Electrochemical biosensor based on PDDA-enhanced cadmium selenide quantum dots and preparation method

Abstract

The present invention discloses an electrochemical biosensor based on PDDA-enhanced cadmium selenide quantum dots and a preparation method thereof, comprising the following steps: S1. obtaining a polished and cleaned glassy carbon electrode; S2. dripping a mixed solution of cadmium selenide quantum dots and PDDA onto the surface of the glassy carbon electrode, naturally drying at room temperature, and then washing to obtain a modified electrode; S3. dripping an aqueous solution of gold nanoparticles modified with hairpin DNA onto the surface of the modified electrode, incubating at room temperature, and then rinsing to obtain an electrochemical biosensor based on PDDA-enhanced cadmium selenide quantum dots, which has a strong and stable light intensity signal and high sensitivity for detecting circulating tumor DNA of lung cancer.

Description

Technical Field

[0001] The present invention relates to the technical field of detecting circulating tumor DNA of lung cancer, and in particular to an electrochemical biosensor based on PDDA-enhanced cadmium selenide quantum dots and a preparation method thereof. Background Art

[0002] Malignant tumors are one of the main causes of rising global mortality rates and have seriously threatened human health and life safety. Lung cancer has become the leading cause of cancer death worldwide due to its high malignancy and lack of effective treatments. According to a survey by the Global Cancer Observatory (GLOBOCAN), there were 19.3 million new cancer cases and nearly 10 million cancer deaths worldwide in 2020, of which 2.2 million were lung cancer patients. Due to the insidious onset of lung cancer, the vast majority of lung cancer patients are already in the late stage of lung cancer when they first visit the doctor. Therefore, the overall survival of patients is not optimistic, with only 400,000 people surviving. In recent years, with the rapid development of modern medicine, cancer patients diagnosed early can be effectively controlled through some medical means, and most patients can even be cured. Therefore, an effective way to improve the cure rate of cancer patients is early diagnosis and timely prevention.

[0003] Electrochemiluminescence (ECL) is an analytical method that combines electrochemistry and chemiluminescence. It combines the advantages of electrochemical analysis, such as strong controllability and rapid analysis, with the high sensitivity and wide linear range of chemiluminescence. With the continuous advancement of nanoscience and technology, nanomaterials of various morphologies and sizes have emerged. Among them, quantum dots (QDs) stand out for their excellent photostability, high quantum yield, and controllable size. Since the discovery of the ECL properties of silicon quantum dots in 2002, QD-based ECL systems have been continuously explored. For example, graphene quantum dots, silicon quantum dots, cadmium sulfide quantum dots, and graphite-like carbon nitride quantum dots have demonstrated excellent analytical performance in ECL analysis. The unique optoelectronic properties of quantum dots can be combined with the high sensitivity of ECL analysis. Using quantum dots as luminescent materials and as carriers for nucleic acid analysis probes, the construction of ECL biosensors with high sensitivity, strong specificity, and low detection limits for nucleic acid analysis is attracting significant attention.

[0004] However, in the prior art, electrochemical biosensors used to detect circulating tumor DNA in lung cancer have low detection sensitivity and unstable light intensity signals. Summary of the Invention

[0005] In view of this, the present application provides an electrochemical biosensor based on PDDA-enhanced cadmium selenide quantum dots and a preparation method, which has a strong and stable light intensity signal and high sensitivity in detecting circulating tumor DNA in lung cancer.

[0006] In order to achieve the above technical objectives, this application adopts the following technical solutions:

[0007] In a first aspect, the present application provides a method for preparing an electrochemical biosensor based on PDDA-enhanced cadmium selenide quantum dots, comprising the following steps:

[0008] S1. Obtain a polished and cleaned glassy carbon electrode;

[0009] S2. A mixture of cadmium selenide quantum dots and PDDA (diethylene glycol phthalate diacrylate) was added dropwise to the surface of a glassy carbon electrode, allowed to dry naturally at room temperature, and then washed to obtain a modified electrode;

[0010] S3. An aqueous solution of hairpin DNA-modified gold nanoparticles was added dropwise to the modified electrode surface, incubated at room temperature, and then rinsed to obtain an electrochemical biosensor based on PDDA-enhanced CdSe quantum dots.

[0011] Preferably, the base sequence of the hairpin DNA (hairpin-DNA) is as follows: 5'-NH2-(CH2)6-GGA AGA CATGAG GAC CAT GTT GTG TCT TCC-(CH2)6-SH-3'.

[0012] Preferably, in step S2, the molar ratio of PDDA to cadmium selenide quantum dots is 1:1.

[0013] Preferably, the particle size of the cadmium selenide quantum dots is 4-5 nm.

[0014] Preferably, the method for preparing cadmium selenide quantum dots comprises the steps of:

[0015] Selenium and NaBH4 are used as raw materials to obtain NaHSe precursor solution;

[0016] After adding thioglycolic acid to the aqueous solution of CdCl2·5H2O, a NaHSe precursor solution is added, and then the mixture is heated under reflux reaction in a nitrogen atmosphere to obtain cadmium selenide quantum dots.

[0017] Preferably, the method for preparing hairpin DNA-modified gold nanoparticles comprises the steps of:

[0018] Under ice bath conditions, HAuCl4·4H2O, K2CO3 solution, and NaBH4 were added to ultrapure water in sequence to obtain AuNPs solution;

[0019] Tris(2-carboxyethyl)phosphine solution is added to the hairpin DNA solution and reacted at room temperature to obtain a mixture. Then, AuNPs solution is added dropwise to the mixture and the reaction is continued at room temperature to obtain hairpin DNA-modified gold nanoparticles. The ends of the hairpin DNA are connected to the gold nanoparticles. During the detection process, the hairpin DNA on the gold nanoparticles is complementary to the ctDNA of lung cancer.

[0020] In the second aspect, the present application provides an electrochemical biosensor based on PDDA-enhanced cadmium selenide quantum dots. On the glassy carbon electrode, the sensor is a first layer of a mixture of PDDA and cadmium selenide quantum dots, and a second layer of gold nanoparticles modified with hairpin DNA.

[0021] In a third aspect, the present application provides an application of an electrochemical biosensor based on PDDA-enhanced cadmium selenide quantum dots as a detection reagent for detecting circulating tumor DNA of lung cancer. According to the mutant DNA sequence of lung cancer cells, the base sequence of circulating tumor DNA (ctDNA) of lung cancer is as follows: 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 CTAT GTC CGGGAA CAC-3'.

[0022] Preferably, the steps of detecting circulating tumor DNA in lung cancer are as follows:

[0023] Standard solutions of target DNA with different concentrations were added dropwise to the electrochemical biosensor based on PDDA-enhanced cadmium selenide quantum dots, and after incubation, the electrochemical biosensor to be tested was obtained.

[0024] A three-electrode system was used, with Ag / AgCl as the reference electrode, a platinum electrode as the counter electrode, and the electrochemical biosensor to be tested as the working electrode, to form an electrochemical workstation. Cyclic voltammetry was used for testing to obtain a photoelectric graph. 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.

[0025] The solution of the DNA to be tested was added dropwise to the electrochemical biosensor based on PDDA enhanced cadmium selenide quantum dots as the working electrode for testing, and the electrochemiluminescence signal intensity value was obtained. The value was inserted into the standard curve to obtain the concentration of the solution of the DNA to be tested.

[0026] Preferably, the buffer system used for the target DNA standard solution and the test DNA solution is a phosphate buffer solution with a pH of 7.4 and a concentration of 0.1 mol / L K2S2O8.

[0027] The beneficial effects of the present application are as follows: This scheme uses cadmium selenide quantum dots as energy donors and hairpin DNA-modified gold nanoparticles as energy acceptors to form an electrochemical energy resonance transfer system. PDDA plays a role in bonding the cadmium selenide quantum dots and the hairpin DNA-modified gold nanoparticles and enhancing the luminescence of the cadmium selenide quantum dots. The ends of the hairpin DNA are connected to the gold nanoparticles to form gold nanoparticles loaded with hairpin DNA modifications. During the detection process, the gold nanoparticles are complementary paired with the ctDNA (circulating tumor DNA) of lung cancer. Compared with the glassy carbon electrode using only cadmium selenide quantum dots, the light intensity after PDDA modification is increased by about 7.6 times. The detection is convenient, fast, low-cost, highly sensitive and stable. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 Working curve of the electrochemical biosensor for this application;

[0029] Figure 2 is the electrochemiluminescence signal intensity after the standard solution of target DNA with different concentrations reacts with the electrochemical biosensor;

[0030] Figure 3 To enhance the electrochemiluminescence signal intensity at different stages of electrochemical biosensor based on PDDA;

[0031] Figure 4 The results of light intensity stability of the electrochemical biosensor in this application;

[0032] Figure 5 This is a comparison chart of the electrochemiluminescence signal intensity of CdSe quantum dot modified electrode and CdS quantum dot modified electrode. DETAILED DESCRIPTION

[0033] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0034] Raw material preparation

[0035] 1. Preparation of hairpin DNA-modified gold nanoparticles, comprising the following steps:

[0036] Measure 200 ml of ultrapure water and cool it to 4°C in a 4°C refrigerator. At 4°C, add 2-5 ml of 10 mmol / L HAuCl4·4H2O to the ultrapure water and stir vigorously. Then, add 1 ml of K2CO3 solution (0.2 mol / L) and continue stirring. Finally, quickly add 9 ml of NaBH4 (0.5 mg / mL) and stir vigorously for 5 minutes. The resulting wine-red solution is stored in a 4°C refrigerator until use to obtain the AuNPs solution.

[0037] Take 8-10 μL of 0.1 mmol / L hairpin DNA solution, then add 0.15-0.3 μL of 10 mmol / L tris(2-carboxyethyl)phosphine solution, the solvent of which is 0.01 mol / L PBS solution containing 0.1 mol / L NaCl, and react for 1 hour. Then, add 100 μL of the obtained AuNPs solution to the above mixture and react for 0.5 hours to obtain hairpin DNA-modified gold nanoparticles.

[0038] 2. Preparation of cadmium selenide quantum dots, comprising the following steps;

[0039] 16 mg of selenium and 151.2 mg of NaBH4 were used to prepare the precursor solution NaHSe. Subsequently, 91.3 mg of CdCl2·2.5H2O was dissolved in 100 ml of deionized water and 50-70 μL of thioglycolic acid (TGA) was added to obtain a cadmium ion solution. NaHSe was added to the above cadmium ion solution and refluxed at 100°C for 1 hour. The reaction was carried out under nitrogen protection to obtain cadmium selenide quantum dots.

[0040] Example 1

[0041] A method for preparing an electrochemical biosensor based on PDDA-enhanced cadmium selenide quantum dots comprises the following steps:

[0042] S1. Polish a 3-5 mm diameter glassy carbon electrode on suede to a mirror finish using 1.0, 0.3, and 0.05 μm Al2O3 slurries, respectively. Ultrasonic cleaning is then performed in nitric acid, ethanol, and distilled water for 5-15 min each, followed by drying with nitrogen to obtain the polished and cleaned glassy carbon electrode.

[0043] S2. Add 7-10 μl of a mixture of CdSe quantum dots and 5-7 μl of 1% PDDA to the surface of a glassy carbon electrode. Allow to dry naturally at room temperature and rinse with PBS to obtain a modified electrode.

[0044] S3. 7-10 μl of an aqueous solution of hairpin DNA-modified gold nanoparticles was added dropwise to the modified electrode surface and incubated at room temperature for 1-2 hours. After a gentle rinse with PBS, an electrochemical biosensor based on PDDA-enhanced cadmium selenide quantum dots was obtained. Cyclic voltammetry was performed with a scan range of -1.6-0 V and a scan rate of 0.1-0.2 V / s. The luminescence intensity was detected by an electrochemiluminescence system.

[0045] The base sequence of the hairpin DNA is as follows: 5'-NH2-(CH2)6-GGA AGA CAT GAG GAC CAT GTT GTGTCT TCC-(CH2)6-SH-3'.

[0046] Example 2

[0047] An electrochemical biosensor based on PDDA-enhanced cadmium selenide quantum dots was used to detect circulating tumor DNA in lung cancer. The steps are as follows:

[0048] Put 10 -17 mol / L, 10 -16 mol / L, 10 -15 mol / L, 10 -14 mol / L, 10 -13 mol / L, 10 -12 mol / L target DNA standard solution was added dropwise to the electrochemical biosensor based on PDDA-enhanced cadmium selenide quantum dots and incubated for 2 h to obtain the electrochemical biosensor to be tested;

[0049] The test was performed using a three-electrode system of an electrochemical workstation. The three-electrode system was used, with Ag / AgCl as the reference electrode, a platinum electrode as the counter electrode, and the electrochemical biosensor to be tested as the working electrode to form an electrochemical workstation. The electrochemical workstation was connected to an ultra-weak luminescence detector. In 10 mL of a phosphate buffer solution with a pH of 7.4 and a concentration of 0.1 mol / L K2S2O8, a photoelectric graph was obtained by using an electrochemiluminescence system and cyclic voltammetry with a scan range of -1.6-0 V and a scan rate of 0.1-0.2 V / s. The logarithm of the concentration of the target DNA was used as the horizontal axis, and the electrochemiluminescence signal intensity was used as the vertical axis for fitting to obtain a standard curve, as shown below: Figure 1 As shown in the figure, it can be seen that the electrochemical biosensor prepared in this application can detect ctDNA concentrations as low as 10 -17mol / L still has a strong signal and high sensitivity. Subsequently, the solution of the DNA to be tested can be added dropwise to the electrochemical biosensor based on PDDA enhanced cadmium selenide quantum dots as the working electrode for testing to obtain the electrochemiluminescence signal intensity value, which is then brought into the standard curve to obtain the concentration of the solution of the DNA to be tested; the electrochemiluminescence signal intensity (ECL) after the standard solution of target DNA with different concentrations reacts with the electrochemical biosensor is as follows Figure 2 shown.

[0050] Test Case

[0051] The electrochemiluminescence signal intensities at different stages of the preparation of the electrochemical biosensor based on PDDA-enhanced cadmium selenide quantum dots in Example 1 were tested respectively, and the steps were as follows:

[0052] A glassy carbon electrode with a diameter of 3-5 mm was polished to a mirror finish on suede using 1.0, 0.3, and 0.05 μm Al2O3 slurries, followed by ultrasonic cleaning in nitric acid, ethanol, and distilled water for 5-15 min, and then dried with nitrogen.

[0053] 7-10 μl of cadmium selenide quantum dots were added dropwise to a glassy carbon electrode. Cyclic voltammetry was performed in a 10 mL phosphate buffer solution with a pH of 7.4 and a concentration of 0.1 mol / L K2S2O8. The scan range was -1.6-0 V and the scan rate was 0.1-0.2 V / s. The electrochemical workstation and electrochemical conditions were the same as those in Example 2 to detect the electrochemiluminescence signal intensity of the cadmium selenide quantum dot / glassy carbon electrode (CdSeQDs / GCE).

[0054] A mixture of 7-10 μl of cadmium selenide quantum dots and 5-7 μl of 1% PDDA was added dropwise to a glassy carbon electrode. The electrochemiluminescence signal intensity of the PDDA / CdSe QDs / GCE was measured using the same electrochemical workstation and electrochemical conditions as in Example 2 in 10 mL of phosphate buffer solution containing 0.1 mol / L K2S2O8 at pH 7.4.

[0055] A mixture of 7-10 ul of cadmium selenide quantum dots and 5-7 ul of 1% PDDA was added dropwise to a glassy carbon electrode, and an aqueous solution of 7-10 ul of hairpin DNA-modified gold nanoparticles was added dropwise to the surface of the modified electrode. The mixture was incubated at room temperature for 1-2 h. After a slight rinse with PBS solution, the electrochemiluminescence signal intensity of the hairpin DNA-modified gold nanoparticles / PDDA / cadmium selenide quantum dots / glassy carbon electrode (AuNPS / Hairpin DNA / PDDA / CdSe QDs / GCE) was detected in 10 mL of a phosphate buffer solution containing 0.1 mol / L K2S2O8 at a pH of 7.4 using the same electrochemical workstation and electrochemical conditions as in Example 2.

[0056] The prepared electrode was rinsed with PBS and then added dropwise for 10 -12 mol / L target DNA was incubated at room temperature for 2 h, and then the electrochemiluminescence signal intensity of lung cancer circulating tumor DNA / hairpin DNA-modified gold nanoparticles / PDDA / cadmium selenide quantum dots / glassy carbon electrode (ctDNA / AuNPS / Hairpin DNA / PDDA / CdSe QDs / GCE) was detected in 10 mL of phosphate buffer solution with a pH of 7.4 and a concentration of 0.1 mol / L K2S2O8 using the same electrochemical workstation and electrochemical conditions as in Example 2;

[0057] The results are as follows Figure 3 As shown by Figure 3 It can be seen that the light intensity after PDDA modification is significantly enhanced compared with the glassy carbon electrode using only CdSe quantum dots.

[0058] Test the electrochemiluminescence signal intensity of the cadmium sulfide quantum dot modified electrode (CdS QDs / GCE) and the PDDA cadmium sulfide quantum dot modified electrode (PDDA / CdS QDs / GCE): 7-10 μl of cadmium sulfide quantum dots were added dropwise to the glassy carbon electrode. In 10 mL of phosphate buffer solution with a pH of 7.4 and a concentration of 0.1 mol / L K2S2O8, cyclic voltammetry was used with a scan range of -1.6-0 V and a scan rate of 0.1-0.2 V / s. The electrochemical workstation and electrochemical conditions were the same as those in Example 2 to detect the electrochemiluminescence signal intensity of the cadmium sulfide quantum dot / glassy carbon electrode (CdS QDs / GCE).

[0059] A mixture of 7-10 μl of cadmium sulfide quantum dots and 5-7 μl of 1% PDDA was added dropwise to a glassy carbon electrode. The electrochemical luminescence signal intensity of the PDDA / CdS QDs / GCE was detected in 10 mL of a phosphate buffer solution containing 0.1 mol / L K2S2O8 at pH 7.4 using the same electrochemical workstation and electrochemical conditions as in Example 2. The comparison of the light intensity results with the cadmium selenide quantum dot modified electrode is shown in the figure below. Figure 5 As shown, it is shown that the effect of PDDA synergistically enhancing the light intensity of the cadmium selenide quantum dot modified electrode is significantly higher than the effect of PDDA synergistically enhancing the light intensity of the cadmium sulfide quantum dot modified electrode.

[0060] The electrochemiluminescence signal intensity of the AuNPS / Hairpin DNA / PDDA / CdSe QDs / GCE was tested within 0-150s. The results are as follows: Figure 4 As shown, the electrochemiluminescence signal intensity remains stable, indicating that the electrochemical biosensor prepared in the present application has strong stability.

[0061] The above are only preferred specific embodiments of the present invention, but the scope of protection of the present invention is not limited thereto. Any changes or replacements that can be easily thought of by any technician familiar with this technical field within the technical scope disclosed by the present invention should be covered by the scope of protection of the present invention.

Claims

1. A method for preparing an electrochemical biosensor based on PDDA-enhanced cadmium selenide quantum dots, characterized in that: The following steps are involved: S1. Obtain a polished and cleaned glassy carbon electrode; S2. A mixture of cadmium selenide quantum dots and PDDA was added dropwise to the surface of the glassy carbon electrode, dried naturally at room temperature, and then washed to obtain a modified electrode; S3. An aqueous solution of hairpin DNA-modified gold nanoparticles was added dropwise to the surface of the modified electrode, incubated at room temperature, and then rinsed to obtain an electrochemical biosensor based on PDDA-enhanced cadmium selenide quantum dots; In step S2, the molar ratio of PDDA to cadmium selenide quantum dots is 1:1; The particle size of the cadmium selenide quantum dots is 4-5 nm; The preparation method of the cadmium selenide quantum dots comprises the steps of: Selenium and NaBH4 are used as raw materials to obtain NaHSe precursor solution; After adding thioglycolic acid to the aqueous solution of CdCl2·5H2O, the NaHSe precursor solution is added, and then heated under reflux reaction in a nitrogen atmosphere to obtain the cadmium selenide quantum dots; The preparation method of the hairpin DNA-modified gold nanoparticles comprises the steps of: Under ice bath conditions, HAuCl4·4H2O, K2CO3 solution, and NaBH4 were added to ultrapure water in sequence to obtain AuNPs solution; Adding tris(2-carboxyethyl)phosphine solution to the hairpin DNA solution and reacting at room temperature to obtain a mixture, then dropwise adding the AuNPs solution to the mixture and continuing the reaction at room temperature to obtain hairpin DNA-modified gold nanoparticles; The first layer of the sensor is a mixture of PDDA and cadmium selenide quantum dots, and the second layer is gold nanoparticles modified with hairpin DNA.

2. The method for preparing an electrochemical biosensor based on PDDA-enhanced cadmium selenide quantum dots according to claim 1, wherein: The base sequence of the hairpin DNA is as follows: 5'-NH2-(CH2)6-GGA AGA CAT GAG GAC CATGTT GTG TCT TCC-(CH2)6-SH-3'.

3. An electrochemical biosensor based on PDDA-enhanced cadmium selenide quantum dots obtained by the preparation method according to any one of claims 1-2.

4. An application of the electrochemical biosensor based on PDDA-enhanced cadmium selenide quantum dots as claimed in claim 3, characterized in that: It is used as a detection reagent for detecting circulating tumor DNA of lung cancer, and the base sequence of the circulating tumor DNA of lung cancer is as follows: 5'-CCA CGT GTG CCG CCT GCT GGG CAT CTG CCT CAC CTC CAC CGT GCA GCT CATCAC GCA GCT CAT GCC CTT CGG CTG CCT CCT GGA CTAT GTC CGG GAA CAC-3'.

5. The use according to claim 4, characterized in that The steps of detecting circulating tumor DNA of lung cancer are as follows: Adding target DNA standard solutions of different concentrations dropwise onto the electrochemical biosensor based on PDDA-enhanced cadmium selenide quantum dots, and incubating to obtain the electrochemical biosensor to be tested; A three-electrode system was used, with Ag / AgCl as the reference electrode, a platinum electrode as the counter electrode, and the electrochemical biosensor to be tested as the working electrode, to form an electrochemical workstation. Cyclic voltammetry was used to obtain a photoelectric graph, and a standard curve was obtained by fitting the graph using the logarithm of the target DNA concentration as the abscissa and the electrochemiluminescence signal intensity as the ordinate. The solution of the DNA to be tested is added dropwise to the electrochemical biosensor based on PDDA enhanced cadmium selenide quantum dots as the working electrode for testing, and the electrochemiluminescence signal intensity value is obtained, which is then inserted into the standard curve to obtain the concentration of the solution of the DNA to be tested.

6. The use according to claim 4, wherein The buffer system used for the target DNA standard solution and the test DNA solution is a phosphate buffer solution with a pH of 7.4 and a concentration of 0.1 mol / L K2S2O8.

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