Electrochemical Biosensor for Detecting EGFRT790M in Non-Small Cell Lung Cancer and Preparation Method

Abstract

The present invention discloses an electrochemical biosensor for detecting EGFRT790M in non-small cell lung cancer constructed by using luminescent material CdSe@ZnS quantum dots, capture probe Au-T790M-30-HairpinDNA, and resonance energy transfer mechanism. The electrochemical biosensor prepared by the present invention has strong electrochemical signals, excellent stability, good reproducibility and accuracy, excellent selective recognition ability for targets, and high detection sensitivity.

Description

Technical Field

[0001] The present invention relates to the technical field of electrochemical sensors, and particularly relates to an electrochemical biosensor for detecting EGFRT790M in non-small cell lung cancer and a preparation method thereof. Background Art

[0002] Lung cancer has become the leading cause of cancer death globally due to its high malignancy and lack of effective treatment methods. According to the survey by the International Agency for Research on Cancer (IARC) under the World Health Organization, in 2020, there were 19.3 million new cancer cases and nearly 10 million cancer deaths globally. Among them, the number of lung cancer deaths was about 1.8 million, ranking first. Due to its insidious onset, the vast majority of lung cancer patients are already in the advanced stage of lung cancer when they first seek medical treatment, and the overall survival situation of patients is not optimistic.

[0003] Lung cancer is divided into small cell carcinoma and non-small cell lung cancer, of which about 80-85% is non-small cell lung cancer, with high malignancy and extremely poor prognosis. Research has shown that nucleic acids are closely related to the occurrence of cancer, and there are mutations in specific DNA base sequences or abnormal expressions of microRNA genes in a large number of cancer tissues. They are caused by the combined action of self-factors and the external environment, but ultimately, almost all cancers are caused by gene mutations. On the other hand, with the in-depth study, not only more diseases have been found to be related to genes, but some of these diseases can be cured through gene therapy. Therefore, the early detection of diseases is crucial. For most diseases, early detection and early treatment can better save the lives of patients.

[0004] The epidermal growth factor receptor (EGFR) is related to regulating cell proliferation, survival, growth, and differentiation. However, if there is overexpression of EGFR, it will be directly related to human cancers. Previous studies have shown that more than 60% of non-small cell lung cancer patients have high expression of the epidermal growth factor receptor and a large number of mutation sites.

[0005] As the treatment progresses, some tumor cells evade the inhibition of drugs through adaptive mutations, generating drug-resistant mutations. Among them, the T790M mutation is the most common drug-resistant mutation in the EGFR gene mutation type of non-small cell lung cancer patients. It usually occurs on the basis of initial mutation sites such as EGFR exon 19 deletion or L858R mutation. By changing the EGFR kinase structure, especially the structure of the ATP-binding site, the binding ability of conventional EGFR inhibitors is greatly reduced, resulting in a weakened therapeutic effect. Circulating tumor DNA (ctDNA) is a linear double-stranded nucleic acid fragment released into the blood circulation during apoptosis or necrosis of tumor cells, usually between 90 and 320 nucleotides (nt). These DNA molecules carry cancer-specific genetic information such as the T790M mutation in EGFR and can distinguish different types of cancer cells. Therefore, rapid and effective detection of T790M mutant DNA is crucial for the early diagnosis and precise treatment of lung cancer patients.

[0006] An electrochemical biosensor is an analytical method combining electrochemistry and molecular biology and has been widely applied in fields such as food and drug inspection, environmental detection, and clinical analysis. In the existing technology, there are problems of poor stability, weak electrochemical signals, and low sensitivity in detecting EGFR-T790M using electrochemical biosensors. Summary of the Invention

[0007] Therefore, the purpose of the present invention is to provide an electrochemical biosensor and a preparation method for detecting EGFRT790M in non-small cell lung cancer, which have high-intensity electrochemical signals, high detection sensitivity, and good stability.

[0008] The above object of the present invention is achieved by the following technical solutions:

[0009] The first aspect of the present invention is to provide a preparation method for an electrochemical biosensor for detecting EGFRT790M in non-small cell lung cancer, comprising the following steps:

[0010] S1. First, drop the luminescent CdSe@ZnS quantum dot solution onto the surface of a glassy carbon electrode, dry it under an infrared lamp, and then drop the PDDA solution and dry it under an infrared lamp to obtain a glassy carbon modified electrode of PDDA / CdSe@ZnS;

[0011] S2. Incubate the hairpin DNA solution and the tris(2-carboxyethyl)phosphine hydrochloride TCEP solution together for 1 to 3 h, and then add the gold nanoparticle solution and incubate for 3 to 12 h to obtain a capture probe solution;

[0012] S3. Drop the capture probe solution onto the surface of the glassy carbon modified electrode of PDDA / CdSe@ZnS, rinse it after incubation for 1 - 12 h under constant temperature conditions to obtain a glassy carbon modified electrode of capture probe / PDDA / CdSe@ZnS, which is the electrochemical biosensor.

[0013] In an optional implementation manner, in step S1, the preparation method of CdSe@ZnS quantum dots is as follows: Add 3-mercaptopropionic acid (MPA) and zinc chloride into ultrapure water, and bubble nitrogen for 30 - 60 min; then add the TGA-CdSe quantum dot solution, heat and react the mixed solution for 2 - 4 h to obtain a CdSe@ZnS quantum dot solution.

[0014] In an optional implementation manner, the preparation method of the TGA-CdSe quantum dot solution is as follows: Mix cadmium chloride and thioglycolic acid (TGA) evenly, adjust the pH to 9 - 11, bubble nitrogen for 30 - 60 min, then inject sodium selenite solution and reducing agent hydrazine hydrate into the mixture, heat and reflux, naturally cool to room temperature after the reaction ends, and dialyze and purify for 24 - 48 h to obtain a TGA-CdSe quantum dot solution.

[0015] In an optional implementation manner, the hairpin DNA consists of 30 bp or 48 bp.

[0016] In an optional implementation manner, the base sequence of the 30 bp hairpin DNA is as follows: T790M-30-Hairpin-DNA: 5'-GGA AGA CAT GAG CTG CAT GAT GAG TCT TCC-(CH2)6-HS-SH-3'.

[0017] In an optional implementation manner, the base sequence of the 48 bp hairpin DNA is as follows: T790M-48-Hairpin-DNA: 5'-GGA AGA GCC GAA GGG CAT GAG CTG CAT GAT GAG CTG CAC GGT TCT TCC-(CH2)6-HS-SH-3'.

[0018] In an optional implementation manner, in step S2, after adding the gold nanoparticle solution and incubating, then add 6-mercaptohexanol (MCH) and continue to incubate for 1 - 12 h to obtain the capture probe solution.

[0019] The second aspect of the present invention is to provide an electrochemical biosensor for detecting EGFRT790M in non-small cell lung cancer.

[0020] The third aspect of the present invention is to provide a method for detecting EGFR T790M in non-small cell lung cancer using an electrochemical biosensor for detecting EGFR T790M in non-small cell lung cancer, comprising the following steps:

[0021] (1) Drop different concentrations of target T100-T790M DNA onto the electrode of the electrochemical biosensor and incubate for 1.0 - 3.0 h;

[0022] (2) Place the sensor in a 0.01M PBS buffer solution containing 0.1M potassium persulfate and measure its electrochemiluminescence intensity;

[0023] (3) Draw a working curve according to the linear relationship between the electrochemiluminescence intensity obtained in step (2) and the logarithm of the concentration of target T100-T790M DNA;

[0024] (4) Detect the sample to be measured using the said sensor, and calculate the concentration of T100-T790M DNA in the sample to be measured through the working curve prepared in step (3).

[0025] In an optional embodiment, the measurement conditions in step (2) are as follows: a standard three-electrode system is adopted, the voltage setting range is 0 to -1.3V, the scanning rate is 100mV / s, and the photomultiplier tube PMT is 900V.

[0026] Compared with the prior art, the technical solution of the present invention has the following advantages:

[0027] In the present invention, a luminescent body CdSe@ZnS is prepared by coating a ZnS shell layer on the outer layer of TGA-CdSe quantum dots, so that the sensor can still maintain a strong luminescence intensity at a low voltage. The sensor constructed by the luminescent body CdSe@ZnS, PDDA, gold nanoparticles AuNPs and hairpin DNA has strong electrochemical signals, excellent stability, good reproducibility and accuracy, excellent selective recognition ability for the target, and high detection sensitivity. Description of the Drawings

[0028] Figure 1 It is the TEM image of MPA-ZnS quantum dots in the embodiment of the present invention;

[0029] Figure 2 It is the UV-visible and fluorescence spectra of MPA-ZnS quantum dots in the embodiment of the present invention;

[0030] Figure 3 It is the TEM image of CdSe@ZnS quantum dots in the embodiment of the present invention;

[0031] Figure 4It is the ultraviolet-visible spectrogram of AuNPs in the embodiment of the present invention;

[0032] Figure 5 It is the working curve graph of the electrochemical biosensor constructed based on hairpin DNA with different lengths and MPA-ZnS quantum dots;

[0033] Figure 6 It is the Nyquist spectrogram of the electrochemical biosensor constructed in the embodiment of the present invention at each stage;

[0034] Figure 7 It is the Zeta potential graph of CdSe@ZnS quantum dots, PDDA and AuNPs-T790M30Hairpin DNA in the electrochemical biosensor constructed by the present invention;

[0035] Figure 8 It is the electrochemical and electrochemiluminescence characterization graph of CdSe@ZnS quantum dots and MPA-ZnS quantum dots of the present invention;

[0036] Figure 9 It is the working curve graph of the electrochemical biosensor of the present invention;

[0037] Figure 10 It is the stability and reproducibility test graph of the electrochemical biosensor of the present invention;

[0038] Figure 11 It is the selectivity test graph of the electrochemical biosensor of the present invention. Detailed implementation manners

[0039] In order to enable those skilled in the art to better understand the solution of this application, the technical solutions in the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings in the embodiments of this application. Obviously, the described embodiments are only a part of the embodiments of this application, rather than all the embodiments. Based on the embodiments in this application, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of this application.

[0040] Raw material preparation

[0041] I. Preparation and characterization of MPA-ZnS quantum dots

[0042] (I) Preparation of MPA-ZnS quantum dots

[0043] Dissolve 0.9 g of Zn(CH3COO)2 and 52 μL of 3-mercaptopropionic acid (MPA) in 50 mL of deionized water. Adjust the pH of the mixture to 10 using NaOH solution and stir for 30 min. Then, under the protection of high-purity nitrogen, continuously stir the mixture at 100 °C for 2 h and dropwise add 25 mL of Na2S (0.2 mol / L) solution to prepare MPA-ZnS quantum dots. After the reaction is completed, precipitate the MPA-ZnS quantum dots with ethanol, centrifuge to collect the solid precipitate at 10000 rpm and wash it, and dry it in a vacuum drying oven at 60 °C for 24 h, and grind it to obtain a powder sample of MPA-ZnS quantum dots.

[0044] (II) Characterization of MPA-ZnS Quantum Dots

[0045] The morphology of the synthesized MPA-ZnS quantum dots was characterized using a transmission electron microscope, and the results are as Figure 1 shown. The prepared MPA-ZnS quantum dots exhibit a uniform elliptical structure, and the particle size distribution is relatively consistent. Although there is slight aggregation, the overall dispersibility is good, indicating that the prepared MPA-ZnS quantum dots have ideal morphology and size characteristics.

[0046] In addition, the optical properties of MPA-ZnS quantum dots were characterized using a UV-visible spectrophotometer, and the obtained absorption spectrum is as Figure 2 (left Y-axis) shown, with an obvious characteristic absorption peak at a wavelength of 303 nm.

[0047] To further explore its optical performance, the fluorescence spectrum of MPA-ZnS quantum dots was also analyzed using a fluorescence spectrophotometer. As Figure 2 (right Y-axis) shown, at an excitation wavelength of 315 nm, an obvious fluorescence emission peak appears at 403 nm, indicating that MPA-ZnS quantum dots have excellent optical properties and strong fluorescence emission ability.

[0048] II. Preparation and Characterization of CdSe@ZnS Quantum Dots

[0049] (I) Preparation of CdSe@ZnS Quantum Dots

[0050] After mixing 20 mL of 5.0 mmol / L CdCl2 with 20 μL of thioglycolic acid uniformly, 1.0 mol / L NaOH solution was added to adjust its pH to 10. Then the solution was diluted to 50 mL and bubbled with high-purity N2 for 30 min. 0.5 mL of 0.1 mol / L Na2SeO3 and 2 mL of hydrazine hydrate (80%) were quickly injected into the mixed solution to obtain a clear and transparent light yellow CdSe quantum dot solution. Then it was refluxed at 100 °C for 4 h to prepare an orange-red TGA-CdSe quantum dot solution. After the reaction, it was naturally cooled to room temperature, and the product was dialyzed and purified using a dialysis bag (molecular weight MW = 1000) for 48 h.

[0051] 4 mmol of 3-mercaptopropionic acid (MPA) and 2 mmol of ZnCl2 were successively added to 40 mL of ultrapure water, and bubbled with high-purity N2 for 30 min to obtain a mixed solution. Then the TGA-CdSe quantum dot solution was added to the mixed solution. MPA was used as the sulfur source, and the solution was heated and reacted for 1 h to obtain a CdSe@ZnS quantum dot solution. After it was naturally cooled, the product was precipitated with ethanol, centrifuged at 10000 rpm to collect the solid precipitate, and washed with ethanol to remove impurities. The obtained sample was redispersed in ultrapure water and stored in a refrigerator at 4 °C for later use.

[0052] (II)Characterization of CdSe@ZnS Quantum Dots

[0053] The morphology of CdSe@ZnS quantum dots was analyzed by high-resolution transmission electron microscopy, and the results are as Figure 3 shown. It was observed that the prepared CdSe@ZnS quantum dots presented a uniform elliptical structure, and the particle size distribution was relatively consistent. Although there was a slight aggregation phenomenon, the overall dispersion was good.

[0054] III. Preparation and Characterization of AuNPs (Gold Nanoparticles)

[0055] (I)Preparation of AuNPs

[0056] 100 mL of ultrapure water was taken and placed at 4 °C for 2 h. 3 mL of 1% HAuCl4·4H2O solution by mass concentration was added to the ice water, and stirred to make it fully mixed and uniformly dispersed. Then 1 mL of K2CO3 solution (0.2 mol / L) was added, and stirred vigorously for 10 min. Then 9 mL of NaBH4 solution (0.5 mg / mL) was quickly injected into the mixed solution, and stirred vigorously for 8 min. Finally, a wine-red AuNPs dispersion was obtained.

[0057] (II)Characterization of AuNPs

[0058] The prepared AuNPs samples were characterized using a UV-visible spectrophotometer, and the results are as follows Figure 4 As shown, the AuNPs solution exhibited a significant characteristic absorption peak at a wavelength of 520 ± 2 nm. This absorption peak is generally considered a typical manifestation of the surface plasmon resonance (SPR) phenomenon of AuNPs. This result not only indicates the successful synthesis of AuNPs but also further confirms its good optical properties.

[0059] Example 1

[0060] I. Design of target DNA

[0061] EGFR-T790M (target DNA) was designed based on the DNA sequence of the EGFR target mutation in non-small cell lung cancer cells. It consists of 100 bp and is used to analyze the detection performance of the sensor designed in this application for EGFR-T790M gene detection. It does not have singularity. The sequence of the EGFR-T790M mutation site is well-known and can be found in the GeneBank nucleotide sequence database. The nucleotide sequence of the target DNA in this scheme is as follows:

[0062] T100-T790M-dsDNA: 5'-CCA CGT GTG CCG CCT GCT GGG CAT CTG CCT CAC CTCCAC CGT GCA GCT CAT CAC GCA GCT CAT GCC CTT CGG CTG CCT CCT GGA CTA TGT CCGGGA ACAC-3'.

[0063] In view of the double-stranded structural characteristics of T100-T790M-dsDNA, heat denaturation treatment is required before detection. Specifically, the sample is heated in a 95°C metal bath for 5 min to dissociate the double-stranded DNA into single-stranded T100-T790M-DNA to ensure the effective hybridization of the probe and the target sequence during subsequent detection.

[0064] II. Design and verification of hairpin DNA

[0065] (1) Design of hairpin DNA

[0066] The hairpin DNA is designed according to the DNA sequence of EGFR-T790M. In the present invention, four hairpin DNAs with different stem-loop lengths (24bp, 30bp, 36bp, 48bp, T790M-24 / 30 / 36 / 48-Hairpin DNA) are systematically designed to explore the differences in their detection performance for T100-T790M DNA. It should be noted that the preparation process of the hairpin DNA is a conventional technique and will not be described herein. The nucleotide sequence information of the four designed hairpin DNAs with different lengths is as follows:

[0067] T790M-24-Hairpin DNA:

[0068] 5'-GGA CAT GAG CTG CAT GAT GAG TCC-(CH2)6-HS-SH-3'.

[0069] T790M-30-Hairpin DNA:

[0070] 5'-GGA AGA CAT GAG CTG CAT GAT GAG TCT TCC-(CH2)6-HS-SH-3'.

[0071] T790M-36-Hairpin DNA:

[0072] 5'-GGC TCA AGA CAT GAG CTG CAT GAT GAG TCT TGA GCC-(CH2)6-HS-SH-3'.

[0073] T790M-48-Hairpin DNA:

[0074] 5'-GGA AGA GCC GAA GGG CAT GAG CTG CAT GAT GAG CTG CAC GGT TCT TCC-(CH2)6-HS-SH-3'.

[0075] (2) Preparation of AuNPs-T790M Hairpin DNA (capture probe solution) with different lengths

[0076] 10 μL of 1 μmol / L T790M-X-Hairpin DNA solutions with different lengths (24 bp, 30 bp, 36 bp, 48 bp) synthesized by the above designs were separately added to 4 1-mL EP tubes. Subsequently, 6 μL (10 mmol / L) of tris(2-carboxyethyl)phosphine solution was added to each tube, and incubated at room temperature for 8 h to reduce disulfide bonds. Then, 100 μL of AuNPs solution was added to each tube, and incubated for 15 h to allow the hairpin DNA to bind to AuNPs through Au-S bonds. To prevent non-specific adsorption, 10 μL of 6-mercaptohexanol (MCH) was added and incubation continued for 1 h. Finally, AuNPs-T790M-24 / 30 / 36 / 48-Hairpin DNA complex solutions were obtained and stored at 4 °C in the dark for later use.

[0077] (3) Preparation of Electrochemical Biosensors Based on Hairpin DNAs with Different Lengths

[0078] After the prepared MPA-ZnS quantum dot solution was shaken and mixed evenly, 6 μL was taken and dropped onto the surface of the pretreated glassy carbon electrode, and dried at room temperature. Subsequently, 6 μL of 1% PDDA solution was dropped onto the electrode surface and dried at room temperature. Then, 6 μL of AuNPs-T790M-24 / 30 / 36 / 48-Hairpin DNA capture probe solutions were separately dropped onto the modified electrode surface and immobilized on the electrode surface through electrostatic interaction, and incubated at 37 °C for 8 h. After incubation, the electrode surface was rinsed with PBS buffer to remove unbound hairpin DNA molecules. Through the above steps, four electrochemical biosensors based on hairpin DNAs with different lengths were successfully prepared.

[0079] (4) Detection of Target T100-T790M DNA by Electrochemical Biosensors Based on MPA-ZnS Quantum Dots Assembled with Hairpin DNAs of Different Lengths

[0080] To systematically study the influence law of hairpin DNA length on the detection performance of the sensor, a series of concentrations of T100-T790M DNA (10 -17 mol / L, 10 -16 mol / L, 10 -15 mol / L, 10 -14 mol / L, 10 -13 mol / L, 10 -12 mol / L) solutions were separately dropped onto the surfaces of these four sensors and incubated in a 37 °C constant temperature drying oven for 1 h to ensure sufficient binding of the target DNA, obtaining four electrochemical biosensors to be measured.

[0081] The test was carried out using a three - electrode system of an electrochemical workstation. A 3 mol / L silver chloride - silver electrode was used as the reference electrode, a platinum sheet electrode (with a size of 1 cm×1 cm) was used as the auxiliary electrode, and the electrochemical biosensor to be tested was used as the working electrode. The electrochemical workstation and an ultra - weak luminescence detector (the voltage of the photomultiplier tube was 900 V) were connected together. In a 10 mL phosphate buffer solution with pH 7.4 containing 0.1 mol / L K2S2O8, through the electrochemiluminescence system, cyclic voltammetry was used with a scanning range from 0 to - 2.0 V and a scanning rate of 100 mV / s to obtain a photoelectric diagram. The logarithm of the target DNA concentration was used as the abscissa, and the electrochemiluminescence signal intensity was used as the ordinate for fitting to obtain a standard curve.

[0082] The results are as Figure 5 shown. It can be seen from Figure 5 that for the sensors constructed based on T790M - 24 / 30 / 36 / 48 - Hairpin DNA (the sensors constructed with 24bp, 30bp, 36bp, and 48bp hairpin DNA correspond to Figures A, B, C, and D in the figure respectively) for the detection of T100 - T790M DNA, the ECL signal intensity increased significantly with the increase in the target DNA concentration. The stem - loop structure design of the hairpin DNA significantly affected the efficiency of the resonance energy transfer system and the detection sensitivity, and the order was 30>48>24>36.

[0083] The capture probe T790M - 30 - Hairpin DNA on the sensor surface specifically binds to the target T100 - T790M DNA, and the sensor shows the best detection performance. Its detection limit is significantly better than the sensing platforms constructed with hairpin DNA of other lengths. Because of its balanced structure, the loop part is long enough to allow binding to some regions of the target T100 - T790M DNA, while the stability of the stem part is moderate. The loop of the 48bp hairpin DNA is longer, which may allow multi - site binding to the target, providing a stronger binding force to overcome the stem stability, so the effect is better than that of the 24bp hairpin. The loop part of the 24bp hairpin is too short to effectively bind to the target, resulting in a poor effect. The 36bp hairpin may have an internal secondary structure (such as in - loop pairing), which hinders binding to the target, so it shows the worst performance. Thus, the 30bp hairpin DNA was selected for subsequent research.

[0084] Example 2

[0085] I. Preparation of the electrochemical biosensor

[0086] This embodiment provides an electrochemical biosensor for detecting EGFRT790M in non-small cell lung cancer. A 30-bp hairpin DNA-modified AuNPs is used as a capture probe (AuNPs-T790M-30Hairpin DNA) to construct the electrochemical biosensor. The preparation process is as follows:

[0087] (1) Preparation of the capture probe solution

[0088] Take 10 μL of 1 μmol / L, 30-bp hairpin DNA solution (T790M-30Hairpin DNA) and add it to a 1-mL EP tube. Then add 10 μL (10 mmol / L) of tris(2-carboxyethyl)phosphine solution to the tube and incubate at room temperature for 6 h to reduce the disulfide bond. Next, add 100 μL of AuNPs solution to the tube and incubate for 15 h to allow the hairpin DNA to bind to AuNPs through Au-S bonds. To prevent non-specific adsorption, add 10 μL of 6-mercaptohexanol (MCH) and continue to incubate for 1 h. Finally, obtain the AuNPs-T790M-30Hairpin DNA complex solution, which is the capture probe solution, and store it at 4°C in the dark for later use.

[0089] (2) Preparation of the electrochemical biosensor

[0090] After oscillating and mixing the prepared CdSe@ZnS quantum dot solution, take 6 μL and drop it onto the surface of the pretreated glassy carbon electrode, and dry it under an infrared lamp. Then, drop 6 μL of 1% PDDA solution onto the electrode surface to fix and increase the ECL intensity of the luminophore, and dry it under an infrared lamp to obtain a glassy carbon modified electrode of PDDA / CdSe@ZnS. Next, drop 6 μL of the AuNPs-T790M-30Hairpin DNA complex solution onto the glassy carbon modified electrode of PDDA / CdSe@ZnS and fix it on the electrode surface through electrostatic interaction, and incubate at 37°C for 8 h. After the incubation is completed, rinse the electrode surface with PBS buffer to remove the unbound hairpin DNA molecules to obtain a glassy carbon modified electrode of AuNPs-T790M-30Hairpin DNA / PDDA / CdSe@ZnS, which is the electrochemical biosensor.

[0091] II. Characterization of the electrochemical biosensor

[0092] The electrochemical impedance spectroscopy (EIS) technique was used to systematically characterize the assembly process of the sensor. During the test, 5 mmol / L [Fe(CN)6]3− / 4− (a mixture of [Fe(CN)6]3− and [Fe(CN)6]4−) was used as the redox probe, and the Nyquist plots were recorded in the frequency range from 100 kHz to 0.01 Hz. By analyzing the change in the charge transfer resistance (Rct) corresponding to the semicircle diameter in the high-frequency region, the step-by-step construction process of the sensor was verified.

[0093] The results are as Figure 6 shown. The bare glassy carbon electrode (bare GCE in the figure) exhibited a large Rct value. After the glassy carbon electrode was modified with CdSe@ZnS quantum dots (CdSe@ZnS QD / GCE in the figure), since the quantum dots are semiconductor materials and the quantum dots and [Fe(CN)6]3− / 4− carry the same negative charge, resulting in a slower electron transfer rate, the Rct increased significantly. After introducing PDDA, the Rct value decreased. This significant change in the interfacial properties was mainly due to the electrostatic interaction between the positively charged quaternary ammonium salt groups in the PDDA molecular chain and [Fe(CN)6]3− / 4−, thus significantly improving the electron transfer efficiency.

[0094] When the negatively charged AuNPs-T790M-30 Hairpin DNA and the positively charged PDDA were immobilized on the electrode surface through electrostatic interaction, due to the excellent electrical conductivity of gold nanoparticles, the Rct further decreased. However, when the target DNA (tDNA in the figure) specifically bound to the hairpin DNA, the loop structure of the hairpin DNA was destroyed, and the hairpin structure changed from the closed state to the open state, resulting in an increase in the distance between the AuNPs and the electrode surface and an increase in the amount of negatively charged primers on the electrode surface, thus causing the Rct value to rise. Through the EIS test results at different modification stages, the step-by-step construction process of the sensor interface could be clearly observed, indicating that the sensor was successfully prepared.

[0095] III. Stability test of the electrochemical biosensor

[0096] To further verify the stability of the electrochemical biosensor constructed by the layer-by-layer assembly technique, the Zeta potential tests of CdSe@ZnS quantum dots, PDDA, and AuNPs-T790M-30 Hairpin DNA were carried out.

[0097] The results are as Figure 7 shown. Figure 7The Zeta potential of CdSe@ZnS quantum dots is shown as -20.0 mV, that of PDDA is +33.5 mV, and that of AuNPs-T790M-30 Hairpin DNA is -21.2 mV. It can be seen that the effective assembly of the sensor is achieved through electrostatic interaction between different layers of substances, forming a stable composite structure, further proving that the prepared sensor has good stability.

[0098] IV. Analysis of the Electrochemiluminescence Resonance Energy Transfer Mechanism of Electrochemical Biosensors

[0099] To deeply study the interaction mechanism between AuNPs and CdSe@ZnS quantum dots, fluorescence spectroscopy technology was used to study the luminescence behavior of CdSe@ZnS quantum dots and their composite system with AuNPs.

[0100] The results are as Figure 8 shown. As can be seen from Figure 8 Figure A in it, the introduction of AuNPs significantly reduces the luminescence intensity of CdSe@ZnS quantum dots, indicating that there is an effective energy transfer process between the two. This phenomenon can be attributed to the action of the fluorescence resonance energy transfer (FRET) mechanism, in which CdSe@ZnS quantum dots act as energy donors and AuNPs act as energy acceptors. Since the FRET efficiency is significantly affected by the spectral overlap degree and spatial distance between the donor and acceptor, in this study, the specific base pairing of 30bp hairpin DNA with the target was used to precisely regulate the nanoscale distance between AuNPs and CdSe@ZnS quantum dots through DNA conformational changes, thereby realizing the regulation of electrochemiluminescence signals.

[0101] Meanwhile, the electrochemical and electrochemiluminescence characterizations of CdSe@ZnS quantum dots and MPA-ZnS quantum dots were carried out using this co-reaction system. As shown in Figure 8 Figure B and Figure C in it, in Figure 8 Figure B, when the CdSe@ZnS quantum dot modified electrode was tested in PBS without K2S2O8, no obvious ECL signal was observed. However, after adding K2S2O8, the CdSe@ZnS quantum dot modified electrode emitted a strong and stable ECL signal, confirming that K2S2O8 acts as a co-reactant to undergo a synergistic redox reaction with the quantum dots. The comparative experiment shows that the bare glassy carbon electrode only produces a weak background signal in PBS containing K2S2O8, further verifying that the reduction product of CdSe@ZnS quantum dots can react with K2S2O8. Similarly, it was also verified from Figure 8 Figure C in it that the reduction product of MPA-ZnS quantum dots can react with K2S2O8. The principle is the same as above and will not be repeated here.

[0102] FromFigure 8 As can be seen from Figures B and C in Figure 8 , the luminescence intensity of CdSe@ZnS quantum dots is enhanced compared with that of MPA-ZnS quantum dots. Especially at low excitation voltages, the luminescence intensity of CdSe@ZnS quantum dots is above 1750 k a.u. at a voltage of about -1.2 V, while the luminescence intensity of MPA-ZnS quantum dots is only about 13000 a.u. at a voltage of -2.0 V. This is because the introduction of CdSe improves the luminescence efficiency of the quantum dots. The CdSe core has strong energy absorption ability, and its relatively large size and small bandgap contribute to enhancing the efficiency of energy absorption and release, reducing the influence of surface defects on the luminescence efficiency, and thus increasing the luminescence intensity. Therefore, in this application, CdSe@ZnS quantum dots are selected as the luminescent material for constructing the electrochemiluminescence sensor.

[0103] Example 3

[0104] I. Detection of Non-Small Cell Lung Cancer EGFR-T790M

[0105] The electrochemiluminescent biosensor prepared in Example 1 was used to detect non-small cell lung cancer EGFR-T790M, which specifically included the following steps:

[0106] S1. Dropwise add standard solutions of the target T100-T790M DNA with concentrations of 10 -17 mol / L, 10 -16 mol / L, 10 -15 mol / L, 10 - 14 mol / L, 10 -13 mol / L, 10 -12 mol / L onto the electrode of the electrochemiluminescent biosensor, incubate for 2 h to obtain the electrochemiluminescent biosensor to be tested;

[0107] S2. Use the three-electrode system of the electrochemical workstation for testing. Use a 3 mol / L silver-silver chloride electrode as the reference electrode, a platinum sheet electrode (with a size of 1 cm × 1 cm) as the auxiliary electrode, and the electrochemiluminescent biosensor to be tested as the working electrode. Connect the electrochemical workstation and the ultra-weak luminescence detector (photomultiplier tube voltage 900 V) together. In a 10 mL phosphate buffer solution with a pH of 7.4 and a concentration of 0.1 mol / L K2S2O8, through the electrochemiluminescence system, use cyclic voltammetry with a scanning range of 0 to -2.0 V and a scanning rate of 100 mV / s to obtain a photoelectric diagram. Use the logarithm of the target DNA concentration (the logarithm of the concentration of T100-T790M DNA) as the abscissa and the electrochemiluminescence signal intensity as the ordinate for fitting to obtain a standard curve.

[0108] The results are as Figure 9As shown, it can be seen that as the target concentration increases, the ECL signal intensity shows a significant upward trend. In the concentration range of 10 aM (amol / L) to 1 pM (pmol / L) (10 -17 mol / L~10 -12 mol / L), there is a good linear correlation between the ECL intensity and the logarithm of the target DNA concentration (logc). The standard curve regression equation is: Y = 122637logc + 2490370, and the correlation coefficient R² reaches 0.9927. The lowest detection limit is 3.2 aM, indicating its excellent detection sensitivity.

[0109] Test Example

[0110] I. Stability and reproducibility tests of the electrochemical biosensor during detection

[0111] In a PBS buffer system containing K2S2O8, when detecting a target with a concentration of 100 fM (fmol / L) by cyclic voltammetry (the same as above), the electrochemical luminescence response curves of CdSe@ZnS quantum dots for 7 consecutive cycles were recorded.

[0112] The results are as shown in Figure A of Figure 10 . As can be seen from Figure A of Figure 10 , the cathodic ECL signal intensity of the quantum dots shows good reproducibility, and its relative standard deviation (RSD) is 1.8%, indicating that the constructed sensor has excellent stability.

[0113] To verify the reproducibility of the sensor, 6 biosensors were prepared according to Example 1 under the same experimental conditions, and the 6 prepared biosensors were incubated with a target with a concentration of 100 fM. The results are as shown in Figure B of Figure 10 . As can be seen from Figure B of Figure 10 , the RSD is 2.3%, indicating that the sensor has good reproducibility and precision.

[0114] II. Selectivity test of the electrochemical biosensor during detection

[0115] To evaluate the selectivity of the sensor, 100 fM wild-type mismatched DNA (T100-T790M-wt DNA) was used as a control for the experiment, and cyclic voltammetry was used for testing in the same process as above.

[0116] The nucleotide sequence of this wild-type mismatched DNA is as follows:

[0117] T100-T790M-wt dsDNA:

[0118] 5'-CCA CGT GTG CCG CCT GCT GGG CAT CTG CCT CAC CTC CAC CGT GCA GCTCAT CAT GCA GCT CAT GCC CTT CGG CTG CCT CCT GGA CTA TGT CCG GGA ACAC-3'.

[0119] In view of the double-stranded structural characteristics of T100-T790M-wt dsDNA, heat denaturation treatment is required before detection. Specifically, the sample is heated in a 95°C metal bath for 5 min to dissociate double-stranded DNA into single-stranded T100-T790M-wt DNA, ensuring effective hybridization between the probe and the target sequence during subsequent detection.

[0120] The results are as Figure 11 shown. It can be seen from Figure 11 that only when the target specifically binds to the hairpin DNA probe (T100-T790M-dsDNA in the figure), the ECL signal is significantly enhanced. This is because the presence of the target disrupts the resonance energy transfer system. The experimental results show that the sensor has excellent selective recognition ability for the target and can effectively distinguish wild-type mismatched DNA from DNA related to the EGFRT-T790M mutation in non-small cell lung cancer.

[0121] III. Detection of actual samples

[0122] To simulate the real detection environment, T100-T790M dsDNA with concentrations of 1 fM (fmol / L), 10 fM (fmol / L), 100 fM (fmol / L), and 1000 fM (fmol / L) was added to healthy human plasma respectively to prepare spiked samples. The samples corresponding to these four concentrations were labeled as Sample 1, Sample 2, Sample 3, and Sample 4.

[0123] The plasma was pretreated to meet the detection requirements of the electrochemiluminescence sensor: precisely measure 20 μL of plasma, mix it evenly with 5 μL of proteinase K buffer system (containing 125 mmol / L Tris-Cl, pH 8.0; 6.25% Tween20; 2.5 mmol / L EDTA), and then incubate it in a 50°C water bath for 20 min to degrade proteins. Finally, the reaction system was heated in a 95°C metal bath for 5 min to inactivate proteinase K and dissociate double-stranded DNA into single-stranded DNA (T100-T790M DNA). The obtained samples can be directly used for subsequent detection and analysis.

[0124] The samples were dropped on the sensor and incubated for 1 h respectively, and the changes in electrochemiluminescence signal values were observed. According to Figure 8The content of T100-T790M DNA in the plasma of each sample was calculated by the linear equation, and the results are shown in Table 1.

[0125] Table 1 shows the detection of T100-T790M DNA in plasma samples

[0126] Sample Added amount (fM) Recovered amount (fM) Recovery rate (%) 1 1 1.108 110.8 2 10 9.47 94.7 3 100 98.3 98.3 4 1000 1065 106.5

[0127] As can be seen from Table 1, the sensor has a good recovery rate for T100-T790M DNA in plasma samples, and the recovery rate ranges from 94.7% to 110.8%, indicating that the electrochemiluminescence biosensor shows excellent feasibility in the detection of actual samples and has strong application potential.

[0128] Although the present invention has been described by using the above preferred embodiments, it is not intended to limit the protection scope of the present invention. Any person skilled in the art can still make various changes and modifications to the above embodiments without departing from the spirit and scope of the present invention, and these still belong to the protection scope of the present invention.

Claims

1. Preparation method of an electrochemical biosensor for detecting EGFRT790M in non-small cell lung cancer, characterized in that, It includes the following steps: S1. First, drop the luminescent CdSe@ZnS quantum dot solution onto the surface of the glassy carbon electrode, dry it under an infrared lamp, then drop the PDDA solution and dry it under an infrared lamp to obtain a glassy carbon modified electrode of PDDA / CdSe@ZnS; S2. Incubate the hairpin DNA solution and tris(2-carboxyethyl)phosphine hydrochloride TCEP solution together for 1 - 3 h, then add the gold nanoparticle solution and incubate for 3 - 12 h to obtain a capture probe solution; S3. Drop the capture probe solution onto the surface of the PDDA / CdSe@ZnS glassy carbon modified electrode, incubate for 1 - 12 h under constant temperature conditions and then rinse to obtain a glassy carbon modified electrode of capture probe / PDDA / CdSe@ZnS, which is the electrochemistry biosensor; Among them, the hairpin DNA consists of 30 bp, and the base sequence of the 30-bp hairpin DNA is as follows: T790M-30-Hairpin-DNA: 5'-GGAAGA CAT GAG CTG CAT GAT GAG TCT TCC-(CH2)6-HS-SH-3'; 2. The preparation method of the electrochemical biosensor for detecting EGFRT790M of non-small cell lung cancer according to claim 1, characterized in that, In the step S1, the preparation method of the CdSe@ZnS quantum dots is as follows: Add 3-mercaptopropionic acid MPA and zinc chloride into ultrapure water, and bubble nitrogen for 30 - 60 min; then add the TGA-CdSe quantum dot solution, heat and react the mixed solution for 2 - 4 h to obtain the CdSe@ZnS quantum dot solution; 3. The preparation method of the electrochemical biosensor for detecting EGFRT790M in non-small cell lung cancer according to claim 2, characterized in that, The preparation method of the TGA-CdSe quantum dot solution is as follows: Mix cadmium chloride and thioglycolic acid TGA evenly, adjust the pH to 9 - 11, bubble nitrogen for 30 - 60 min, then inject sodium selenite solution and reducing agent hydrazine hydrate into the mixture, heat and reflux, naturally cool to room temperature after the reaction ends, and dialyze and purify for 24 - 48 h to obtain the TGA-CdSe quantum dot solution; 4. The preparation method of the electrochemical biosensor for detecting EGFRT790M in non-small cell lung cancer according to claim 1, wherein, In the step S2, after incubating with the gold nanoparticle solution, add 6-mercaptohexanol MCH and continue to incubate for 1 - 12 h to obtain the capture probe solution; 5. An electrochemistry biosensor for detecting EGFRT790M of non-small cell lung cancer obtained by the preparation method according to any one of claims 1 - 4.

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