Electrochemiluminescence resonance energy transfer aptamer sensor for HER2-ECD detection in breast cancer

Abstract

The present invention provides an electrochemiluminescence energy resonance transfer aptamer sensor for detecting HER2-ECD in breast cancer. The present invention uses Au NPs / g-C3N4 / PDDA composite luminescent material as a substrate and AuPt@ZIF-67 / pDNA substrate signal quenching solution, and utilizes the energy resonance transfer (ECL-RET) effect to achieve ultrasensitive detection of HER2-ECD. Compared with the traditional HER2-ECD detection method, the method of the present invention has the advantages of high sensitivity, strong specificity, rapid detection, convenient operation, low price of equipment and materials, and no pollution, thereby providing a new analytical method for the detection of HER2-ECD.

Description

Technical Field

[0001] The present invention relates to the technical field of electrochemical detection, and in particular to an electrochemiluminescence energy resonance transfer aptamer sensor for detecting HER2-ECD of breast cancer and a detection method thereof. Background Art

[0002] Breast Cancer (BC) is a malignant tumor that occurs in the mammary epithelium or ductal epithelium. Over the years, the incidence of breast cancer has been increasing worldwide, with more than 1 million new cases reported each year. Compared with other cancers, breast cancer has a high prevalence in women and poses a huge threat to women's life and health. If not diagnosed early, the disease can lead to death in the most serious cases. Therefore, it is necessary to develop an economical, sensitive and rapid breast cancer detection method to give correct and timely treatment in the early stages of breast cancer development, thereby inhibiting the further development of breast cancer. Human epidermal growth factor receptor 2 (HER-2) is an important prognostic factor for breast cancer. It encodes a transmembrane receptor-like protein with a relative molecular weight of 185KD and has tyrosine kinase activity. Human epidermal growth factor receptor 2 (HER2) consists of an extracellular domain (ECD), a lipophilic transmembrane region and an intracellular domain (ICD). The most commonly used method to determine HER2 status is immunohistochemistry. However, false negative results are sometimes given, which can cause some patients to lose the opportunity for anti-HER2 treatment.

[0003] In addition to cellular HER2, levels of the HER2 extracellular domain (HER2-ECD), which is released into the circulation through ADAM (a disintegrin and metalloproteinase) protease-mediated cleavage of membrane-bound HER2, have been shown to correlate with tumor burden in patients with primary breast cancer and metastatic cancer. Analysis of circulating levels of HER2-ECD has been proposed as a minimally invasive alternative to traditional methods for assessing HER-2 levels, predicting treatment response, and monitoring treatment response.

[0004] At present, the analysis technology of HER2-ECD is mainly concentrated on enzyme-linked immunosorbent assay (ELISA). Although it is widely used, it also has some shortcomings, such as ELISA often has false positives and is easily affected by interferences. Therefore, with the improvement of the clinical practicality of serum HER2-ECD, it is of great significance to develop a sensitive, economical and rapid detection method and establish its accurate quantitative analysis method. Summary of the invention

[0005] In order to solve the problems in the prior art, the present invention provides an electrochemiluminescence energy resonance transfer aptamer sensor for breast cancer HER2-ECD detection. The present invention uses a graphene phase carbon nitride (g-C3N4) composite luminescent material functionalized by polydiallyldimethylammonium chloride (PDDA) modified by gold nanoparticles (Au NPs) as a base signal material, and a metal organic framework material (MOF) modified by gold platinum (AuPt) as a signal quenching material. A large amount of partially complementary chain 2 (pDNA) of the HER2-ECD binding aptamer chain is loaded through the bonding action between metal nanoparticles and amino groups, and unbound active sites are blocked with 6-mercaptohexanol (MCH), and finally the AuPt@ZIF-67 / pDNA base signal quenching solution is prepared. It is worth noting that the UV spectrum of ZIF-67 overlaps with the ECL spectrum of g-C3N4, and the ECL signal of g-C3N4 is quenched due to the ECL-RET effect. After AuPt is modified on the surface of ZIF-67, the stability of MOF can be effectively improved, and AuPt serves as an active binding site for the partial complementary chain 2 (pDNA) of the HER2-ECD binding aptamer chain, thereby increasing the pDNA loading capacity. Therefore, the present invention uses Au NPs / g-C3N4 / PDDA composite luminescent material as the substrate and AuPt@ZIF-67 / pDNA substrate signal quenching solution, and utilizes the energy resonance transfer (ECL-RET) effect to achieve ultra-sensitive detection of HER2-ECD, providing a new diagnostic approach for early diagnosis of breast cancer patients.

[0006] Unless otherwise specified, the parts described in the present invention are all parts by weight and the percentages described are all mass percentages.

[0007] To achieve the above object, the technical solution of the present invention is:

[0008] An electrochemiluminescence energy resonance transfer aptamer sensor for breast cancer HER2-ECD detection, characterized in that the method for constructing the electrochemiluminescence energy resonance transfer aptamer sensor for breast cancer HER2-ECD detection is: dropping a substrate material Au NPs / g-C3N4 / PDDA dispersion onto a clean glassy carbon electrode surface, drying at room temperature, then dropping cDNA onto the electrode surface, and incubating at 4-5°C for 12-13 h; then dropping an MCH solution onto the electrode, and incubating at room temperature for 30-35 min; then dropping APT onto the electrode, and incubating at room temperature for 1-2 h; finally dropping an AuPt@ZIF-67 / pDNA substrate signal quenching solution onto the electrode, and incubating at room temperature for 3-4 h, to obtain the electrochemiluminescence aptamer sensor for HER2-ECD detection.

[0009] The preparation method of the base material Au NPs / g-C3N4 / PDDA dispersion is as follows: adding the g-C3N4 / PDDA dispersion into a colloidal gold nanoparticle (Au NPs) solution, stirring for 6-7 hours, centrifuging, washing, and then dispersing the precipitate in ultrapure water to obtain a uniformly dispersed Au NPs / g-C3N4 / PDDA dispersion.

[0010] The g-C3N4 / PDDA dispersion: g-C3N4 is dissolved in ultrapure water, uniformly dispersed by ultrasonication, PDDA solution (5wt%) is added, magnetic stirring is performed for 12-13h, centrifuged and washed, and the precipitate is dispersed in ultrapure water to obtain a uniformly dispersed g-C3N4 / PDDA dispersion.

[0011] The preparation method of the AuPt@ZIF-67 / pDNA substrate signal quenching solution is as follows: weighing AuPt@ZIF-67 nanocomposite powder, dissolving it in ultrapure water, uniformly dispersing it by ultrasonication, adding a partial complementary chain 2 (pDNA) of the HER2-ECD binding aptamer, stirring it in an ice bath for 12-13 hours, centrifuging it, washing it with water, re-dispersing the precipitate in ultrapure water, adding a 6-mercaptohexanol (MCH) solution, stirring it for 1-2 hours, centrifuging it, washing it with water, and re-dispersing the precipitate in ultrapure water to obtain the AuPt@ZIF-67 / pDNA substrate signal quenching solution.

[0012] The preparation method of the AuPt@ZIF-67 nanocomposite is as follows: cobalt nitrate hexahydrate (Co(NO3)2·6H2O) and 2-methylimidazole (C4H6N2) are weighed and dissolved in anhydrous methanol, and the C4H6N2 solution is slowly added under the action of ultrasound, and the ultrasonic reaction is continued for 1-2 hours; then K2PtCl6 (0.3 wt%) and HAuCl4 (0.2 wt%) are added to the mixed solution, and the reaction is stirred for 3-4 hours; finally, the product is separated, washed with anhydrous methanol, centrifuged, and vacuum dried at 80-85°C to obtain the AuPt@ZIF-67 nanocomposite.

[0013] The preparation method of the colloidal gold nanoparticle (Au NPs) solution is as follows: 1% HAuCl4 is added to ultrapure water, heated to boiling, and then 1% trisodium citrate is added to the boiling solution. When the color of the solution changes from blue to red, the mixture is kept boiling for 15-20 minutes, cooled to room temperature, and ultrapure water is added to disperse to obtain a colloidal gold nanoparticle (Au NPs) solution.

[0014] The method for preparing the AuPt@ZIF-67 / pDNA substrate signal quenching solution comprises the following steps:

[0015] 1) AuPt@ZIF-67:

[0016] First, cobalt nitrate hexahydrate (Co(NO3)2·6H2O) and 2-methylimidazole (C4H6N2) were weighed and dissolved in anhydrous methanol. Under the action of ultrasound, the C4H6N2 solution was slowly poured into the Co(NO3)2·6H2O solution, and then the ultrasonic reaction was continued for 1 hour. K2PtCl6 (0.3 wt%) and HAuCl4 (0.2 wt%) were added to the mixed solution, and the reaction was stirred for 3 hours. Finally, the mixture was washed with anhydrous methanol for 3 times, centrifuged, and dried in vacuum at 80°C to obtain AuPt@ZIF-67 nanocomposites.

[0017] 2) AuPt@ZIF-67 / pDNA: Weigh the AuPt@ZIF-67 powder obtained in step 1), dissolve it in ultrapure water, disperse it evenly by ultrasonication, add the partial complementary chain 2 (pDNA) of the HER2-ECD binding aptamer, stir it in an ice bath for 12 h, centrifuge it, wash it with water, and redisperse the precipitate in ultrapure water. Add 1 mM 6-mercaptohexanol (MCH) solution, stir it for 1 h, centrifuge it, wash it with water, and redisperse the precipitate in ultrapure water to obtain the AuPt@ZIF-67 / pDNA substrate signal quenching solution.

[0018] Specifically, an electrochemiluminescence resonance energy transfer aptamer sensor for breast cancer HER2-ECD detection is characterized in that the method for constructing the electrochemiluminescence resonance energy transfer aptamer sensor for breast cancer HER2-ECD detection comprises the following steps:

[0019] (1) Preparation of substrate materials;

[0020] 1) Au NPs: Add 1 ml (1%) HAuCl4 to 100 ml ultrapure water and heat to boiling, then quickly add 2.5 ml (1%) trisodium citrate to the boiling solution; when the color of the solution changes from blue to red, keep the mixture boiling for 15 minutes, cool to room temperature, add ultrapure water to make up to 100 ml, and obtain colloidal gold nanoparticles (Au NPs);

[0021] 2) g-C3N4 / PDDA dispersion: Weigh 2 mg of g-C3N4, dissolve it in 2 ml of ultrapure water, disperse it evenly by ultrasonication, add 600 ml of PDDA solution (5 wt%), stir it magnetically for 12 h, centrifuge and wash it, and then disperse the precipitate in 2 ml of ultrapure water to obtain a uniformly dispersed g-C3N4 / PDDA dispersion;

[0022] 3) Au NPs / g-C3N4 / PDDA dispersion: Take 2 ml of the g-C3N4 / PDDA dispersion prepared in step 2), add 600 ul of Au NPs prepared in step 1), stir for 6 h, centrifuge, wash, and then disperse the precipitate in 1 ml of ultrapure water to obtain a uniformly dispersed Au NPs / g-C3N4 / PDDA dispersion;

[0023] (2) Preparation of signal quenching materials;

[0024] 1) AuPt@ZIF-67:

[0025] First, cobalt nitrate hexahydrate (Co(NO3)2·6H2O, 0.582 g) and 2-methylimidazole (C4H6N2, 0.656 g) were weighed and dissolved in 25 mL of anhydrous methanol respectively; under the action of ultrasound, the C4H6N2 solution was slowly poured into the Co(NO3)2·6H2O solution, and then the ultrasonic reaction was continued for 1 h; secondly, 2 mL of K2PtCl6 (0.3 wt%) and 3 mL of HAuCl4 (0.2 wt%) were added to the mixed solution, and the reaction was stirred for 3 hours; finally, the mixture was washed with anhydrous methanol for 3 times, centrifuged, and dried in vacuum at 80 °C to obtain the AuPt@ZIF-67 nanocomposite;

[0026] 2) AuPt@ZIF-67 / pDNA: Weigh 2 mg of the AuPt@ZIF-67 powder obtained in step 1), dissolve it in 1 ml of ultrapure water, disperse it evenly by ultrasonication, add 200 μL of the partial complementary chain 2 (pDNA) of the 2 μM HER2-ECD binding aptamer, stir it in an ice bath for 12 h, centrifuge it, wash it with water, and redisperse the precipitate in 1 ml of ultrapure water. Add 200 μL of 1 mM 6-mercaptohexanol (MCH) solution, stir it for 1 h, centrifuge it, wash it with water, and redisperse the precipitate in 500 μL of ultrapure water to obtain the AuPt@ZIF-67 / pDNA substrate signal quenching solution;

[0027] (3) Construction of electrochemiluminescence resonance energy transfer (ECL-RET) aptamer sensor for HER2-ECD detection:

[0028] 1) Treat HER2-ECD binding aptamer (APT), partial complementary strand 1 of APT (cDNA), and partial complementary strand 2 of APT (pDNA) with 20 mM Tris-HCl (pH=7.4) buffer at room temperature and store at 4°C for future use;

[0029] 2) Soak the glassy carbon electrode in piranha solution (98% H2SO4 / 30% H2O2 = 3:1, v / v) for 30 min and then rinse with ultrapure water for later use;

[0030] 3) The electrode obtained in step 2) was polished to a mirror surface with 0.3 μm and 0.05 μm Al2O3 powders, and then the electrode was ultrasonically treated in the order of ultrapure water, anhydrous ethanol, and ultrapure water, and dried for later use;

[0031] 4) The electrode obtained in step 3) was electrochemically activated in 0.5 M H2SO4, then rinsed with ultrapure water and dried;

[0032] 5) 10 μL of the substrate material Au NPs / g-C3N4 / PDDA solution was dropped onto the cleaned glassy carbon electrode surface in step 4) and dried at room temperature;

[0033] 6) Add 10 μL of cDNA prepared in step 1) to the electrode prepared in step 5) and incubate at 4°C for 12 h;

[0034] 7) Add 10 μL of 1 mM MCH solution onto the electrode obtained in step 6) and incubate at room temperature for 30 min;

[0035] 8) Add 10 μL of APT prepared in step 1) onto the electrode prepared in step 7) and incubate at room temperature for 1 h;

[0036] 9) 15 μL of the AuPt@ZIF-67 / pDNA substrate signal quenching solution was added dropwise to the electrode prepared in step 8) and incubated at room temperature for 3 h to obtain an electrochemiluminescent aptamer sensor for HER2-ECD detection.

[0037] The present invention also provides a method for detecting HER2-ECD using the electrochemiluminescence resonance energy transfer (ECL-RET) aptamer sensor.

[0038] A method for detecting HER2-ECD using the electrochemiluminescence resonance energy transfer (ECL-RET) aptamer sensor is characterized by comprising the following steps:

[0039] 1) Add different concentrations of the target human epidermal growth factor receptor 2 extracellular domain (HER2-ECD) to the electrode of the above sensor;

[0040] 2) The electrode was placed in a 0.1 M PBS (pH = 7.0) solution containing 8 mM K2S2O8 for characterization, and its electrochemiluminescence intensity was measured;

[0041] 3) According to the linear relationship between the luminescence intensity obtained in step 2) and the logarithmic value of the HER2-ECD concentration, a working curve is drawn;

[0042] 4) The sample to be tested is detected by the sensor, and the obtained current value is calculated through the working curve prepared in step 3) to obtain the HER2-ECD concentration of the sample to be tested.

[0043] Compared with the prior art, the preparation method and application of the electrochemiluminescence resonance energy transfer (ECL-RET) aptamer sensor for detecting HER2-ECD of the present invention have the following outstanding features:

[0044] The present invention prepares a composite luminescent material of graphite phase carbon nitride (g-C3N4) functionalized by polydiallyldimethylammonium chloride (PDDA) modified by gold nanoparticles (Au NPs) and a signal quenching material of metal organic framework (MOF) modified by gold platinum (AuPt), and loads a large amount of partial complementary chain 2 (pDNA) of HER2-ECD binding aptamer through the bonding action between metal particles and amino groups, and finally prepares the AuPt@ZIF-67 / pDNA substrate signal quenching solution. The composite luminescent material of graphite phase carbon nitride (g-C3N4) functionalized by polydiallyldimethylammonium chloride (PDDA) modified by gold nanoparticles (Au NPs) prepared by the present invention has strong and stable cathode ECL activity. At the same time, the ultraviolet spectrum of the signal quenching material is highly overlapped with the ECL spectrum of Au NPs / g-C3N4 / PDDA. The addition of AuPt increases the stability of ZIF-67 and serves as an active binding site for the partial complementary chain 2 (pDNA) of the HER2-ECD binding aptamer, thereby increasing the pDNA loading amount. In the present invention, polydiallyldimethylammonium chloride-graphite phase carbon nitride (g-C3N4 / PDDA) and gold nanoparticles (Au NPs) are used to synthesize Au NPs / g-C3N4 / PDDA composite materials by stirring as a sensing interface, and a large amount of partial complementary chain 1 (cDNA) of the HER2-ECD binding aptamer is captured to further achieve signal amplification. By the above means, the prepared electrochemiluminescence energy resonance transfer aptamer sensor is successfully used for ultrasensitive detection of HER2-ECD. Compared with the traditional HER2-ECD detection method, the advantages of the present invention are high sensitivity, strong specificity, rapid detection, convenient operation, low price of equipment and materials, and no pollution, thereby providing a new analytical method for the detection of HER2-ECD.

[0045] The beneficial effects of the present invention are:

[0046] 1) The graphene-phase carbon nitride (g-C3N4) composite luminescent material functionalized with polydiallyldimethylammonium chloride (PDDA) modified with gold nanoparticles (Au NPs) can greatly improve the characteristics of g-C3N4, such as few loading sites, low luminescence efficiency, and easy electrode passivation, thereby improving the sensor performance and achieving signal amplification, while also improving the detection sensitivity.

[0047] 2) The electrochemiluminescence resonance energy transfer (ECL-RET) aptamer sensor prepared by the present invention has high specificity for identifying the target and can improve the selectivity of the sensor, thereby providing a new research direction and analysis method for the detection of trace HER2-ECD.

[0048] 4) The materials involved in the present invention can all be synthesized under laboratory conditions, with simple operation, low raw material price, low toxicity, environmental friendliness, and extremely small amount used each time, thus reducing experimental costs.

[0049] 5) The entire detection and analysis method of the present invention has clear and simple steps, high sensitivity and rapid signal response.

[0050] 6) The electrochemical aptasensor prepared by this method can provide a new method for the detection of HER2-ECD. BRIEF DESCRIPTION OF THE DRAWINGS

[0051] Figure 1 The time-electrochemiluminescence intensity diagrams of different modified electrodes in 0.1 M PBS (pH 7.0) containing 8 mM K2S2O8 in the voltage range from 0 to -1.2 V at a scan rate of 200 mV / s (A) and the continuous scanning of Au NPs / g-C3N4 / PDDA (B).

[0052] Figure 2 The time-electrochemiluminescence intensity diagram (A) of different modified electrodes scanned in 0.1 M PBS (pH 7.0) containing 8 mM K2S2O8 in the voltage range from 0 to -1.2 V at a scan rate of 200 mV / s, the UV spectrum of AuPt@ZIF-67 / pDNA (b) and the ECL spectrum of Au NPs / g-C3N4 / PDDA (a).

[0053] Figure 3 Cyclic voltammetry (A) and impedance characterization (B) of different modified electrodes in 5 mM K3[Fe(CN)6] / K4[Fe(CN)6] solution with a voltage range of -0.2 to 0.7 V and a scan rate of 100 mV / s.

[0054] Figure 4Figure 2 is the result of detecting different concentrations of HER2-ECD by the sensor of the present invention, wherein Figure (A) is a time-electrochemiluminescence intensity graph of the sensor scanning 0.0001, 0.001, 0.01, 0.1, 1, 10 and 100 ng / mL HER2-ECD in 0.1 M PBS (pH 7.0) containing 8 mM K2S2O8; Figure (B) is a calibration curve of the electrochemiluminescence intensity of the sensor and the logarithmic value of different concentrations of HER2-ECD.

[0055] Figure 5 is the sensor stability test result, which is the time-electrochemiluminescence intensity graph obtained after the sensor incubated with 1 ng / mL HER2-ECD was scanned continuously for 12 cycles;

[0056] Figure 6 The figure shows the reproducible results of the sensors obtained by incubating 10 ng / mL HER2-ECD with five different glassy carbon electrodes at the same time and scanning under the same conditions.

[0057] Figure 7 It is the specific detection graph of HER2-ECD aptamer sensor, in which the interferents are neuroenolase (NSE, 10 ng / mL), human epididymis protein 4 (HE4, 10 ng / mL), prostate specific antigen (PSA, 10 ng / mL) carcinoembryonic antigen (CEA, 10 ng / mL), human serum albumin (HAS, 10 ng / mL) and soluble fragment of cytokeratin 19 (CYFRA21-1, 10 ng / mL). DETAILED DESCRIPTION

[0058] The present invention will be further described below in conjunction with embodiments, but the present invention is not limited thereto.

[0059] The main chemical reagents used in the embodiments of the present invention are as follows:

[0060] The human epidermal growth factor receptor 2 ligand binding domain (HER2-ECD) ELISA kit was purchased from Jiangsu Jingmei Biotechnology Co., Ltd.; polydiallyldimethylammonium chloride (PDDA, 30%) was purchased from Solebol (Beijing, China); graphite phase carbon nitride (g-C3N4) was purchased from Nanjing Pioneer Nano Co., Ltd. (Nanjing, China); cobalt nitrate hexahydrate and 2-methylimidazole were purchased from Aladdin Biochemical Technology Co., Ltd. (Shanghai, China); chloroauric acid (HAuCl4) and potassium chloroplatinate (K2PtCl6) were purchased from Sigma (USA); 6-mercaptohexanol (MCH) and potassium persulfate (K2S2O8) were purchased from J&K Scientific Ltd (Beijing, China);

[0061] The aptamer involved was synthesized by Shanghai Bioengineering Co., Ltd. The specific sequence is as follows:

[0062] Sequence of HER2-ECD binding aptamer chain (APT): 5'-TCCACCTTTCCGTCTAACTCCCCACTTTAT-3'

[0063] The sequence of the complementary strand 1 (cDNA) to the APT part: 5'-NH2-(CH2)6-ATAAAGTGGGGAGTT-3'

[0064] Sequence of the complementary strand 2 (pDNA) to the APT part: 5'- AGACGGAAAGGTGGA -(CH2)6-NH2-3'

[0065] Equipment and technical parameters used:

[0066] Instrumentation: Time / voltage-electrochemiluminescence intensity was measured using an MPI-EⅡ electrochemiluminescence workstation (Xi'an, China). Cyclic voltammetry (CV) and impedance spectroscopy (EIS) were performed using a Metrohm Autolab BV electrochemical workstation (Modular Instruments, Switzerland). A three-electrode system was used for electrochemiluminescence detection: a modified glassy carbon electrode (4 mm in diameter) was used as the working electrode, a platinum wire was used as the counter electrode, and silver-silver chloride (saturated KCl) was used as the reference electrode. A three-electrode system was used for electrochemical detection: a modified glassy carbon electrode (4 mm in diameter) was used as the working electrode, a platinum wire was used as the counter electrode, and a saturated calomel electrode (SCE) was used as the reference electrode. The pH value was monitored by a pH meter (S210 SevenCompact, Mettler-Toledo, Shanghai, China). The UV-visible absorption spectrum was measured using a UV spectrophotometer (UV-2600, Shimadzu, Japan). The electrochemiluminescence three-electrode system was scanned at 200 mV / s in 0.1 M PBS (pH 7.0) containing 8 mM K2S2O8. The electrochemical three-electrode system was scanned at 100 mV / s in 5 mM K3[Fe(CN)6] / K4[Fe(CN)6] solution.

[0067] Example 1

[0068] The electrochemiluminescence resonance energy transfer aptamer sensor for breast cancer HER2-ECD detection is constructed, including the following steps:

[0069] (1) Preparation of substrate materials;

[0070] 1) Au NPs: Add 1 ml (1%) HAuCl4 to 100 ml ultrapure water and heat to boiling, then quickly add 2.5 ml (1%) trisodium citrate to the boiling solution; when the color of the solution changes from blue to red, keep the mixture boiling for 15 minutes, cool to room temperature, add ultrapure water to make up to 100 ml, and obtain colloidal gold nanoparticles (Au NPs);

[0071] 2) g-C3N4 / PDDA dispersion: Weigh 2 mg of g-C3N4, dissolve it in 2 ml of ultrapure water, disperse it evenly by ultrasonication, add 600 ml of PDDA solution (5 wt%), stir it magnetically for 12 h, centrifuge and wash it, and then disperse the precipitate in 2 ml of ultrapure water to obtain a uniformly dispersed g-C3N4 / PDDA dispersion;

[0072] 3) Au NPs / g-C3N4 / PDDA dispersion: Take 2 ml of the g-C3N4 / PDDA dispersion prepared in step 2), add 600 ul of Au NPs prepared in step 1), stir for 6 h, centrifuge and wash, and then disperse the precipitate in 1 ml of ultrapure water to obtain a uniformly dispersed Au NPs / g-C3N4 / PDDA dispersion.

[0073] (2) Preparation of signal quenching materials;

[0074] 1) AuPt@ZIF-67:

[0075] First, cobalt nitrate hexahydrate (Co(NO3)2·6H2O, 0.582 g) and 2-methylimidazole (C4H6N2, 0.656 g) were weighed and dissolved in 25 mL of anhydrous methanol. Under the action of ultrasound, the C4H6N2 solution was slowly poured into the Co(NO3)2·6H2O solution, and then the ultrasonic reaction was continued for 1 h. 2 mL K2PtCl6 (0.3 wt%) and 3 mL HAuCl4 (0.2 wt%) were added to the mixed solution, and the reaction was stirred for 3 hours. Finally, it was washed with anhydrous methanol for 3 times, centrifuged, and dried in vacuum at 80 °C to obtain the AuPt@ZIF-67 nanocomposite.

[0076] 2) AuPt@ZIF-67 / pDNA: Weigh 2 mg of the AuPt@ZIF-67 powder obtained in step 1), dissolve it in 1 ml of ultrapure water, disperse it evenly by ultrasonication, add 200 μL of the partial complementary chain 2 (pDNA) of the 2 μM HER-ECD binding aptamer, stir it in an ice bath for 12 h, centrifuge it, wash it with water, and redisperse the precipitate in 1 ml of ultrapure water. Add 200 μL of 1 mM 6-mercaptohexanol (MCH) solution, stir it for 1 h, centrifuge it, wash it with water, and redisperse the precipitate in 500 μL of ultrapure water to obtain the AuPt@ZIF-67 / pDNA substrate signal quenching solution.

[0077] (3) Construction of electrochemiluminescence resonance energy transfer aptamer sensor for HER2-ECD detection:

[0078] 1) Treat the HER2-ECD binding aptamer chain (APT), partial complementary chains 1 (cDNA) and 2 (pDNA) of APT with 20 mM Tris-HCl (pH=7.4) buffer at room temperature and store at 4°C for future use;

[0079] 2) Soak the glassy carbon electrode in piranha solution (98% H2SO4 / 30% H2O2 = 3:1, v / v) for 30 min and then rinse with ultrapure water for later use;

[0080] 3) The electrode obtained in step 2) was polished to a mirror surface with 0.3 μm and 0.05 μm Al2O3 powders, and then the electrode was ultrasonically treated in the order of ultrapure water, anhydrous ethanol, and ultrapure water, and dried for later use;

[0081] 4) The electrode obtained in step 3) was electrochemically activated in 0.5 M H2SO4, then rinsed with ultrapure water and dried;

[0082] 5) 10 μL of the substrate material Au NPs / g-C3N4 / PDDA solution was dropped onto the cleaned glassy carbon electrode surface in step 4) and dried at room temperature;

[0083] 6) Add 10 μL of cDNA prepared in step 1) to the electrode prepared in step 5) and incubate at 4°C for 12 h;

[0084] 7) Add 10 μL of 1 mM MCH solution onto the electrode obtained in step 6) and incubate at room temperature for 30 min;

[0085] 8) Add 10 μL of APT prepared in step 1) onto the electrode prepared in step 7) and incubate at room temperature for 1 h;

[0086] 9) 15 μL of the AuPt@ZIF-67 / pDNA substrate signal quenching solution was added dropwise to the electrode prepared in step 8) and incubated at room temperature for 3 h to obtain an electrochemiluminescent aptamer sensor for HER2-ECD detection.

[0087] Example 2

[0088] Detection of HER2-ECD using electrochemiluminescence resonance energy transfer aptamer sensor

[0089] The electrochemiluminescence resonance energy transfer aptamer sensor constructed in Example 1 was used to detect HER2-ECD in the following steps:

[0090] 1. Draw the working curve

[0091] 1) The modified electrodes from step 5) to step 9) of Example 2 were placed in 0.1 M PBS (pH=7.0) containing 8 mM K2S2O8 for characterization, and their electrochemiluminescence response signals were measured. The results are as follows: Figure 1 As shown in A: (a) Adding substrate material AuNPs / g-C3N4 / PDDA; (b) Adding cDNA; (c) Adding MCH solution for sealing; (d) Adding APT; (e) Adding signal quenching material AuPt@ZIF-67 / pDNA; (f) Adding HER2-ECD; Figure 1 B: Base material Au NPs / g-C3N4 / PDDA.

[0092] 2) Different modified electrodes were placed in 0.1 M PBS (pH=7.0) containing 8 mM K2S2O8 for characterization, and their electrochemiluminescence response signals were measured. The results are shown in Figure 2 A shows: (a) Au NPs / g-C3N4 / PDDA; (b) g-C3N4 / PDDA; (c) AuPt@ZIF-67 / g-C3N4 / PDDA. The ECL spectra of Au NPs / g-C3N4 / PDDA and the UV spectra of AuPt@ZIF-67 / pDNA were measured respectively. The results are shown in Figure 2 B shows: (a) ECL spectrum of Au NPs / g-C3N4 / PDDA; (b) UV spectrum of AuPt@ZIF-67 / pDNA.

[0093] 3) The modified electrodes from step 5) to step 9) of Example 2 were placed in 5 mM K3[Fe(CN)6] / K4[Fe(CN)6] solution for CV and EIS characterization. The current response signals were measured. The results are as follows: Figure 3As shown in A: (a) Adding substrate material Au NPs / g-C3N4 / PDDA; (b) Adding cDNA; (c) Adding MCH solution for sealing; (d) Adding APT; (e) Adding signal quenching material AuPt@ZIF-67 / pDNA; (f) Adding HER2-ECD. The impedance response signal is measured, and the results are shown in Figure 3 As shown in B: (a) Adding substrate material Au NPs / g-C3N4 / PDDA; (b) adding cDNA; (c) adding MCH solution for sealing; (d) adding APT; (e) adding signal quenching material AuPt@ZIF-67 / pDNA; (f) adding HER2-ECD.

[0094] 4) 10 μL of target HER2-ECD of different concentrations was added to the electrode of the aptamer sensor prepared in Example 1, and the luminescence intensity was measured respectively. Figure 4 As shown in A: The concentrations from bottom to top are: 0.0001, 0.001, 0.01, 0.1, 1, 10 and 100 ng / mL.

[0095] 5) Based on the linear relationship between the obtained luminescence intensity value and the logarithmic value of HER2-ECD concentration, draw a working curve (such as Figure 4 B). The results showed that the luminescence intensity response value and the logarithmic value of HER2-ECD concentration had a good linear relationship in the range of 100 fg / mL-100 ng / mL, with a linear correlation coefficient of 0.9989 and a detection limit of 17.3 fg / mL. Figure 4 shown.

[0096] 2. Sensor stability test: After the sensor prepared in Example 1 was subjected to 12 consecutive cycles of ECL measurement under optimal conditions, its luminous intensity only decreased by 5.1% (e.g. Figure 5 The sensor has good stability.

[0097] 3. Sensor reproducibility test: The sensor prepared in Example 1 by incubating the same concentration of HER-2 (10 ng / mL) with five different glassy carbon electrodes was subjected to ECL measurement (e.g. Figure 6 The relative standard deviation (RSD) was 3.6%, indicating that the sensor had good reproducibility.

[0098] 4. Sensor specificity test: In order to study the specificity of the proposed adaptive sensor, the interfering substances that may exist in serum were used: neuroenolase (NSE, 10 ng / mL), human epididymis protein 4 (HE4, 10 ng / mL), prostate specific antigen (PSA, 10 ng / mL) carcinoembryonic antigen (CEA, 10 ng / mL), human serum albumin (HAS, 10 ng / mL) and soluble fragment of cytokeratin 19 (CYFRA21-1, 10 ng / mL). The electrochemiluminescence intensity response values ​​of different interfering substances were measured at the same concentration and conditions in 0.1 M PBS (pH=7.0) containing 8 mM K2S2O8. The results showed that (e.g. Figure 7 As shown in the figure), the proposed aptamer sensor based on the high specific reaction of HER2-ECD~HBA has good specificity.

Claims

1. An electrochemiluminescence energy resonance transfer aptamer sensor for detecting the extracellular domain of human epidermal growth factor receptor 2 HER2-ECD in breast cancer, characterized in that: The construction method of the electrochemiluminescence energy resonance transfer aptamer sensor for HER2-ECD detection is as follows: the substrate material Au NPs / g-C3N4 / PDDA dispersion is added dropwise to the clean glassy carbon electrode surface, and then the complementary chain 1cDNA of the aptamer chain APT that binds to HER2-ECD is added dropwise to the electrode surface after drying at room temperature, and incubated at 4-5°C for 12-13h; then the 6-mercaptohexanol MCH solution is added dropwise to the electrode, and incubated at room temperature for 30-35min; then APT is added dropwise to the electrode, and incubated at room temperature for 1-2h; finally, the AuPt@ZIF-67 / pDNA substrate signal quenching solution is added dropwise to the electrode, and incubated at room temperature for 3-4h, thereby obtaining the electrochemiluminescence aptamer sensor for HER2-ECD detection; The preparation method of the substrate material Au NPs / g-C3N4 / PDDA dispersion is as follows: adding the g-C3N4 / PDDA dispersion into the colloidal gold nanoparticle Au NPs solution, stirring for 6-7 hours, centrifuging, washing, and then dispersing the precipitate in ultrapure water to obtain a uniformly dispersed AuNPs / g-C3N4 / PDDA dispersion; The g-C3N4 / PDDA dispersion: g-C3N4 is dissolved in ultrapure water, uniformly dispersed by ultrasonication, PDDA solution is added, magnetic stirring is performed for 12-13 hours, centrifuged, washed, and the precipitate is dispersed in ultrapure water to obtain a uniformly dispersed g-C3N4 / PDDA dispersion; The preparation method of the AuPt@ZIF-67 / pDNA substrate signal quenching solution is as follows: weighing AuPt@ZIF-67 nanocomposite powder, dissolving it in ultrapure water, uniformly dispersing it by ultrasonication, adding 2pDNA, a partial complementary chain of the HER2-ECD binding aptamer, stirring it in an ice bath for 12-13 hours, centrifuging it, washing it with water, re-dispersing the precipitate in ultrapure water, adding MCH solution, stirring it for 1-2 hours, centrifuging it, washing it with water, and re-dispersing the precipitate in ultrapure water to obtain the AuPt@ZIF-67 / pDNA substrate signal quenching solution; The preparation method of the AuPt@ZIF-67 nanocomposite is as follows: cobalt nitrate hexahydrate and 2-methylimidazole are weighed and dissolved in anhydrous methanol, and C4H6N2 solution is slowly added under the action of ultrasound, and the ultrasonic reaction is continued for 1-2 hours; then K2PtCl6 and HAuCl4 are added to the mixed solution, and the reaction is stirred for 3-4 hours; Finally, the product was separated, washed with anhydrous methanol, centrifuged, and dried under vacuum at 80-85 °C to obtain the AuPt@ZIF-67 nanocomposite; The sequence of the HER2-ECD binding aptamer chain APT: 5′-TCCACCTTTCCGTCTAACTCCCCACTTTAT-3′; The sequence of the cDNA complementary to the APT part 1: 5'-NH2-(CH2)6-ATAAAGTGGGGAGTT-3'; The sequence of the 2pDNA chain complementary to the APT part is: 5'-AGACGGAAAGGTGGA-(CH2)6-NH2-3'.

2. The body-fitting sensor according to claim 1, wherein: The preparation method of the colloidal gold nanoparticle Au NPs solution is as follows: 1% HAuCl4 is added to ultrapure water, heated to boiling, and then 1% trisodium citrate is added to the boiling solution. When the color of the solution changes from blue to red, the mixture is kept boiling for 15-20 minutes, cooled to room temperature, and ultrapure water is added to disperse to obtain the colloidal gold nanoparticle AuNPs solution.

3. The body-fitting sensor according to claim 1, wherein: The preparation method of the AuPt@ZIF-67 / pDNA substrate signal quenching solution comprises the following steps; 1) AuPt@ZIF-67: First, cobalt nitrate hexahydrate and 2-methylimidazole were weighed and dissolved in anhydrous methanol; under the action of ultrasound, the 2-methylimidazole solution was slowly poured into the cobalt nitrate hexahydrate solution, and then the ultrasonic reaction was continued for 1 hour; K2PtCl6 and HAuCl4 were added to the mixed solution, and the reaction was stirred for 3 hours; finally, the mixture was washed with anhydrous methanol for 3 times, centrifuged, and vacuum dried at 80°C to obtain the AuPt@ZIF-67 nanocomposite; 2) AuPt@ZIF-67 / pDNA: Weigh the AuPt@ZIF-67 powder obtained in step 1), dissolve it in ultrapure water, disperse it evenly by ultrasonication, add the partially complementary chain 2pDNA of the HER2-ECD binding aptamer, stir it in an ice bath for 12 h, centrifuge it, wash it with water, and redisperse the precipitate in ultrapure water. Add 1 mM MCH solution, stir it for 1 h, centrifuge it, wash it with water, and redisperse the precipitate in ultrapure water to obtain the AuPt@ZIF-67 / pDNA substrate signal quenching solution.

4. An electrochemiluminescence resonance energy transfer aptamer sensor for detecting HER2-ECD in breast cancer, characterized in that: The method for constructing an electrochemiluminescence resonance energy transfer aptamer sensor for detecting HER2-ECD in breast cancer comprises the following steps: (1) Preparation of substrate materials; 1) Au NPs: Add 1 ml of 1% HAuCl4 to 100 ml of ultrapure water and heat to boiling, then quickly add 2.5 ml of 1% trisodium citrate to the boiling solution; when the color of the solution changes from blue to red, keep the mixture boiling for 15 minutes, cool to room temperature, add ultrapure water to make up to 100 ml, and obtain colloidal gold nanoparticles Au NPs; 2) g-C3N4 / PDDA dispersion: Weigh 2 mg of g-C3N4, dissolve in 2 ml of ultrapure water, disperse evenly by ultrasonication, add 600 ml of 5 wt% PDDA solution, stir magnetically for 12 h, centrifuge, wash, and then disperse the precipitate in 2 ml of ultrapure water to obtain a uniformly dispersed g-C3N4 / PDDA dispersion; 3) Au NPs / g-C3N4 / PDDA dispersion: Take 2 ml of the g-C3N4 / PDDA dispersion prepared in step 2), add 600 ul of Au NPs prepared in step 1), stir for 6 h, centrifuge, wash, and then disperse the precipitate in 1 ml of ultrapure water to obtain a uniformly dispersed AuNPs / g-C3N4 / PDDA dispersion; (2) Preparation of signal quenching materials; 1) AuPt@ZIF-67: First, 0.582 g of cobalt nitrate hexahydrate and 0.656 g of 2-methylimidazole were weighed and dissolved in 25 mL of anhydrous methanol respectively; under the action of ultrasound, the 2-methylimidazole solution was slowly poured into the cobalt nitrate hexahydrate solution, and then the ultrasonic reaction was continued for 1 hour; secondly, 2 mL of K2PtCl6 with a concentration of 0.3 wt% and 3 mL of HAuCl4 with a concentration of 0.2 wt% were added to the mixed solution, and the reaction was stirred for 3 hours; finally, the mixture was washed with anhydrous methanol for 3 times, centrifuged, and dried in vacuum at 80°C to obtain the AuPt@ZIF-67 nanocomposite; 2) AuPt@ZIF-67 / pDNA: Weigh 2 mg of the AuPt@ZIF-67 powder obtained in step 1), dissolve it in 1 ml of ultrapure water, disperse it evenly by ultrasonication, add 200 μL of 2 μM HER2-ECD binding aptamer partial complementary chain 2pDNA, stir it in an ice bath for 12 h, centrifuge it, wash it with water, and redisperse the precipitate in 1 ml of ultrapure water, add 200 μL of 1 mM MCH solution, stir it for 1 h, centrifuge it, wash it with water, and redisperse the precipitate in 500 μL of ultrapure water to obtain the AuPt@ZIF-67 / pDNA substrate signal quenching solution; (3) Construction of electrochemiluminescence resonance energy transfer aptamer sensor for HER2-ECD detection: 1) treating the HER2-ECD binding aptamer APT, the partial complementary chain 1 cDNA of APT and the partial complementary chain 2 pDNA of APT with 20 mM Tris-HCl buffer at pH 7.4 at room temperature and storing at 4° C. for future use; 2) Soak the glassy carbon electrode in piranha wash solution (98% H2SO4 / 30% H2O2=3:1 by volume) for 30 minutes and then rinse with ultrapure water for later use; 3) The electrode obtained in step 2) was polished to a mirror surface with 0.3 μm and 0.05 μm Al2O3 powders, and then the electrode was ultrasonically treated in the order of ultrapure water, anhydrous ethanol, and ultrapure water, and dried for later use; 4) electrochemically activating the electrode obtained in step 3) in 0.5M H2SO4, then rinsing with ultrapure water and drying; 5) dropping 10 μL of the substrate material Au NPs / g-C3N4 / PDDA solution onto the surface of the cleaned glassy carbon electrode in step 4) and drying at room temperature; 6) 10 μL of the cDNA prepared in step 1) was added dropwise to the electrode prepared in step 5) and incubated at 4° C. for 12 h; 7) Add 10 μL of 1 mM MCH solution dropwise onto the electrode obtained in step 6) and incubate at room temperature for 30 min; 8) 10 μL of the APT prepared in step 1) was dripped onto the electrode prepared in step 7) and incubated at room temperature for 1 h; 9) 15 μL of the AuPt@ZIF-67 / pDNA substrate signal quenching solution was added dropwise to the electrode prepared in step 8) and incubated at room temperature for 3 h to obtain an electrochemiluminescent aptamer sensor for HER2-ECD detection.

5. A method for detecting HER2-ECD using an electrochemiluminescence resonance energy transfer aptamer sensor, characterized in that: The steps include: 1) adding different concentrations of the target human epidermal growth factor receptor 2 extracellular domain HER2-ECD to the electrode of the sensor according to any one of claims 1 to 4; 2) The electrode was placed in a 0.1M PBS solution containing 8 mM K2S2O8 at pH = 7.0 for characterization, and its electrochemiluminescence intensity value was measured; 3) drawing a working curve based on the linear relationship between the luminescence intensity obtained in step 2) and the logarithmic value of the HER2-ECD concentration; 4) The sample to be tested is detected by the sensor, and the obtained current value is calculated through the working curve prepared in step 3) to obtain the HER2-ECD concentration of the sample to be tested.

Images