Method for detecting miRNA-107 by using AIECL sensor based on resonance energy transfer

Abstract

The invention relates to a method for detecting miRNA-107 by using an AIECL sensor based on resonance energy transfer. The method comprises the following steps: modifying the surface of GCE with prepared Pdots; then, HP-BHQ is modified to the surface of Pdots through an amidation reaction; due to the fact that resonance energy transfer exists between the Pdots and the BHQ, ECL signals of the Pdots can be effectively quenched by the BHQ; the obtained electrode acts on miRNA-107, then DSN is used for cutting off DNA fragments in DNA-RNA hybrid double strands, BHQ leaves the surface of the electrode along with the cut-off DNA fragments while the miRNA-107 realizes cyclic amplification, ECL signals are changed, the logarithm of the concentration of delta ECL and the concentration of the miRNA-107 shows a linear relation, and a corresponding fitted equation is obtained. The detection method established by the invention shows low detection limit and good stability and selectivity in the detection of the miRNA-107.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of detection, in particular to an AIECL sensor based on resonance energy transfer and its application in miRNA-107 detection. BACKGROUND

[0002] MicroRNA (miRNA) is a class of endogenous non-coding small molecule RNA composed of 19-24 nucleotides. It regulates the expression level of oncogenes and tumor suppressor genes by complementary binding with the untranslated region (3'-UTR) of mRNA, and then induces potential carcinogenic changes, and is considered as a diagnostic marker for various cancers. Among them, the expression disorder of miRNA-107 in gastric cancer, lung cancer, hepatocellular carcinoma, pancreatic ductal adenocarcinoma and castration-resistant prostate cancer seriously affects the occurrence and development of diseases. Traditional miRNA detection methods include Northern blot and quantitative reverse transcription polymerase chain reaction (qRT-PCR), etc., but they are complex to operate and time-consuming, and are subject to many limitations in practical application. New miRNA detection methods include electrochemical method, fluorescence method and surface enhanced Raman method, etc., but their sensitivity is low, and it is difficult to meet the needs of ultra-low concentration miRNA detection. Therefore, it is of great significance to establish an accurate and sensitive miRNA-107 detection method for early diagnosis and prognosis monitoring of cancer.

[0003] Electrochemiluminescence (ECL) as a powerful analytical method, has the advantages of high sensitivity, high selectivity, low background interference and wide linear range, and is widely used in the field of biosensing. Electrochemiluminescence-resonance energy transfer (ECL-RET) occurs when the emission spectrum of the donor and the absorption spectrum of the acceptor overlap sufficiently and are close in distance. Compared with traditional ECL systems, ECL-RET shows higher sensitivity and lower background signal by transferring resonance energy from the donor to the acceptor. For example, there is a ECL sensor for dopamine detection based on the energy transfer system of carboxylated g-C3N4 / CuO, with a detection limit of 8.2nM (Li MS, Wang CX, Liu DF. A novel“off-on”electrochemiluminescence sensor based on highly efficient resonance energy transfer in C-g-C3N4 / CuO nanocomposite[J]. Analytica Chimica Acta, 2020, 1138: 30-37.); an ECL sensor for acetylcholinesterase detection is constructed by using energy transfer between luminol and gold nanoparticle decorated covalent organic framework, with a detection limit of 0.17nM (Wang XY, Wang J, Ding HC, et al. Detection of acetylcholinesterase based on ECL resonance energy transfer between luminol and gold nanoparticle decorated covalent organic framework[J]. Microchemical Journal, 2025, 209: 112774.). In addition, the combination of ECL with signal amplification strategy can further improve the sensitivity of detection.

[0004] Aggregation-induced electrochemiluminescence (AIECL) refers to a way that luminescent groups in the aggregate state, the molecules are tightly stacked to produce spatial constraints, limiting the intramolecular free rotation, thereby reducing energy consumption and enhancing ECL emission. Therefore, AIECL has higher detection sensitivity. In recent years, polymer dots (Pdots) have become a new hotspot in the field of AIECL due to their non-toxicity, easy synthesis, strong light stability, good biocompatibility and high fluorescence quantum yield. In 2018, Sun et al. innovatively applied AIE active polymer dots to the ECL-RET system and proposed an ECL aptamer sensor for detecting trace Pb 2+ (Sun F, Wang ZY, Feng YQ, et al. Electrochemiluminescent resonance energy transfer of polymer dots for aptasensing [J]. Biosensors and Bioelectronics, 2018, 100: 28-34). SUMMARY

[0005] The purpose of the present application is to provide a method for detecting miRNA-107 by using an AIECL sensor based on resonance energy transfer. The present application provides a novel, sensitive, stable and selective method for detecting miRNA-107, which has a wide application prospect in the field of disease diagnosis and biosensing.

[0006] The purpose of the present application is achieved by the following technical solutions:

[0007] A method for detecting miRNA-107 by using an AIECL sensor based on resonance energy transfer, which comprises the following steps:

[0008] Step 1. Preparation of Pdots: Synthesis of polymer dots Pdots by nano-coprecipitation method using polymer THF mother liquor;

[0009] Step 2. The Pdots obtained in step 1 are modified to the surface of a bare glassy carbon electrode (GCE); then HP-BHQ is modified to the surface of the Pdots by an amidation reaction between the carboxyl groups on the surface of the Pdots and the amino groups at the ends of HP-BHQ; at this time, the ECL signal is quenched due to the resonance energy transfer (RET) between the Pdots and BHQ; the obtained electrode is used to react with different concentrations of miRNA-107 standard solution to form DNA-RNA hybrid double-stranded; then an excess of DSN is added, and the DSN cuts the DNA fragments in the DNA-RNA hybrid double-stranded, and the miRNA-107 realizes cyclic amplification while the BHQ leaves the electrode surface with the cut DNA fragments, and the ECL signal is restored;

[0010] Step 3. The ECL signal change before and after the introduction of miRNA-107 is measured by cyclic voltammetry to obtain ΔECL, and ΔECL and the logarithm of the concentration of miRNA-107 show a good linear relationship and obtain the corresponding fitting equation.

[0011] The electrochemiluminescence (ECL) sensor constructed in step 2 can be used to detect the test sample, so as to realize qualitative and quantitative detection of miRNA-107 in the test sample. Specifically, if the ECL changes before and after the introduction of the test sample (i.e. the miRNA-107 standard solution is replaced by the test sample), it indicates that the test sample contains miRNA-107, realizing qualitative detection of miRNA-107, and the ECL change value before and after the introduction of the test sample, i.e. ΔECL, is substituted into the fitting equation to calculate the concentration of miRNA-107 in the test sample, realizing quantitative detection of miRNA-107.

[0012] The specific operation method of step 1 is as follows:

[0013] First, prepare a 100 μg / mL polymer THF stock solution; then, under ultrasonic conditions, quickly inject 2 mL of the polymer THF stock solution into 10 mL of ultrapure water and further ultrasonicate for 3 min; in this case, the hydrophobic polymer chains collapse due to the difference in polarity in the aqueous solvent, quickly aggregate to form nanospheres; then, use a rotary evaporator to distill under reduced pressure to remove excess THF and water, obtain 2 mL of Pdots concentrated solution; finally, filter the Pdots concentrated solution through a 0.22 μm polyether sulfone (PES) needle filter, and finally obtain uniformly dispersed carboxylated Pdots, and store at 4°C for standby.

[0014] The specific operation method of step 2 includes the following steps:

[0015] Step 2.1 Electrode pretreatment: A small amount of alumina powder was poured on the suede and wetted with ultrapure water to obtain an alumina slurry; the glassy carbon electrode was polished with the alumina slurry and then ultrasonically cleaned in ultrapure water and ethanol in sequence; the pretreated electrode was dried with nitrogen and immersed in an electrolyte containing 0.1 M KCl, 5 mM K3[Fe(CN)6] and K4[Fe(CN)6]; the cyclic voltammogram (CV) was measured in a scanning potential range of -0.2 V to 0.6 V using a standard three-electrode system (the glassy carbon electrode as the working electrode, the Ag / AgCl electrode (saturated KCl) as the reference electrode, and the platinum wire electrode as the counter electrode); if the peak potential difference (ΔEp) of the redox pair is less than 90 mV, it proves that the electrode surface is clean and active, and can be further used for quantitative detection. If ΔEp is too large, it will introduce experimental error and affect the accuracy of the experiment; finally, the qualified electrode surface was rinsed with ultrapure water, and then the water on the electrode surface was dried with nitrogen to obtain an electrode with mirror-like luster; 3- / 4- The peak potential difference (ΔEp) of the redox pair is less than 90 mV, which proves that the electrode surface is clean and active, and can be further used for quantitative detection. If ΔEp is too large, it will introduce experimental error and affect the accuracy of the experiment; finally, the qualified electrode surface was rinsed with ultrapure water, and then the water on the electrode surface was dried with nitrogen to obtain an electrode with mirror-like luster;

[0016] Step 2.2 Preparation of nucleic acid solution: The miRNA-107 lyophilized powder was placed in a high-speed refrigerated centrifuge and centrifuged at 12000 rpm / min at 4°C for 2-3 min to fully settle the powder; according to the requirements of the instructions, a specified amount of diethyl pyrocarbonate treated ultrapure water (DEPC water) was added and gently mixed to prepare a 100 μM stock solution; different concentrations of working solutions required for subsequent experiments (such as different concentrations of miRNA-107 standard solution) were obtained according to the principle of stepwise dilution; the rest of the nucleic acid processing steps are the same as above;

[0017] Step 2.3 Construction of ECL sensor: 10 μL of Pdots prepared in step 1 was dropped on the electrode surface pretreated in step 2.1 and naturally air-dried at room temperature (obtained the electrode modified with Pdots, named GCE / Pdots); then, the electrode was incubated in a mixed solution of 20 mM NHS and 10 mM EDC at 37°C for 1 h to activate the carboxyl groups on the Pdots; then, 1 μM HP-BHQ was added to the electrode and incubated at 4°C for 12 h; then, the electrode was rinsed with 10×PBS (pH 7.4) to remove the unbound HP, and the non-specific binding sites on the electrode surface were blocked with 100 μM BSA; then, the electrode was placed in 80 μL of different concentrations of miRNA-107 and incubated at 37°C for 2 h; at this time, HP-BHQ and miRNA-107 complementary pairing formed DNA-RNA hybrid double-stranded; then, an excess of DSN was added and incubated at 37°C for 2 h to completely cut off the DNA fragments in the DNA-RNA hybrid double-stranded, while releasing miRNA-107 into the circulation.

[0018] The method for measuring ECL in step 3 is: scanning from 0 to 1.4V by cyclic voltammetry (CV) and recording ECL curve in MPI-E multifunctional electrochemical and chemiluminescence analysis system.

[0019] The result obtained in step 3 is: when the concentration of miRNA-107 is gradually increased in the range of 1fM-10pM, the response intensity of ECL shows an obvious upward trend; ΔECL shows a good linear relationship with the logarithm of the concentration of miRNA-107, and the fitting equation is ΔECL = 33227.669 + 2179.83 x lgC miRNA-107 (R 2 = 0.997), and the LOD calculated based on signal-to-noise ratio (S / N) = 3 is 0.82fM, wherein ΔECL refers to the change value of ECL before and after introducing miRNA-107, and C miRNA-107 refers to the concentration of miRNA-107.

[0020] The application constructs an AIECL sensor for ultra-sensitive detection of miRNA-107 based on ECL-RET mechanism. Figure 1 As shown in A, Pdots with AIECL activity are prepared by nano-coprecipitation method. Figure 1 As shown in B, due to the resonance energy transfer between BHQ and Pdots, the ECL signal of Pdots can be effectively quenched by HP-BHQ, thereby causing the signal to be "turned off". The target miRNA-107 and HP are complementary paired to form a hybrid double strand. In combination with the signal amplification strategy assisted by DSN, miRNA-107 is released from the hybrid double strand into the cycle, and at the same time, BHQ falls off from the electrode surface with the DNA fragments cut off, and the signal is "turned on". With the increase of the concentration of miRNA-107, the ECL signal is continuously enhanced. The results show that the sensor has a good linear response in the range of 1fM-10pM of the concentration of miRNA-107, and the detection limit is as low as 0.82fM.

[0021] Compared with the prior art, the application has the following advantages:

[0022] 1. The Pdots obtained by the present application have a clear emission peak near 500 nm, and the absolute PL quantum yield reaches 23.29% measured by an integrating sphere. In addition, the fluorescence lifetime of the Pdots is calculated to be 2.75 ns by collecting the PL transient spectrum of the Pdots. In order to study the electrochemiluminescence behavior of the Pdots, the present application further analyzes the Pdots by CV and ECL. The CV curve has two irreversible oxidation peaks at +0.9 V and +1.16 V, respectively, wherein the peak at +0.9 V corresponds to the oxidation process of the coreactant TEA, and the peak at +1.16 V is derived from the oxidation reaction of the Pdots themselves. This is consistent with the observation of a significant ECL signal of the Pdots at a potential of +1.2 V.

[0023] 2. The sensor established by the present application has superior stability and reproducibility: the inventors constructed three groups of ECL sensors with exactly the same conditions, and the relative standard deviation (RSD) of ΔECL was calculated to be 2.9%, indicating that the sensor has superior stability and reproducibility.

[0024] 3. The sensor established by the present application has good selectivity: the present application uses the sensor to detect miRNA-122, miRNA-141, miRNA-21 and miRNA-107, respectively, and the results show that only when miRNA-107 exists, the ECL intensity will change significantly, and the signal changes of miRNA-122, miRNA-141 and miRNA-21 can be ignored. This result shows that the sensor has good selectivity, and only has ECL response to miRNA-107.

[0025] 4. The sensor established by the present application has good linear response and sensitivity: when the concentration of miRNA-107 is gradually increased in the range of 1 fM-10 pM, the response intensity of ECL shows a clear upward trend. ΔECL shows a good linear relationship with the logarithm of the concentration of miRNA-107, and the fitting equation is ΔECL = 33227.669 + 2179.83 x lgC miRNA-107 (R 2 = 0.997), and the LOD calculated based on the signal-to-noise ratio (S / N) = 3 is 0.82 fM, which is significantly lower than the previous report. BRIEF DESCRIPTION OF DRAWINGS

[0026] Figure 1 is a preparation process schematic diagram of the Pdots and the AIECL sensor of the present application; wherein, Figure 1 A is the synthesis diagram of the Pdots with AIECL activity, Figure 1 B is the preparation diagram of the AIECL sensor.

[0027] Figure 2are part of the characterization of Pdots; wherein 2A is the TEM image of Pdots (inset: particle size distribution); 2B is the UV-Vis spectrum (black line) and PL spectrum (red line) of Pdots; 2C is the CV curve of Pdots in TEA co-reactant; 2D is the ECL intensity-potential curve of Pdots in TEA co-reactant.

[0028] Figure 3 are the fluorescence lifetime images of Pdots.

[0029] Figure 4 are the PAGE results, wherein 4A is the schematic diagram of DSN signal amplification reaction; 4B is the PAGE reaction system: (1) miRNA-107, (2) HP, (3) miRNA-107 + HP, (4) miRNA-107 + HP + DSN, M is Marker.

[0030] Figure 5 are the performance analysis of ECL sensor, wherein 5A is the ECL response of 1 fM-10 pM miRNA-107. 5B is the relationship between 1 fM-10 pM miRNA-107 and ΔECL intensity change (inset: linear relationship). 5C is the reproducibility of ECL sensor for detecting 10 fM miRNA-107. 5D is the ECL signal comparison of 10 pM miRNA-107 and other miRNAs (error bar represents the standard deviation of three parallel experiments). DETAILED DESCRIPTION

[0031] The content of the present application will be described in detail below in combination with the drawings and examples of the specification:

[0032] 1. Instruments and reagents

[0033] 1.1 Instruments

[0034] The instruments used in the experiment are shown in Table 1-1.

[0035] Table 1-1 Experimental instruments

[0036]

[0037] 1.2 Reagents

[0038] The nucleic acid sequences used in the experiment were synthesized by Shengong Bioengineering (Shanghai) Co., Ltd., and are shown in Table 1-2. The remaining reagents are shown in Table 1-3.

[0039] Table 1-2 Nucleic acid sequences

[0040]

[0041] Table 1-3 Experimental reagents

[0042]

[0043]

[0044] 2 Experimental methods

[0045] 2.1 Preparation of polymer dots (Pdots)

[0046] According to previous research (Gao H, Zhang N, Hu J, et al. Molecular Engineering of Polymer Dots for Electrochemiluminescence Emission [J]. ACS APPLIED NANOMATERIALS, 2021, 4(7): 7244-7252), polymer dots (Pdots) were synthesized by nanoprecipitation method. First, a 100 pg / mL polymer THF stock solution was prepared. Then, 2 mL of the polymer THF stock solution was quickly injected into 10 mL of ultrapure water under ultrasonic conditions, and further ultrasonicated for 3 min. In this case, the hydrophobic polymer chains collapsed due to the difference in polarity in the aqueous solvent, quickly aggregated to form nanospheres. Next, rotary evaporation was used to distill under reduced pressure to remove excess THF and water, obtaining 2 mL of Pdots concentrate. Finally, the above solution was filtered through a 0.22 pm polyether sulfone (PES) syringe filter, and finally obtained uniformly dispersed carboxylated Pdots, which were stored at 4°C for standby use.

[0047] 2.2 Construction of electrochemiluminescence (ECL) sensor

[0048] 2.2.1 Electrode pretreatment

[0049] A small amount of alumina powder was poured on the suede and wetted with ultrapure water to obtain an alumina slurry. After polishing the glassy carbon electrode with the alumina slurry, it was ultrasonicated in ultrapure water and ethanol in turn. The pretreated electrode was blown dry with nitrogen, immersed in an electrolyte containing 0.1 M KCl, 5 mM K3[Fe(CN)6] and K4[Fe(CN)6], and a standard three-electrode system (glassy carbon electrode as working electrode, Ag / AgCl electrode (saturated KCl) as reference electrode, platinum wire electrode as counter electrode) was used to measure the cyclic voltammogram (CV) in the scan potential of -0.2 V ~ 0.6 V.

[0050] If [Fe(CN)6] 3- / 4-The peak potential difference (ΔΕρ) of the redox couple is less than 90 mV, which proves that the electrode surface is clean and active, and can be further used for quantitative detection. If ΔΕρ is too large, it will introduce experimental error and affect the accuracy of the experiment. Finally, the qualified electrode surface is rinsed with ultrapure water, and then the water on the electrode surface is blown dry with nitrogen, obtaining an electrode in a mirror-like state.

[0051] 2.2.2 Preparation of nucleic acid solution

[0052] The miRNA-107 freeze-dried powder was placed in a high-speed centrifuge, centrifuged at 12000 rpm / min at 4°C for 2-3 min, so that the powder was fully settled. According to the requirements of the instructions, a specified amount of diethyl pyrocarbonate treated ultrapure water (DEPC water) was added and mixed gently to prepare a 100 μΜ stock solution. Different concentrations of working solutions required for subsequent experiments (such as different concentrations of miRNA-107 standard solution) will be obtained according to the principle of step-by-step dilution. The rest of the nucleic acid processing steps are the same as above.

[0053] In order to obtain the relationship between ECL and the concentration of miRNA-107, the inventors prepared miRNA-107 standard solutions with a concentration range of 1 fM-10 pM. In order to explore the selectivity of the constructed sensor, the inventors also prepared several different miRNA solutions, such as miRNA-122, miRNA-141, and miRNA-21.

[0054] 2.2.3 Construction of ECL sensor

[0055] 10 μL of Pdots was dropped on the electrode surface and naturally air-dried at room temperature. Then, the electrode was incubated in a mixed solution of 20 mM NHS and 10 mM EDC at 37°C for 1 h to activate the carboxyl groups on the Pdots. Then, 1 μΜ HP-BHQ was added to the electrode and incubated at 4°C for 12 h. Then, the electrode was rinsed with 10x PBS (pH 7.4) to remove the unbound HP, and the non-specific binding sites on the electrode surface were blocked with 100 μΜ BSA. Then, the electrode was placed in 80 μL of different concentrations of miRNA-107 and incubated at 37°C for 2 h. At this time, HP-BHQ and miRNA-107 complementary pairing form DNA-RNA hybrid double-stranded. Next, an excess of DSN was added and incubated at 37°C for 2 h to completely cut off the DNA fragment in the DNA-RNA hybrid double-stranded, while releasing miRNA-107 into the cycle.

[0056] 2.3 ECL measurement

[0057] The ECL of the constructed ECL sensor before and after the introduction of miRNA-107 is measured by cyclic voltammetry. The measurement method of ECL is as follows: the MPI-E multifunctional electrochemical and chemiluminescence analysis system is used to scan and record the ECL curve by cyclic voltammetry (CV) from 0 to 1.4V.

[0058] 2.4 Characterization of Pdots

[0059] In order to study the morphology of Pdots, the Pdots obtained in step 2.1 are analyzed by transmission electron microscopy (TEM).

[0060] 2.4.1 Analysis of optical physical properties

[0061] In order to study the optical physical properties of Pdots, the present application analyzes them by ultraviolet-visible absorption spectrum (UV-Vis) and photoluminescence spectrum (PL).

[0062] 2.4.2 Electrochemical and ECL measurement

[0063] In order to study the electrochemiluminescence behavior of Pdots, the present application further analyzes them by CV and ECL. The specific method is as follows:

[0064] A 25mM TEA solution prepared with 10x PBS is used as a co-reactant, and the Pdots-modified electrode (GCE / Pdots) is used as a working electrode. The MPI-E multifunctional electrochemical and chemiluminescence analysis system is used to scan and record the CV and ECL curves by cyclic voltammetry (CV) from 0 to 1.4V, in order to explore the electrochemical and electrochemiluminescence behavior of Pdots.

[0065] 2.5 Non-denaturing polyacrylamide gel electrophoresis (PAGE)

[0066] 2.5.1 Nucleic acid solution treatment

[0067] 0.5μM and 2.5μM miRNA-107 solutions, and 0.5μM, 1μM and 5μM HP solutions are prepared. Then, all the nucleic acid solutions are annealed at 95℃ for 5min, and then slowly cooled to room temperature.

[0068] 2.5.2 Hybridization system construction

[0069] Two groups of experimental groups were set. Experimental group 1 mixed 0.5 μM of miRNA-107 and 1 μM of HP uniformly at a volume ratio of 1:1 (final concentration: 0.25 μM miRNA-107 + 0.5 μM HP), and experimental group 2 mixed 2.5 μM of miRNA-107 and 5 μM of HP uniformly at a volume ratio of 1:1 (final concentration: 1.25 μM miRNA-107 + 2.5 μM HP). Both groups of experimental groups were incubated at 37°C for 2 h to ensure complete reaction.

[0070] 2.5.3 Enzymatic cleavage reaction

[0071] Take 2 μL of the hybridization product of experimental group 2, mix it with 6 μL of ultrapure water, 1 U of DSN, and 1 μL of 10x DSN buffer, and continue to incubate at 37°C for 2 h to ensure that the DSN completes the enzymatic cleavage reaction, and set it as experimental group 3 (final concentration: 0.25 μM miRNA-107 + 0.5 μM HP).

[0072] 2.5.4 Preparation of electrophoresis samples

[0073] Take 0.5 μM miRNA-107 (sample 1), 0.5 μM HP (sample 2), 10 μL of the hybridization product of experimental group 1 (sample 3), and 10 μL of the hybridization product of experimental group 3 (sample 4), and add 2 μL of 6x loading buffer to mix uniformly.

[0074] 2.5.5 Configuration of electrophoresis liquid

[0075] Take 1 package of electrophoresis liquid dry powder, add 500 mL of ultrapure water to dissolve completely, and configure it into 1x electrophoresis buffer.

[0076] 2.5.6 Electrophoresis analysis

[0077] Use 15% non-denaturing polyacrylamide gel, first pre-electrophorese at 80V for 30 min to ensure field balance, then add 2 μL Marker and 10 μL of the sample to be tested in the sample well in turn, and then electrophorese at 80V for 30 min to separate nucleic acids, which can avoid nucleic acid diffusion caused by high voltage, and finally electrophorese at 120V for 12 min to speed up the separation speed and make the bands completely separated. After electrophoresis, the gel is placed in an electrophoresis buffer containing 10 μL Gel-Green for staining for 10 min, and finally the electrophoresis image is collected and analyzed in the gel imaging system.

[0078] 2.6 Performance test of ECL sensor

[0079] 2.6.1 Linearity of ECL sensor

[0080] The ECL signal changes of the constructed ECL sensor before and after introducing different concentrations of miRNA-107 standard solution were measured respectively, and the corresponding ΔECL was obtained. The linear relationship between ΔECL and the concentration of miRNA-107 was obtained, and the corresponding fitting equation was obtained. Among them, the concentration range of the miRNA-107 standard solution was 1 fM-10 pM.

[0081] 2.6.2 Stability and reproducibility performance test of ECL sensor

[0082] Three groups of ECL sensors with the same conditions were constructed according to step 2.2, and were acted with the same concentration of miRNA-107. The ECL of the three groups of ECL sensors (including the ECL before and after introducing miRNA-107 respectively) was measured respectively, and the corresponding ΔECL was obtained. The relative standard deviation of the ΔECL of the three groups of ECL sensors was calculated.

[0083] 2.6.3 Selectivity performance test of ECL sensor

[0084] The different concentrations of miRNA-107 in step 2.2.3 were replaced by other miRNAs such as miRNA-122, miRNA-141 and miRNA-21 respectively, and the corresponding ECL was tested to observe whether the ECL intensity changed before and after introducing miRNA.

[0085] 3 Experimental results

[0086] 3.1 Characterization of Pdots

[0087] To study the morphology of Pdots, we analyzed them by transmission electron microscopy (TEM). We can clearly observe that the prepared Pdots have a spherical morphology, the particles are well dispersed, there is no obvious agglomeration, and the overall size has good uniformity (see Figure 2 A). To accurately determine the particle size distribution characteristics of Pdots, we statistically analyzed 100 nanoparticles, and the results showed that the average particle size of Pdots was 3.3 nm (see Figure 2 A inset).

[0088] To study the optical properties of Pdots, we analyzed them by ultraviolet-visible absorption spectrum (UV-Vis) and photoluminescence spectrum (PL). The UV-Vis spectrum of Pdots has obvious absorption peaks near 250 nm and 355 nm, the former is attributed to the π-π* transition of the side chain p-hydroxybenzoic acid unit, and the latter is attributed to the π-π* transition of the polymer main chain[ 22] (see Figure 2 B). The PL spectrum shows that Pdots have a obvious emission peak near 500 nm, and the absolute PL quantum yield measured by the integrating sphere is up to 23.29% (seeFigure 2 B). In addition, by collecting the PL transient spectrum of Pdots and calculating, we obtained the fluorescence lifetime of 2.75 ns (see Figure 3 ).

[0089] To study the electrochemiluminescence behavior of Pdots, we further analyzed them by CV and ECL. The CV curve appeared two irreversible oxidation peaks at +0.9 V and +1.16 V, respectively, in which the peak at +0.9 V corresponded to the oxidation process of the co-reactant TEA, while the peak at +1.16 V originated from the oxidation reaction of Pdots itself (reference Xu ZH, Weng X, Wang SS, et al. Highly sensitive aggregation-induced electrochemiluminescence sensor for cadmium detection in Ganoderma lucidum [J]. Food chemistry, 2025, 470: 142661) (see Figure 2 C). This is consistent with the observation of significant ECL signal of Pdots at +1.2 V potential (see Figure 2 D). According to previous studies (such as Chen MM, Gao H, Ge ZB, et al. Ultrasensitive Electrochemiluminescence Sensor Utilizing Aggregation-Induced Emission Active Probe for Accurate Arsenite Quantification in Rice Grains [J]. Journal of agricultural and food chemistry, 2024, 72(5): 2826-2833), the ECL luminescence mechanism is speculated as follows:

[0090] TEA-e - →TEAH ·+ (1)

[0091] TEAH ·+ -H + →TEA · (2)

[0092] Pdot-e - →Pdot ·+ (3)

[0093] Pdot ·+ +TEA · →Pdot* (4)

[0094] Pdot * →Pdot+hv (5)3.2 Feasibility of Sensing Strategy

[0095] The signal amplification mechanism of this sensor is based on the following principle: When miRNA-107 and HP interact, they bind due to their highly complementary base sequences, generating a new DNA-RNA hybrid chain. Based on the characteristic that DSN can specifically cleave DNA in DNA-RNA hybrids but has no effect on free DNA or RNA, with the cleavage of DSN, miRNA-107 is released from the hybrid chain and enters the next cycle, thereby achieving signal amplification (see...). Figure 4 A).

[0096] To verify the feasibility of the DSN-assisted signal amplification strategy, we analyzed the reaction system using PAGE. Lane 1 (miRNA-107) and lane 2 (HP) showed typical single-stranded nucleic acid migration bands. Compared with lanes 1 and 2, lane 3 (miRNA-107 and HP hybrid strand) showed a new band with a slower migration rate, indicating that miRNA-107 and HP successfully reacted to form a hybrid double strand. Compared with lane 3, the band corresponding to the hybrid double strand disappeared in lane 4 (dSN-treated miRNA-107 and HP hybrid strand), while the band corresponding to miRNA-107 reappeared. This indicates that DSN can specifically cleave the DNA fragment in the hybrid double strand, thereby releasing the complete miRNA-107. miRNA-107 enters the cycle and continues to bind with the remaining HP to form a hybrid double strand, which is then cleaved by DSN until it disappears completely. Therefore, the above results show that the signal amplification strategy is reasonable and feasible (see...). Figure 4 B).

[0097] 3.3 Analytical Performance

[0098] To evaluate the analytical performance of the ECL sensor, we examined its linearity, reproducibility, and selectivity. As the concentration of miRNA-107 gradually increased within the range of 1 fM to 10 pM, the response intensity of the ECL sensor showed a significant upward trend (see...). Figure 5 A). ΔECL showed a good linear relationship with the logarithm of miRNA-107 concentration, and the fitted equation was ΔECL=33227.669+2179.83×lgC miRNA-107 (R 2 =0.997), and the LOD calculated based on a signal-to-noise ratio (S / N) of 3 is 0.82fM (see Figure 5B), which is significantly lower than the previous reports (see Table 3-1). The results show that the sensor has higher accuracy and sensitivity for the detection of miRNA-107.

[0099] Table 3-1 Comparison of the analysis performance of the sensor with other sensors

[0100]

[0101] Among them, the references

[11] ,

[24] -

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[0112] The present application constructs three groups of ECL sensors with exactly the same conditions, and the relative standard deviation (RSD) of ΔECL is 2.9% after calculation, indicating that the sensor has superior stability and reproducibility (see Figure 5 C). Moreover, using the sensor to detect miRNA-122, miRNA-141, miRNA-21 and miRNA-107 respectively, we observed that only when miRNA-107 exists, the ECL intensity will change obviously, while the signal changes of miRNA-122, miRNA-141 and miRNA-21 are negligible. This result shows that the sensor has good selectivity, and only miRNA-107 has ECL response (see Figure 5 D).

[0113] In summary, this invention prepared polymer dots (Pdots) with AIECL activity via a nano-coprecipitation method, and characterized their morphology, photophysical properties, electrochemical properties, and ECL properties. Non-denaturing polyacrylamide gel electrophoresis (PAGE) was used to verify the feasibility of a double-stranded specific endonuclease (DSN)-assisted signal amplification strategy. Based on the resonance energy transfer between Pdots and BHQ, an AIECL sensor for miRNA-107 detection was established, and the linearity, reproducibility, and selectivity of the sensor were analyzed and evaluated. Results: The Pdots exhibited a typical spherical structure with uniform size and good photophysical and electrochemiluminescence properties. ΔECL and lgC miRNA-107 It exhibits good linearity in the range of 1.0fM to 10.0pM, with the linear equation being ΔECL = 33227.669 + 2179.83 × lgC miRNA-107 (R 2 =0.997), the detection limit is 0.82 fM (S / N=3); the relative standard deviation (RSD) of the three sensors prepared under the same experimental conditions is 2.9%; in addition, the sensor has a strong ECL signal response only for miRNA-107, while the response to other miRNAs is weak.

[0114] Therefore, this invention constructs an aggregation-induced electrochemiluminescence (AIECL) sensor based on resonant energy transfer, achieving accurate and sensitive detection of miRNA-107. The principle of the sensor constructed in this invention is as follows:

[0115] Pdots are highly efficient and stable ECL luminescent emitters. When drop-coated onto an electrode surface, they exhibit a strong initial ECL signal. Upon introduction of HP, Pdots with carboxyl-functionalized surfaces and HP with an amino group modified at the 5′ end are linked via a condensation reaction. Simultaneously, BHQ, modified at the 3′ end of HP, exhibits a highly similar absorption spectrum to the emission spectrum of Pdots. Due to resonance energy transfer, BHQ quenches the initial signal of Pdots, resulting in a "shutdown" signal. Upon addition of the target miRNA-107, miRNA-107 and HP complementaryly pair to form a DNA-RNA hybrid double strand. Under DSN cleavage, miRNA-107 is released from the hybrid double strand and enters the cycle, causing BHQ to move away from the electrode surface, thus restoring the signal. As the concentration of miRNA-107 increases, the amount of BHQ on the electrode surface decreases, and the ECL signal significantly strengthens. Therefore, the content of miRNA-107 can be determined by monitoring changes in ECL signal intensity.

[0116] The AIECL sensor constructed in the application has wide linear range, low detection limit, good stability and selectivity in the detection of miRNA-107, and has wide application prospects in the fields of disease diagnosis and biosensing.

Claims

1. A method for miRNA-107 detection using an AIECL sensor based on resonance energy transfer, characterized in that: It comprises the following steps: Step 1. Preparation of Pdots: polymer dots Pdots are synthesized by nanoco-precipitation method using polymer THF mother liquor; Step 2. Modification of Pdots obtained in step 1 to the surface of a bare glassy carbon electrode (GCE); then HP-BHQ is modified to the surface of Pdots by an amidation reaction between the carboxyl group on the surface of Pdots and the amino group at the end of HP-BHQ; due to the resonance energy transfer (RET) between Pdots and BHQ, the ECL signal is quenched at this time; the obtained electrode is reacted with different concentrations of miRNA-107 standard solution to form DNA-RNA hybrid double-stranded; then an excess of DSN is added, which cuts the DNA fragment in the DNA-RNA hybrid double-stranded, and miRNA-107 realizes cyclic amplification while BHQ leaves the electrode surface with the cut DNA fragment, and the ECL signal is restored; Step 3. The change of ECL signal before and after the introduction of miRNA-107 is measured by cyclic voltammetry to obtain ΔECL, which shows a good linear relationship with the logarithm of the concentration of miRNA-107 and obtains the corresponding fitting equation.

2. The method of claim 1, wherein: The specific operation method of step 1 is as follows: First, prepare 100 μg / mL of polymer THF mother liquor; then, under ultrasonic conditions, quickly inject 2 mL of polymer THF mother liquor into 10 mL of ultrapure water and further ultrasonic for 3 min; in this case, the hydrophobic polymer chain collapses due to the difference in polarity in the aqueous solvent, quickly aggregates to form nanospheres; then, use a rotary evaporator to distill under reduced pressure to remove excess THF and water, obtain 2 mL of Pdots concentrated solution; finally, filter the Pdots concentrated solution through a 0.22 μm polyether sulfone needle filter, and finally obtain uniformly dispersed carboxylated Pdots, and store at 4°C.

3. The method of claim 1, wherein: The specific operation method of step 2 includes the following steps: Step 2.1 electrode pretreatment: polish the glassy carbon electrode with alumina paste and then ultrasonic in ultrapure water and ethanol; dry the pretreated electrode with nitrogen, immerse it in an electrolyte containing 0.1 M KCl, 5 mM K3[Fe(CN)6] and K4[Fe(CN)6], and measure the cyclic voltammogram in a standard three-electrode system at a scan potential of -0.2 V-0.6 V; finally, wash the potassium ferricyanide on the surface of the qualified electrode with ultrapure water, then dry the water on the electrode surface with nitrogen, and obtain the electrode in a mirror finish state; Step 2.2 preparation of nucleic acid solution: freeze-dried miRNA-107 powder is placed in a high-speed centrifuge, centrifuged at 12000 rpm / min at 4°C for 2-3 min, so that the powder is fully settled; add a specified amount of diethyl pyrocarbonate treated ultrapure water and mix gently to obtain a 100 μM stock solution; different concentrations of working solution required for subsequent experiments will be obtained according to the principle of stepwise dilution method; Step 2.3 Construction of ECL sensor: 10 μL Pdots prepared in step 1 were dropped on the electrode surface pretreated in step 2.1 and naturally dried at room temperature; then, the electrode was incubated in a mixed solution of 20 mM NHS and 10 mM EDC at 37 °C for 1 h to activate the carboxyl groups on Pdots; after that, 1 μM HP-BHQ was added to the electrode and incubated at 4 °C for 12 h; then, the electrode was rinsed with 10x PBS to remove the unbound HP and blocked with 100 μM BSA to seal the non-specific binding sites on the electrode surface; then, the electrode was placed in 80 μL miRNA-107 with different concentrations and incubated at 37 °C for 2 h; at this time, the HP-BHQ and miRNA-107 complementary pairing formed DNA-RNA hybrid double-stranded; then, an excess of DSN was added and incubated at 37 °C for 2 h to completely cut off the DNA fragment in the DNA-RNA hybrid double-stranded, while releasing miRNA-107 into the cycle.

4. The method of claim 1, wherein: The method for measuring ECL in step 3 is: the ECL curve is scanned and recorded by cyclic voltammetry (CV) from 0 to 1.4 V in MPI-E multifunctional electrochemical and chemiluminescence analysis system.

5. The method of claim 1, wherein: The results obtained in Step 3 are as follows: when the concentration of miRNA-107 is gradually increased in the range of 1 fM-10 pM, the response intensity of ECL shows a clear upward trend; the change in ECL (ΔECL) and the logarithm of the concentration of miRNA-107 show a good linear relationship, and the fitting equation is ΔECL = 33227.669 + 2179.83 x lgC miRNA-107 (R 2 = 0.997), and the LOD calculated based on the signal-to-noise ratio (S / N) = 3 is 0.82 fM, wherein ΔECL refers to the change in ECL before and after the introduction of miRNA-107, and C miRNA-107 refers to the concentration of miRNA-107.

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