free CRISPR / Cas12a system is activated by a toehold-mediated strand displacement reaction. The present invention also uses PFN NPs to modify a glassy carbon electrode (GCE) and incubate it with a dopamine (DA)-labeled DNA hairpin structure H1 to obtain a quenched ECL signal. After the modified GCE is incubated with the PFOA-activated CRISPR / Cas12a system, the hairpin H1 is cut off and released from the electrode surface, restoring the ECL signal. The degree of signal recovery is related to the PFOA concentration, thereby achieving quantitative detection of PFOA. The limit of detection (LOD) is as low as 1.97×10-15 M, outperforms existing methods.

[0139] 3. The present invention not only develops a simple and convenient method to reduce the ECL triggering potential of the polyfluorene-based luminescent group, but also pioneers the application of aptamer-triggered nucleic acid amplification strategy in PFOA detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0140] Figure 1 TEM image of PFN NPs;

[0141] Figure 2 This is the statistical result diagram of the average particle size of PFN NPs;

[0142] Figure 3 is the FT-IR absorption curve;

[0143] Figure 4 It is a three-dimensional ECL spectrum diagram;

[0144] Figure 5 This is the ECL spectrum diagram in SP trigger mode;

[0145] Figure 6 This is the ECL spectrum diagram in CV trigger mode;

[0146] Figure 7 This is the ECL spectrum diagram in SP trigger mode;

[0147] Figure 8 It is a three-dimensional ECL spectrum diagram in CV trigger mode;

[0148] Fig. 9 This is the DPV test result diagram;

[0149] Fig.10 This is the result diagram of the effect of active oxygen and oxygen on ECL reaction;

[0150] Fig.11 The results of the effect of DA-labeled H1 on the ECL response in SP triggering mode are shown in the figure;

[0151] Fig.12 This is the result diagram of the effect of DA on CV behavior;

[0152] Fig.13 The results of the effects of DA and oxidized DA on the UV-visible absorption spectrum and the ECL spectrum of PFN NPs are shown in the figure;

[0153] Fig.14 The SP-triggered ECL results of PFN NPs / GCE detected at different initial potentials;

[0154] Fig.15This is the result diagram of the effect of initial potential duration on ECL emission;

[0155] Fig.16 This is the result diagram of the effect of pulse potential on ECL emission;

[0156] Fig.17 The graph shows the change of potential, current and ECL intensity over time on PFN NPs / GCE;

[0157] Fig.18 It is the CV result diagram under different marks;

[0158] Fig.19 The EIS results under different labels are shown;

[0159] Fig. 20 Image analysis of gel electrophoresis of different molecules;

[0160] Fig.21 The results of the effects of different conditions on the ECL response of the biosensor containing PFOA are shown in the figure;

[0161] Fig. 22 This is the result diagram of the effect of PFOA concentration on ECL signal intensity;

[0162] Fig.23 It is a calibration graph of ECL intensity and logarithmic value of PFOA concentration;

[0163] Fig.24 This is the ECL reaction result diagram under 10 consecutive potential scans;

[0164] Fig.25 This is a graph showing the biosensor's response to different substances. DETAILED DESCRIPTION

[0165] The principles and features of the present invention are described below, and the examples are only used to explain the present invention and are not used to limit the scope of the present invention. If no specific conditions are specified in the embodiments, they are carried out according to normal conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments is not specified, they are all conventional products that can be purchased commercially.

[0166] Example 1

[0167] The method for detecting perfluorooctanoic acid based on aptamer-triggered nucleic acid amplification strategy and electrochemiluminescence comprises the following steps:

[0168] S1. Add poly[9,9-bis(3′-(N,N-dimethylamino)propyl)-2,7-fluorene]-alkyl-2,7-(9,9-dioctylfluorene)] to tetrahydrofuran and ultrasonicate for 5 h to obtain a mixed solution 1, wherein the concentration of poly[9,9-bis(3′-(N,N-dimethylamino)propyl)-2,7-fluorene]-alkyl-2,7-(9,9-dioctylfluorene)] in the mixed solution 1 is 1 mg / mL, and then add poly(styrene) 5 mL of the mixed solution 1 and 1 mL of the mixed solution 2 were mixed for 2 h until they were uniformly mixed, and then 10 mL of ultrapure water was added and ultrasonicated for 5 min, and the mixture was stirred at 80° C. until the tetrahydrofuran evaporated, and then filtered with a 0.44 μm membrane filter to obtain a nanoparticle dispersion (PFN NPs dispersion);

[0169] S2, incubate 10 μL of the corresponding linker chain (L) with a concentration of 2 μM and 10 μL of the corresponding aptamer chain (A) with a concentration of 2 μM at 37°C for 1 h to form a duplex (S1 / S2), take Jialing River water as the test sample, add 10 μL of Jialing River water to the 10 μL duplex and react at 37°C for 1 h, so that A in the duplex specifically binds to perfluorooctanoic acid to release L, then add 5 μL of 2 μM padlock probe and 10 U T4 DNA ligase and react at 37°C for 1 h to form a circular template, heat at 65°C for 15 min to inactivate T4 DNA ligase, then add 1 μL of 10 mM dNTPs, 8 U restriction endonuclease, 5 U DNA polymerase and 1× polymerase buffer, perform rolling circle amplification reaction at 37°C for 1 h, and finally heat at 65°C for 15 min to obtain rolling circle amplification products;

[0170] S3, incubate 10 μL of the corresponding single-stranded 1 (S1) with a concentration of 2 μM and 10 μL of the corresponding single-stranded 2 (S2) with a concentration of 2 μM at 37°C for 1 h to form a duplex (S1 / S2), and then react with 10 μL of the rolling circle amplification product obtained in step S2 at 37°C for 1 h, and then add 1×N-ethylmaleimide buffer, 1.5 μL of crRNA with a concentration of 50 nM and 1 μL of Cas12a with a concentration of 50 nM to activate the CRISPR / Cas12a system;

[0171] S4. Mix 10 μL of 0.02 g / mL 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide and 10 μL of 0.005 g / mL N-hydroxysuccinimide to obtain a coupling agent. Use the coupling agent to activate the carboxyl group of 10 μL of 2 μM DNA hairpin structure H1 at 4°C for 2 h. Add 2.5 μL of 10 mM dopamine and stir at 4°C for 2 h to obtain dopamine-labeled H1. Then polish the glassy carbon electrode with alumina powder and Ultrasonicate with water, anhydrous ethanol and water for 5 minutes each time, add 10 μL of the nanoparticle dispersion obtained in step S1 and dry naturally to obtain a modified glassy carbon electrode, activate with a coupling agent for 20 minutes, incubate with 10 μL of dopamine-labeled H1 at 4°C overnight, then add 10 μL of 1 mM hexanediol, and finally place the activated CRISPR / Cas12a system obtained in step S3 on the glassy carbon electrode and incubate at 37°C for 1 hour. After the end, wash off the excess solution with phosphate buffer;

[0172] S5. The step pulse method was used for measurement. The modified glassy carbon electrode obtained in step S4 was used as the working electrode, the Ag / AgCl electrode was used as the reference electrode, and the Pt electrode was used as the counter electrode. The pulse voltage was set to +0.8 V, the pulse time was set to 1 s, the photomultiplier tube was set to 600 V, and the amplification level was set to 3. The content of perfluorooctanoic acid was measured to be 1.13×10 -10 M.

[0173] The sequence information of the oligonucleotides used in the examples is shown in Table 1.

[0174] Table 1 Sequence information of oligonucleotides

[0175]

[0176]

[0177] Example 2

[0178] The method for detecting perfluorooctanoic acid based on aptamer-triggered nucleic acid amplification strategy and electrochemiluminescence comprises the following steps:

[0179] S1. Add poly[9,9-bis(3′-(N,N-dimethylamino)propyl)-2,7-fluorene]-alkyl-2,7-(9,9-dioctylfluorene)] to tetrahydrofuran and ultrasonicate for 4 hours to obtain a mixed solution 1, wherein the concentration of poly[9,9-bis(3′-(N,N-dimethylamino)propyl)-2,7-fluorene]-alkyl-2,7-(9,9-dioctylfluorene)] in the mixed solution 1 is 0.5 mg / mL, and then add poly(styrene- 4.5 mL of mixed solution 1 and 0.5 mL of mixed solution 2 were mixed for 1.5 h until they were uniformly mixed, and then 9 mL of ultrapure water was added and ultrasonicated for 4 min, and the mixture was stirred at 75 ° C until the tetrahydrofuran evaporated, and then filtered with a 0.44 μm membrane filter to obtain a nanoparticle dispersion (PFN NPs dispersion);

[0180] S2, incubate 9 μL of the corresponding link chain with a concentration of 1 μM and 9 μL of the corresponding aptamer chain with a concentration of 1 μM at 36°C for 0.5 h to form a duplex, take Jialing River water as the test sample, add the test sample to the 9 μL duplex and react at 36°C for 0.5 h, then add 4 μL of 1 μM padlock probe and 9U T4 DNA ligase and react at 36°C for 0.5 h to form a circular template, heat at 60°C for 10 min to inactivate T4 DNA ligase, then add 1 μL of 1 mM dNTPs, 7U restriction endonuclease, 4U DNA polymerase and 1× polymerase buffer, perform rolling circle amplification reaction at 36°C for 0.5 h, and finally heat at 60°C for 10 min to obtain rolling circle amplification products;

[0181] S3, incubate 9 μL of the corresponding single-stranded 1 with a concentration of 1 μM and 9 μL of the corresponding single-stranded 2 with a concentration of 1 μM at 36°C for 0.5 h to form a duplex, and then react with 9 μL of the rolling circle amplification product obtained in step S2 at 36°C for 0.5 h, and then add 1×N-ethylmaleimide buffer, 1 μL of crRNA with a concentration of 45 nM and 0.5 μL of Cas12a with a concentration of 45 nM to activate the CRISPR / Cas12a system;

[0182] S4, 9 μL of 0.01 g / mL 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide and 9 μL of 0.004 g / mL N-hydroxysuccinimide were mixed evenly to obtain a coupling agent, and 9 μL of 1 μM DNA hairpin structure H1 carboxyl group was activated with the coupling agent at 3°C ​​for 1 h, 2 μL of 9 mM dopamine was added and stirred at 3°C ​​for 1 h to obtain dopamine-labeled H1, and then the glassy carbon electrode was polished with alumina powder and Sonicate with water, anhydrous ethanol and water in turn, each time for 4 minutes, add 9 μL of the nanoparticle dispersion obtained in step S1 and dry naturally to obtain a modified glassy carbon electrode, activate with a coupling agent for 15 minutes, incubate with 9 μL of dopamine-labeled H1 at 3°C ​​overnight, then add 9 μL of 0.5 mM hexanediol, and finally place the activated CRISPR / Cas12a system obtained in step S3 on the glassy carbon electrode for incubation. After the end, wash off the excess solution with phosphate buffer;

[0183] S5. The step pulse method was used for measurement. The modified glassy carbon electrode obtained in step S4 was used as the working electrode, the Ag / AgCl electrode was used as the reference electrode, and the Pt electrode was used as the counter electrode. The pulse voltage was set to +0.8 V, the pulse time was set to 1 s, the photomultiplier tube was set to 500 V, and the amplification level was set to 2. The content of perfluorooctanoic acid was measured to be 1.09×10 -10 M.

[0184] Example 3

[0185] The method for detecting perfluorooctanoic acid based on aptamer-triggered nucleic acid amplification strategy and electrochemiluminescence comprises the following steps:

[0186] S1. Add poly[9,9-bis(3′-(N,N-dimethylamino)propyl)-2,7-fluorene]-alkyl-2,7-(9,9-dioctylfluorene)] to tetrahydrofuran and ultrasonicate for 6 hours to obtain a mixed solution 1, wherein the concentration of poly[9,9-bis(3′-(N,N-dimethylamino)propyl)-2,7-fluorene]-alkyl-2,7-(9,9-dioctylfluorene)] in the mixed solution 1 is 1.5 mg / mL, and then add poly(styrene-co- Maleic anhydride) was added to tetrahydrofuran and ultrasonicated for 6 hours to obtain a mixed solution 2, wherein the concentration of poly(styrene-co-maleic anhydride) in the mixed solution 2 was 1.5 mg / mL, 4.5 mL of the mixed solution 1 and 0.5 mL of the mixed solution 2 were mixed for 2.5 hours until they were uniformly mixed, and then 11 mL of ultrapure water was added and ultrasonicated for 6 minutes, and stirred at 85°C until the tetrahydrofuran evaporated, and then filtered with a 0.44 μm membrane filter to obtain a nanoparticle dispersion (PFN NPs dispersion);

[0187] S2, incubate 11 μL of the corresponding link chain with a concentration of 3 μM and 11 μL of the corresponding aptamer chain with a concentration of 3 μM at 38°C for 1.5 h to form a duplex, take Jialing River water as the test sample, add 11 μL of the test sample to the 11 μL duplex, react at 38°C for 1.5 h, then add 6 μL of 3 μM padlock probe and 11U T4 DNA ligase at 38°C for 1.5 h to form a circular template, heat at 70°C for 20 min to inactivate T4 DNA ligase, then add 1.5 μL of 11 mM dNTPs, 9U restriction endonuclease, 6U DNA polymerase and 1× polymerase buffer, perform rolling circle amplification reaction at 38°C for 1.5 h, and finally heat at 70°C for 20 min to obtain rolling circle amplification products;

[0188] S3, incubate 11 μL of the corresponding single-stranded 1 with a concentration of 3 μM and 11 μL of the corresponding single-stranded 2 with a concentration of 3 μM at 38 ° C for 1.5 h to form a duplex, and then react with 11 μL of the rolling circle amplification product obtained in step S2 at 38 ° C for 1.5 h, and then add 1×N-ethylmaleimide buffer, 2 μL of crRNA with a concentration of 55 nM and 1.5 μL of Cas12a with a concentration of 55 nM to activate the CRISPR / Cas12a system;

[0189] S4. Mix 11 μL of 0.03 g / mL 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide and 11 μL of 0.006 g / mL N-hydroxysuccinimide to obtain a coupling agent. Use the coupling agent to activate the carboxyl group of 11 μL of 3 μM DNA hairpin structure H1 at 5°C for 3 h. Add 3 μL of 11 mM dopamine and stir at 5°C for 3 h to obtain dopamine-labeled H1. Then polish the glassy carbon electrode with alumina powder and Sonicate with water, anhydrous ethanol and water in turn, each time for 6 minutes, add 11 μL of the nanoparticle dispersion obtained in step S1 and dry naturally to obtain a modified glassy carbon electrode, activate with a coupling agent for 25 minutes, incubate with 11 μL of dopamine-labeled H1 at 5°C overnight, then add 11 μL of 1.5 mM hexanediol, and finally place the activated CRISPR / Cas12a system obtained in step S3 on the glassy carbon electrode for incubation. After the end, wash off the excess solution with phosphate buffer;

[0190] S5. The step pulse method was used for measurement. The modified glassy carbon electrode obtained in step S4 was used as the working electrode, the Ag / AgCl electrode was used as the reference electrode, and the Pt electrode was used as the counter electrode. The pulse voltage was set to +0.9 V, the pulse time was set to 2 s, the photomultiplier tube was set to 700 V, and the amplification level was set to 4. The content of perfluorooctanoic acid was measured to be 1.1×10 -10 M.

[0191] Test Example 1

[0192] The nanoparticles (PFN NPs) prepared in Example 1 were observed, and the results were as follows: Figure 1 The average particle size of PFN NPs was statistically analyzed, and the results are shown in Figure 2 shown.

[0193] Depend on Figure 1 and Figure 2 It can be seen that the morphology of PFN NPs is a spherical structure with an average particle size of 125.0 nm, which basically conforms to the normal distribution.

[0194] Test Example 2

[0195] Fourier transform infrared spectroscopy (FI-IR) was used to characterize the PFN NPs, and the original poly [9,9-di(3′-(N,N-dimethylamino)propyl)-2,7-fluorene]-alkyl-2,7-(9,9-dioctylfluorene)] (PFN) was used as a control. Figure 3 As shown, curve a is the absorption peak of PFN, and curve b is the absorption peak of PFN NPs.

[0196] Depend on Figure 3 It can be seen that on the PFN alkyl chain, the antisymmetric stretching vibration of the CH bond produces 2918.19 cm -1 and 2852.61cm -1 On the alkane chain, the out-of-plane deformation vibration of the CH bond produces an absorption peak of 815.85 cm -1 In addition, the vibration of the aromatic ring skeleton caused the absorption peak at 1456.20 cm -1 For PFN functionalized with styrene-co-maleic anhydride (PSMA), i.e., PFN NPs, the vibration of -COOH produced an absorption peak at 1714.65 cm -1 FT-IR confirmed that PSMA successfully functionalized PFN NPs with carboxyl groups.

[0197] Test Example 3

[0198] Under the conditions of pH 7.4 and phosphate buffer solution (PBS) concentration of 0.1 M, the ECL spectra of PFN NPs were collected by PFN NPs / GCE method in step pulse (SP) trigger mode with a pulse potential of +0.80 V. The results are shown in Figure 4 shown.

[0199] The comparison of photoluminescence (PL) spectrum and two-dimensional (ECL) spectrum under the conditions of pH 7.4 and 0.1M PBS is shown in Figure 2. Figure 5 As shown in Figure 2, when the wavelength is 600, the curve below is the PL (λex =390nm) spectrum; the curve on the top is the ECL spectrum of PFN NPs / GCE in SP trigger mode.

[0200] Depend on Figure 4 and Figure 5 It can be seen that the maximum emission wavelength of ECL is 530nm, and the luminescence spectrum shows that the maximum emission wavelength is 436nm. In comparison, the ECL spectrum is red-shifted by 94nm.

[0201] Test Example 4

[0202] Under the conditions of pH 7.4 and 0.1 M PBS, the ECL signals at PFN NPs / GCE were detected by cyclic voltammetry (CV) and step pulse method (SP) as electrochemical excitation methods, respectively. The results of CV are shown in Figure 2. Figure 6 As shown, the results of SP are as follows Figure 7 shown.

[0203] Depend on Figure 6 and Figure 7 It can be seen that at +1.25 V, the ECL intensity detected by the CV trigger mode is about 9000 a.u., and at +0.80 V, the ECL intensity detected by the SP trigger mode is 14000 a.u., which indicates that the SP trigger mode produces stronger ECL emission than the CV trigger mode at a lower trigger potential.

[0204] Meanwhile, under the conditions of pH 7.4 and phosphate buffer solution concentration of 0.1 M, the ECL spectra of PFN NPs were collected in CV trigger mode using PFN NPs / GCE under 0-+1.25 V potential scanning, and the results are shown in Figure S8.

[0205] Depend on Figure 8 It can be seen that the maximum emission wavelength of ECL is 530 nm, which is consistent with the SP triggering mode in Experimental Example 3. The results show that neither CV nor SP as the electrochemical triggering method affects the optical properties of PFN nanoparticles.

[0206] Test Example 5

[0207] To investigate the ECL emission mechanism at low trigger potentials, differential pulse voltammetry (DPV) behaviors of bare GCE and PFN NPs / GCE were studied at a scanning potential of 0-+1.00 V in 0.1 M PBS at pH 7.4. Fig. 9 As shown, when the potential is 0.6 V, the curve at the bottom is the differential pulse voltammetry (DPV) detection result on GCE, and the curve at the top is the DPV detection result on PFN NPs / GCE.

[0208] Depend on Fig. 9 It can be seen that no oxidation peak was detected for bare GCE. For GCE / PFN NPs, two obvious oxidation peaks can be observed, appearing at +0.69V and +0.93V, respectively. The oxidation peak of +0.69V comes from the oxidation of the trimethylamine (TA) group of PFN NPs, and the peak of +0.93V comes from the oxidation of the conjugated skeleton of PFN NPs. According to the DPV behavior, it can be inferred that the emission of ECL is attributed to the oxidation of the TA group and the conjugated skeleton of PFN NPs.

[0209] Test Example 6

[0210] The effects of reactive oxygen species (ROS) and dissolved oxygen in PBS on the ECL reaction were investigated. Under the conditions of pH 7.4 and 0.1 M PBS, PBS was saturated with N2 to remove O2. In order to remove OH - and O 2- , free radical scavengers L-cysteine ​​(L-Cys) and superoxide dismutase (SOD) were added to the solution, and then the SP-triggered ECL emission of PFN NPs / GCE was tested under air saturation, N2 saturation, PBS with L-cys and SOD added, respectively. Fig.10 shown.

[0211] Depend on Fig.10 It can be seen that PFN NPs / GCE showed almost the same ECL signals in air-saturated PBS and N2-saturated PBS, indicating that O2 had nothing to do with the ECL luminescence of PFN NPs. This suggests that ROS and O2 were not involved in the ECL reaction.

[0212] Trimethylamine groups (TA) are part of PFN nanoparticles (PFN NPs), which can be expressed as PFN NPs-TA. In ECL emission, the TA groups in PFN NPs are oxidized to cationic radicals PFN NPs-TA + , and then deprotonated to the intermediate PFN NPs-TA - , and then further oxidized to free radical PFN NPs-+-TA - Subsequently, it was converted into PFN NPs*-TA via intramolecular electron transfer. + Finally, PFN NPs-TA and PFN NPs*-TA + Reaction to form excited state Thus, ECL emission is generated. The specific reaction equation is shown below.

[0213] PFN NPs-TA-e - →PFN NPs-TA +

[0214] PFN NPs-TA + -H + →PFN NPs-TA ·

[0215] PFN NPs-TA · -e - →PFN NPS ·+ -TA ·

[0216] PFN NPs ·+ -TA · →PFN NPs * -TA +

[0217]

[0218] Compared with CV-triggered ECL, SP-triggered ECL exhibits stronger ECL emission at a lower trigger potential, which can be reasonably explained as follows. Compared with CV, SP, as an instantaneous electrochemical method, can release more photons in the same time. In addition, the free radical reaction time of SP is shorter than that of CV, which can effectively reduce the decay of free radicals.

[0219] Test Example 7

[0220] The quenching mechanism of ECL was investigated. Under the conditions of pH 7.4 and 0.1 M PBS, PFN NPs labeled with dopamine (DA) and H1 were used as a control. In the SP trigger mode, the luminescence intensity of PFN NPs under a pulse voltage of +0.80 V changed with time. Fig.11 As shown, at a time of 1 s, the lower curve is the result of PFNNPs with dopamine-labeled H1 added, and the upper curve is the result of PFN NPs.

[0221] Depend on Fig.11 It can be seen that PFN NPs obtained strong ECL emission under a pulse voltage of +0.80 V. When DA-labeled H1 was added, a significant decrease in the ECL intensity was observed, indicating that DA can significantly quench the ECL emission of PFN NPs. This is because DA is easily oxidized to phthalates, which then act as an acceptor to quench the ECL signal through resonance energy transfer (RET).

[0222] Test Example 8

[0223] The electrochemical oxidation of DA was studied by measuring the CV behavior. The CV behavior of bare GCE without DA and bare GCE with DA was tested under a potential scan of +0.80 V at pH 7.4 and 0.1 M PBS. The results are shown in Fig.12 As shown, when the potential is 0 V, the curve at the bottom is the CV behavior of the bare GCE containing DA, and the curve at the top is the CV behavior of the bare GCE without DA.

[0224] Depend on Fig.12 As shown, when the potential scanning range is 0-+0.80V, an obvious oxidation peak can be obtained at +0.44V, and the starting potential is +0.22V.

[0225] To investigate whether DA can undergo RET with PFN NPs, the UV-visible absorption spectra and ECL emission spectra of DA, oxidized DA, and PFN NPs were shown in Figure 2 . Fig.13 As shown, the absorption peaks at a wavelength of 600 nm are the results of DA, oxidized DA and PFNNPs from bottom to top.

[0226] Depend on Fig.13 As shown, compared with DA, oxidized DA has obvious light absorption at wavelengths of 400 nm to 800 nm. It can be seen that the ECL emission spectra of oxidized DA and PFN NPs have obvious overlap, indicating that PFN NPs and oxidized DA can effectively quench the ECL signal through RET.

[0227] Test Example 9

[0228] The optimal electrochemical parameters of SP-triggered ECL of PFN NPs / GCE were investigated, including initial potential, initial potential duration, and pulse potential duration.

[0229] Under the conditions of pH 7.4, 0.1 M PBS, and when the initial potential duration and pulse potential duration were both set to 1 s, the SP-triggered ECL of PFN NPs / GCE was detected with different initial potentials ranging from +0.40 V to -0.10 V. The results are shown in Fig.14 shown.

[0230] Depend on Fig.14 It can be seen that the ECL emission is almost undetectable when the initial potential exceeds +0.30 V. As the initial potential decreases from +0.30 V to 0 V, the ECL intensity increases rapidly and reaches a peak when the initial potential is 0 V.

[0231] Under the conditions of pulse potential duration of 1s and initial potential of 0V, the effect of initial potential duration on ECL emission was investigated. The results are as follows Fig.15 shown.

[0232] Depend on Fig.15 It can be seen that as the initial potential duration increases from 1s to 5s, the ECL emission intensity shows a slight downward trend.

[0233] The ECL emission intensity was detected when the pulse potential duration was from 1s to 5s, and the effect of the pulse potential on the ECL emission was explored when the initial potential duration was 1s and the initial potential was 0V. The results are as follows Fig.16 shown.

[0234] Depend on Fig.16 It can be seen that the ECL intensity decreases slightly with the increase of the pulse potential duration. Therefore, a pulse potential duration of 1 s was used as the optimal parameter.

[0235] When the electrochemical parameters are optimal, the potential, current and ECL intensity of PFN NPs / GCE change with time as shown in Figure 2. Fig.17 As shown, from top to bottom are the changes in potential, current and ECL intensity of PFN NPs / GCE over time.

[0236] Depend on Fig.17 It can be seen that at each initial potential duration (0-1s, 2-3s, 4-5s, etc.), no current and ECL emission were observed. Strong current and ECL emission were detected for each instantaneous pulse from the initial potential 0V to the pulse potential +0.80V (1s, 3s, 5s, etc.). In addition, when pulsing from the pulse potential to the next initial potential (2s, 4s, 6s, etc.), a strong negative current was observed, but no ECL emission.

[0237] Test Example 10

[0238] In 5 mM [Fe(CN)6] 3- / 4- In solution, the scan rate during CV measurement was 0.1 V / s and the operating voltage was -0.20-+0.6 V. The biosensor assembly was verified by CV and electrochemical impedance spectroscopy (EIS). Fig.18 As shown. Fig.18 In the figure, when the potential is 0.3 V, the curves in the upper part are, from bottom to top, the CV graphs of HT (ethylene glycol) / DA-labeled H1 / PFN NPs / GCE, activated Cas12a / HT / DA-labeled H1 / PFN NPs / GCE, DA-labeled H1 / PFN NPs / GCE, PFN NPs / GCE and bare GCE.

[0239] Depend on Fig.18It can be seen that the peak current of PFN NPs / GCE decreased significantly compared with bare GCE, because the poor conductivity of PFN NPs hindered electron transfer. Afterwards, the redox current peak gradually decreased with the incubation of DA-labeled H1 and HT, because their conductivity was weak. When activated Cas12a was incubated on GCE, the redox current increased due to the shearing effect of Cas12a on the hairpin.

[0240] The EIS graph under the same conditions as the CV measurement is as follows: Fig.19 As shown, in 5 mM [Fe(CN)6] 3- / 4- In solution, the frequency of EIS measurement is 10 -1 -10 5 Hz, the operating voltage is 0.22V, and the amplitude is 5mV. Fig.19 In the figure, when the resistance is 300 ohm, from bottom to top are the EIS graphs of DA-labeled H1 / PFN NPs / GCE, PFN NPs / GCE, activated Cas12a / HT / DA-labeled H1 / PFN NPs / GCE, bare GCE, and HT / DA-labeled H1 / PFN NPs / GC. The semicircle diameter in the EIS graph represents the resistance electron transfer (R et )

[0241] Depend on Fig.19 It can be seen that the R of bare GCE et As PFN NPs, DA-labeled H1, and HT were gradually incubated on GCE, R et Gradually increases, due to the shearing effect of Cas12a on the hairpin, R et decline.

[0242] The above conclusions show that the biosensor has been assembled successfully.

[0243] Test Example 11

[0244] To verify the feasibility of nucleic acid signal amplification, polyacrylamide gel electrophoresis (PAGE) analysis was performed using 16% PAGE. Fig. 20 As shown, in A, from left to right, lane M and lanes 1-7 correspond to DNA Marker, aptamer, L, aptamer+L, A-L+PFOA, padlock probe, padlock probe+L+T4 DNA ligase, and circular template+dNTPs+NB.BbvCI endonuclease+Phi29 polymerase, respectively. In B, lane M and lanes 1-5 correspond to DNA Marker, S1, S2, S1+S2, H, and S1+S2+H+RCA product+CRISPR / Cas12a system, respectively.

[0245] Depend on Fig. 20 As can be seen from lane A, when L was mixed with the aptamer, a new band with a slow migration speed was observed, indicating that the aptamer hybridized with L to form a double-stranded AL. After adding PFOA, the same band as L appeared again, indicating that PFOA hybridized with the aptamer in the duplex AL, releasing L from the duplex AL. Compared with lane 5, a new band with a slower migration speed appeared in lane 6, indicating that the circular template has been assembled. In lane 7, phi29 polymerase, dNTPs and Nb.BbvCI endonuclease were added to the circular template, and an obvious RCA reaction occurred, and a new band corresponding to the single-stranded S0 was observed.

[0246] Depend on Fig. 20 As shown in B, when S1 was mixed with S2, a slow-migrating band was observed compared with lanes 1 and 2, indicating that the S1 / S2 double strand was successfully formed, and the band of H1 in lane 3 disappeared, indicating that the CRISPR / Cas12a system had been activated to cut the DNA hairpin structure H1.

[0247] Test Example 12

[0248] To ensure excellent analytical performance, the RCA reaction time, CRISPR / Cas12a system shearing time, and the amount of Phi29 polymerase and Nb.BbvCI endonuclease were optimized using a biosensor containing 1 nM PFOA. Fig.21 As shown, AD represents the effects of RCA reaction time, CRISPR / Cas12a system shearing time, Nb.BbvCI endonuclease dosage and Phi29 polymerase dosage on the ECL reaction of the biosensor containing 1 nM PFOA in PBS (0.10 M pH 7.4), respectively.

[0249] Depend on Fig.21 It can be seen that when the RCA reaction time increases from 30min to 150min, the ECL intensity increases rapidly and stabilizes after 60min; as the CRISPR / cas12a system shearing time is extended from 30min to 150min, the ECL intensity increases significantly at the beginning and reaches a plateau after 60min. Therefore, in subsequent experiments, the RCA reaction time and the CRISPR / cas12a system shearing time are set to 60min as the optimal time; when the dosage of Nb.BbvCI endonuclease increases from 2U to 10U, the ECL intensity increases rapidly and reaches a peak at 8U, so 8U is the optimal dosage of Nb.BbvCI endonuclease; as the dosage of Phi29 polymerase increases from 3U to 5U, the ECL intensity increases gradually, and there is no obvious change after 5U.

[0250] Test Example 13

[0251] When the RCA reaction time and the CRISPR / Cas12a system shearing time were both 60 min, and the dosages of Phi29 polymerase and Nb.BbvCI endonuclease were 5 U and 8 U, respectively, the constructed ECL photosensor was used to detect PFOA in 0.10 M PBS (pH 7.4). The ECL reaction results from 10 fM to 1 μM were as follows: Fig. 22 As shown in the figure, the calibration graph of ECL intensity and PFOA concentration logarithm is as follows Fig.23 The ECL reaction results under 10 consecutive potential scans are shown in Fig.24 shown.

[0252] Depend on Fig. 22 It can be seen that when the concentration of PFOA is between 10.0fM and 1.0μM, the ECL signal increases with the increase of PFOA concentration, and the ECL intensity is significantly linearly correlated with the logarithm of PFOA concentration.

[0253] Depend on Fig.23 It can be seen that Fig.23 The corresponding calibration curve is described and can be expressed as I = 9665.38 + 495.20 lg c. The detection limit (LOD) is calculated from the existing data. The specific method is as follows: the ECL intensity of the blank sample is measured three times in parallel, and the average value of the ECL intensity (I B ) and standard deviation (S B ); then according to formula I L =I B +k×S B Calculate the limit detection signal (I L ), the numerical factor k is selected as 3, indicating an expected confidence level of 99.86%; adding I to the linear regression equation L The LOD can be obtained. According to the ECL measurement results of the blank sample, calculate I B and S B They are 2213.33 and 56.54 respectively, and the LOD is further calculated to be 1.97×10 -15 M (S / N=3). Compared with other detection methods, the biosensor prepared by the present invention exhibits extremely low LOD and a wider linear range, which is attributed to the efficient nucleic acid amplification strategy and the excellent ECL performance of PFN NPs.

[0254] Depend on Fig.24 It can be seen that the ECL signals of the sensitive sensor containing 10fM and 1nM PFOA were collected under continuous scanning for 10 circles to demonstrate the stability of the sensitive sensor. The relative standard deviation (RSD) of the ECL signal was 2.9% and 2.4%, respectively, showing the excellent stability of the sensitive sensor.

[0255] Test Example 14

[0256] To explore the specificity of the sensor prepared by the present invention, various environmental pollutants were used as interfering substances, including perfluoropentanoic acid (PFPeA), perfluoroheptanoic acid (PFHpA), 2,2,3,3,4,4,4-heptafluoro-1-butanol (C4H3F7O) and perfluorobutane-1-sulfonic acid (PFBS), as well as a mixture containing all of the above substances, and a blank group was used as a control. The concentration of perfluorooctanoic acid (PFOA) was 1×10 -6 M, the concentration of other interfering substances is 100 times that of PFOA. The ECL results are shown in the figure.

[0257] As can be seen from the figure, when only interfering substances are present, the corresponding ECL signal is basically the same as the blank sample. However, in the presence of PFOA, the ECL signal increases significantly in both the PFOA mixed group and the PFOA group, showing the excellent specificity of the biosensor.

[0258] Test Example 15

[0259] The Jialing River water (Chongqing, China) was used as an actual sample, and a recovery experiment was conducted by the standard addition method to explore the practical application of the biosensor. Three parallel determinations were performed using the ECL biosensor and ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS / MS). The ECL biosensor was used to detect the Jialing River unadded sample as a control, and the PFOA concentration was 1.13×10 -10 M, the relative standard deviation (RSD) was 5.3%. Due to the insufficient sensitivity of UPLC-MS, PFOA was not detected in the unspiked samples. The detection results of the three spiked samples are shown in Table 2.

[0260] Table 2 Results of PFOA detection in samples using UPLC-MS and ECL biosensor

[0261]

[0262] As shown in Table 2, in the ECL test, the recovery rate of PFOA is between 99% and 105%. The RSD of the two methods is less than 5%. Taking the test results of UPLC-MS / MS as the standard, the relative error of the ECL test results was calculated, and the error was less than ±3%. This shows that the biosensor prepared by the present invention has good accuracy and applicability, and the biosensor has great application potential in environmental detection.

[0263] In summary, the present invention developed an aptamer-triggered nucleic acid amplification strategy for ultrasensitive and specific monitoring of perfluorooctanoic acid. SP was used instead of CV as the electrochemical excitation method to adjust the low ECL trigger potential of PFN NPs. At a voltage of +0.80 V, PFN NPs could emit a strong ECL signal without exogenous core reactants. The SP-triggered ECL mode created a new method for modulating ECL with low trigger potential. PFN NPs coupled with RCA-assisted CRISPR / cas12a system constructed an ECL sensitive sensor for detecting PFOA with a low LOD of 1.97×10 -15 M. This method establishes an ultrasensitive method for monitoring trace amounts of PFOS in real water samples, which can more effectively ensure water quality.

Claims

1. A method for detecting perfluorooctanoic acid based on an aptamer-triggered nucleic acid amplification strategy and electrochemiluminescence, characterized in that: The following steps are involved: S1. Add poly[9,9-bis(3′-(N,N-dimethylamino)propyl)-2,7-fluorene]-alkyl-2,7-(9,9-dioctylfluorene)] to tetrahydrofuran and perform ultrasonication to obtain a mixed solution 1. Then add poly(styrene-comaleic anhydride) to tetrahydrofuran and perform ultrasonication to obtain a mixed solution 2. After uniformly mixing the mixed solution 1 and the mixed solution 2, add ultrapure water and perform ultrasonication. Stir at high temperature until the tetrahydrofuran evaporates, and then filter to obtain a nanoparticle dispersion. S2, incubate the corresponding link chain and the corresponding aptamer chain to form a duplex, then add the sample to be tested for reaction, then add the padlock probe and T4 DNA ligase to react to form a circular template, heat to inactivate the T4 DNA ligase, then add dNTPs, restriction endonuclease, DNA polymerase and polymerase buffer to perform rolling circle amplification reaction, and obtain the rolling circle amplification product after heating; S3, incubate the corresponding single strand 1 and the corresponding single strand 2 to form a duplex, then react with the rolling circle amplification product obtained in step S2, and then add N-ethylmaleimide buffer, crRNA and Cas12a to activate the CRISPR / Cas12a system; S4, activate the carboxyl group of the DNA hairpin structure H1 with a coupling agent, add dopamine and stir to obtain dopamine-labeled H1, then polish the glassy carbon electrode with alumina powder and perform ultrasonic cleaning, add the nanoparticle dispersion obtained in step S1 and dry to obtain a modified glassy carbon electrode, activate it with a coupling agent, incubate it with dopamine-labeled H1, then add hexylene glycol, and finally drop the activated CRISPR / Cas12a system obtained in step S3 on the glassy carbon electrode for incubation, and wash off the excess solution with phosphate buffer after completion; S5. Use the step pulse method to measure, using the modified glassy carbon electrode obtained in step S4 as the working electrode, the Ag / AgCl electrode as the reference electrode, and the Pt electrode as the counter electrode to measure the content of perfluorooctanoic acid.

2. The aptamer-triggered nucleic acid amplification strategy and electrochemiluminescence detection method for perfluorooctanoic acid according to claim 1, characterized in that: In step S1, the concentration of the poly [9,9-bis (3'- (N, N-dimethylamino) propyl) -2,7-fluorene] -alkyl -2,7- (9,9-dioctylfluorene)] in the mixed solution one is 0.5-1.5 mg / mL, and the concentration of the poly (styrene - co-maleic anhydride) in the mixed solution two is 0.5-1.5 mg / mL, and the mixed solution one and the mixed solution two are mixed for 1.5-2.5 hours, and the volume ratio of the mixed solution one to the mixed solution two is 4.5-5.5:0.5-1.

5.

3. The aptamer-triggered nucleic acid amplification strategy and electrochemiluminescence detection method for perfluorooctanoic acid according to claim 1, characterized in that: In step S2, the nucleotide sequence of the corresponding linker strand is shown in SEQ ID NO: 1, the nucleotide sequence of the corresponding aptamer strand is shown in SEQ ID NO: 2, and the nucleotide sequence of the padlock probe is shown in SEQ ID NO: 3; In step S3, the nucleotide sequence corresponding to single strand 1 is shown as SEQ ID NO: 4, and the nucleotide sequence corresponding to single strand 2 is shown as SEQ ID NO: 5; In step S4, the nucleotide sequence of the DNA hairpin structure H1 is shown in SEQ ID NO:

6.

4. The method for detecting perfluorooctanoic acid based on aptamer-triggered nucleic acid amplification strategy and electrochemiluminescence according to claim 1, characterized in that: In step S2, the restriction endonuclease is NB.BbvCI, the DNA polymerase is Phi29 DNA polymerase, and the polymerase buffer is Phi29 polymerase buffer.

5. The aptamer-triggered nucleic acid amplification strategy and electrochemiluminescence detection method for perfluorooctanoic acid according to claim 1, characterized in that: In step S3, the volume ratio of the single strand 1 to the rolling circle amplification product obtained in step S2 is 9-11:9-11.

6. The aptamer-triggered nucleic acid amplification strategy and electrochemiluminescence detection method for perfluorooctanoic acid according to claim 1, characterized in that: In step S3, the volume ratio of single-stranded 1, crRNA and Cas12a is 9-11:1-2:0.5-1.

5.

7. The method for detecting perfluorooctanoic acid based on aptamer-triggered nucleic acid amplification strategy and electrochemiluminescence according to claim 1, characterized in that: In step S4, the coupling agent includes a 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide solution and an N-hydroxysuccinimide solution, the concentration of the 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide solution is 0.01-0.03 g / mL, the concentration of the N-hydroxysuccinimide solution is 0.004-0.006 g / mL, and the volume ratio of the 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide solution to the N-hydroxysuccinimide solution is 9-11:9-11.

8. The aptamer-triggered nucleic acid amplification strategy and electrochemiluminescence detection method for perfluorooctanoic acid according to claim 1, characterized in that: In step S4, the volume ratio of the DNA hairpin structure H1 to dopamine is 9-11:2-3.

9. The aptamer-triggered nucleic acid amplification strategy and electrochemiluminescence detection method for perfluorooctanoic acid according to claim 1, characterized in that: In step S4, the volume ratio of the nanoparticle dispersion obtained in step S1, dopamine-labeled H1 and hexylene glycol is 9-11:9-11:9-11.

10. The aptamer-triggered nucleic acid amplification strategy and electrochemiluminescence detection method for perfluorooctanoic acid according to claim 1, characterized in that: In step S5, the pulse voltage is +0.8-+0.9V, the pulse time is 1-2s, the photomultiplier tube is 500-700V, and the amplification level is 2-4.

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