Preparation method and application of faraday cage type electrochemical sensor for detecting circulating tumor cells

Abstract

This invention discloses a method for preparing and applying a Faraday cage electrochemical sensor for detecting circulating tumor cells. The method includes the following steps: synthesis of the capture unit Fe3O4-APTs; synthesis of the signal unit GO@PTCA-APTs; adding the capture and signal units to a cell standard solution, incubating at 37°C for 1–1.5 h, magnetically separating and redispersing them in PBS solution, and then dropping them onto a magnetic glassy carbon electrode surface to prepare the Faraday cage electrochemical sensor for detecting circulating tumor cells in one step; using the Faraday cage electrochemiluminescence biosensor as the working electrode, the ECL intensity corresponding to different concentrations of circulating tumor cells is measured using electrochemiluminescence immunoassay, and the concentration of circulating tumor cells in the test solution is obtained based on the ECL signal value; the advantages are high sensitivity, high specificity, reliable detection results, and simple and rapid operation.

Description

Technical Field

[0001] This invention relates to an electrochemical sensor, and more particularly to a method for preparing and applying a Faraday cage electrochemical sensor for detecting circulating tumor cells. Background Technology

[0002] Breast cancer (BC) is the most common cancer among women worldwide, and the direct cause of death in most BC patients is the metastasis of tumor cells to other vital sites via the bloodstream. These tumor cells that detach from solid tumor lesions and enter the peripheral blood are called circulating tumor cells (CTCs). CTCs can monitor cancer progression and provide new avenues for personalized treatment of cancer patients. However, the number of CTCs in peripheral blood is scarce, less than 10%. 6 ~10 7 Only one CTC is present in a white blood cell. Therefore, developing a rapid, sensitive, and accurate method for detecting CTCs is of great significance.

[0003] Several techniques for detecting CTCs have been proposed, including inductively coupled plasma mass spectrometry (ICP-MS), flow cytometry (FCM), fluorescence imaging, microfluidic systems, and surface-enhanced pulled scattering. However, these methods typically require specialized equipment and personnel, are complex to operate, time-consuming, and costly, failing to meet the demands for simple, rapid, sensitive, and accurate detection. In contrast, electrochemiluminescence (ECL) combines the advantages of electrochemical detection and chemiluminescence technologies, such as high sensitivity, good selectivity, low cost, fast response, and simple equipment operation, showing great promise for CTC detection.

[0004] However, current electrochemiluminescence biosensors for detecting CTCs generally employ a traditional sandwich design. As CTCs are micrometer-sized targets, the resulting complex, due to its distance from the electrode, hinders electron tunneling, preventing the electrochemiluminescence reaction from occurring. In contrast, the Faraday cage construction method allows for a higher loading of signal markers, all of which can participate in the electrode reaction. Electrons can flow freely between the electrode and the signal unit without being obstructed by the large circulating tumor cell target, resulting in a significant improvement in sensitivity. Currently, there are no publicly available reports, either domestically or internationally, on the use of Faraday cage construction methods for electrochemiluminescence biosensors to detect circulating tumor cells (MCF-7). Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide a method for preparing and applying a Faraday cage electrochemical sensor for detecting circulating tumor cells that is highly sensitive, highly specific, provides reliable detection results, and is simple and rapid to operate.

[0006] The technical solution adopted by the present invention to solve the above-mentioned technical problems is as follows:

[0007] 1. A method for preparing a Faraday cage electrochemical sensor for detecting circulating tumor cells, comprising the following steps:

[0008] (1) Synthesis of the trapping unit Fe3O4-APTs

[0009] a. First, dissolve 0.5–0.7 g of anhydrous FeCl3 and 0.2–0.3 g of sodium citrate in 15–20 mL of ethylene glycol, then add 0.9–1.2 g of anhydrous sodium acetate. After stirring for 30 min, transfer the mixed solution to a stainless steel high-pressure reactor lined with polytetrafluoroethylene and place it in a constant temperature drying oven at 200–220 °C for 10 h. After cooling to room temperature, wash twice with ultrapure water and ethanol, and disperse in 15–20 mL of water after magnetic separation to obtain a carboxylated Fe3O4 (Fe3O4-COOH) dispersion.

[0010] b. Add 1-2 mL of EDC / NHS mixed solution to 1-2 mL of Fe3O4-COOH dispersion obtained in step (1)a. Stir at room temperature for 6 h, then add 0.6-0.8 mL of 5-7 μmol / L aptamer MUC1 (Apt1) and 0.6-0.8 mL of 5-7 μmol / L nucleolin aptamer AS1411 (Apt2). Incubate at 4 °C for 6-8 h, then add 0.2-0.4 mL of 2 wt% bovine serum albumin (BSA) solution. Incubate at room temperature for 2-3 h to block non-specific active sites. Finally, magnetically separate and redisperse in 1-3 mL of water to obtain the Fe3O4-APTs dispersion of the capture unit.

[0011] (2) Synthesis of signal unit GO@PTCA-APTs

[0012] a. Add 10–20 mg of 3,4,9,10-perylenetetracarboxylic acid dianhydride (PTCDA) to 2–5 mL of freshly prepared 4–5 mol / L NaOH solution. Heat and stir at 70–80 °C until PTCDA is completely dissolved. After the solution turns yellow-green, continue to add 1–2 mol / L HCl dropwise until the mixture turns completely red. Centrifuge at 4400–5000 × g and wash the precipitate 3–5 times alternately with anhydrous ethanol and ultrapure water. Redisperse the precipitate in 3–8 mL of water to obtain a 3,4,9,10-perylenetetracarboxylic acid (PTCA) dispersion.

[0013] b. Add 0.1–0.3 mL of the 2–4 mg / mL PTCA dispersion obtained in step (2)a to 2–5 mL of 0.5 mg / mL graphene oxide (GO), and stir at room temperature in the dark for 12–16 h to obtain the GO@PTCA dispersion; add 1–2 mL of EDC / NHS mixed solution to 1–3 mL of GO@PTCA dispersion, stir at room temperature in the dark for 6–10 h, then add 0.4–0.7 mL of 5–7 μmol / L aptamer MUC1 (Apt1) and 0.4 mL of 5–7 μmol / L nucleolin aptamer AS1411 (Apt2), incubate at 4 °C for 6–8 h, and then add 0.2–0.4 mL of... After incubating a 2wt% bovine serum albumin (BSA) solution at room temperature for 2–3 h to block non-specific active sites, centrifuge at 4400–5000 × g and redisperse in 1–3 mL of water to obtain the signaling unit GO@PTCA-APTs.

[0014] (3) Construction of Faraday cage-type electrochemiluminescence biosensor

[0015] Add 20–30 μL of the capture unit and 20–30 μL of the signal unit to 0.8–1 mL of cell standard solution, incubate at 37 °C for 1–1.5 h, and then magnetically separate. The resulting complex of capture unit-circulating tumor cell-signal unit is redispersed in 20–40 μL of PBS solution and dropped onto the surface of a magnetic glassy carbon electrode (MGCE). This completes the one-step preparation of a Faraday cage electrochemical sensor for detecting circulating tumor cells.

[0016] Furthermore, the concentration of EDC in the EDC / NHS mixed solution is 0.4 mol / L, and the concentration of NHS is 0.1 mol / L.

[0017] Furthermore, the sequence of the aptamer MUC1 is: 5′-NH2-GCAGTTGATCCTTTGGATACCCTGG-3′; the sequence of the nucleolin aptamer AS1411 is: 5′-NH2-TTTGGTGGTGGTGGTTGTGGTGGTGGTG-3′.

[0018] Furthermore, the circulating tumor cells are MCF-7.

[0019] 2. A method for detecting circulating tumor cells using the above-mentioned Faraday cage electrochemiluminescence biosensor, which is not intended for diagnosis or treatment, includes the following steps: using the prepared Faraday cage electrochemiluminescence biosensor for detecting circulating tumor cells as the working electrode, a platinum electrode as the auxiliary electrode, and an Ag / AgCl electrode as the reference electrode, placing it in a 0.1 mol / L Tris-HCl buffer solution containing 0.1 mol / L co-reaction reagent K2S2O8 at pH=8.7, measuring the ECL intensity corresponding to different concentrations of circulating tumor cells using electrochemiluminescence method, and obtaining the concentration of circulating tumor cells in the test solution based on the ECL signal value.

[0020] Furthermore, the electrochemiluminescence method has a potential range of -1.9V to 0.0V, a potential scan rate of 0.1V / s, and a photomultiplier tube voltage of 800V.

[0021] Invention Principle: This invention utilizes a Faraday cage construction model to construct a rapid, simple, accurate, and sensitive electrochemiluminescent biosensor for detecting circulating tumor cells (CTCs). Fe3O4-COOH contains a large number of dual aptamers (APTs), namely the MUC1 aptamer Apt1 and the nucleolin aptamer Apt2, linked by amide bonds to form a capture unit. Using GO (Gastrointestinal oxidase) as a substrate, which is flexible, has a large surface area, and good conductivity, a large number of electrochemiluminescent signal markers (PTCA) are assembled onto the GO surface through π-π conjugation. The dual aptamers (APTs) are then bound to PTCA via amide bonds, forming a signal unit. In the presence of the target, thanks to the high affinity and specificity of the APTs, the capture unit and signal unit can rapidly bind to CTCs, ultimately forming a "capture unit-circulating tumor cell-signal unit" complex. This complex is then rapidly assembled on the electrode surface under magnetic force, constituting a Faraday cage-type electrochemiluminescent biosensor. Higher CTC cell concentrations result in more complexes and a stronger electrochemiluminescent signal.

[0022] Compared with the prior art, the advantages of the present invention are as follows:

[0023] 1. High sensitivity: Compared with the traditional method of labeling electrochemiluminescence signal markers on nanomaterials or aptamers, which has a limited number of markers, this invention adopts a Faraday cage construction mode, which allows for a large loading of signal markers and all of them to participate in the electrode reaction. Electrons can flow freely between the electrode and the signal unit without being hindered by the huge target MCF-7, thus achieving a significant improvement in sensitivity.

[0024] 2. High selectivity: By utilizing two nucleic acid aptamers with high affinity and high specificity, MCF-7 cells can be isolated and enriched simultaneously in actual sample detection, achieving specific recognition and capture of MCF-7 cells.

[0025] 3. Simple operation: In the MCF-7 cell solution to be tested, the "capture unit-MCF-7-signal unit" complex can be formed in one step and rapidly assembled onto the electrode surface under magnetic force to form a Faraday cage electrochemiluminescence biosensor, which is fast, convenient and facilitates rapid experimental operation. Attached Figure Description

[0026] Figure 1 Flowchart of a Faraday cage-type electrochemiluminescence biosensor for detecting circulating tumor cells MCF-7;

[0027] Figure 2 The graph shows the relationship between ECL intensity and MCF-7 concentration.

[0028] Figure 3 A graph showing the linear relationship between different MCF-7 concentrations and ECL intensity;

[0029] Figure 4 The sensor was used to test blank and 10 samples respectively. 5 cervical cancer cells / mL (HeLa), 10 4 ECL signal map measured from cells / mL of breast cancer cells (MCF-7) and a mixture of two circulating tumor cells;

[0030] Figure 5 The images show the ECL signal measured by the sensor on a blank sample, two healthy individuals, and two breast cancer patients. Detailed Implementation

[0031] The present invention will be further described in detail below with reference to the accompanying drawings and embodiments. I. Specific Implementation Methods

[0033] Example 1

[0034] A method for preparing a Faraday cage-type electrochemical sensor for detecting circulating tumor cells includes the following steps:

[0035] (1) Synthesis of the trapping unit Fe3O4-APTs

[0036] a. First, dissolve 0.6g of anhydrous FeCl3 and 0.25g of sodium citrate in 18mL of ethylene glycol, then add 1g of anhydrous sodium acetate. After stirring for 30min, transfer the mixed solution to a stainless steel high-pressure reactor lined with polytetrafluoroethylene and place it in a constant temperature drying oven at 210℃ for 10h. After cooling to room temperature, wash twice with ultrapure water and ethanol in sequence, and disperse in 15-20mL of water after magnetic separation to obtain a carboxylated Fe3O4 (Fe3O4-COOH) dispersion.

[0037] b. Add 1.5 mL of EDC / NHS mixed solution to 1.5 mL of Fe3O4-COOH dispersion obtained in step (1)a. Stir at room temperature for 6 h, then add 0.7 mL of 6 μmol / L aptamer MUC1 (Apt1) and 0.7 mL of 6 μmol / L nucleolin aptamer AS1411 (Apt2). Incubate at 4 °C for 6–8 h, then add 0.3 mL of... A 2 wt% bovine serum albumin (BSA) solution was incubated at room temperature for 2–3 h to block non-specific active sites. Finally, the solution was magnetically separated and redispersed in 2 mL of water to obtain the Fe3O4-APTs dispersion of the capture unit. The EDC / NHS mixed solution contained 0.4 mol / L EDC and 0.1 mol / L NHS. The sequence of aptamer MUC1 (Apt1) was 5′-NH2-GCAGTTGATCCTTTGGATACCCTGG-3′; the sequence of nucleolin aptamer AS1411 (Apt2) was 5′-NH2-TTTGGTGGTGGTGGTTGTGGTGGTGGTGG-3′.

[0038] (2) Synthesis of signal unit GO@PTCA-APTs

[0039] a. Add 15 mg of 3,4,9,10-perylenetetracarboxylic acid dianhydride (PTCDA) to 3 mL of freshly prepared 4.5 mol / L NaOH solution. Heat and stir at 75 °C until PTCDA is completely dissolved. After the solution turns yellow-green, continue to add 1.5 mol / L HCl dropwise until the mixture turns completely red. Centrifuge at 4400–5000 × g and wash the precipitate 45 times alternately with anhydrous ethanol and ultrapure water. Redisperse the precipitate in 5 mL of water to obtain a 3,4,9,10-perylenetetracarboxylic acid (PTCA) dispersion.

[0040] b. Add 0.2 mL of 2-4 mg / mL PTCA dispersion obtained in step (2)a to 3 mL of 0.5 mg / mL graphene oxide (GO), stir at room temperature in the dark for 14 h to obtain GO@PTCA dispersion; add 1.5 mL of EDC / NHS mixed solution to 2 mL of GO@PTCA dispersion, stir at room temperature in the dark for 8 h, add 0.6 mL of 6 μmol / L aptamer MUC1 (Apt1) and 0.4 mL of 6 μmol / L nucleolin aptamer AS1411 (Apt2), incubate at 4 °C for 7 h, add 0.3 mL of 2 wt% bovine serum albumin (BSA) solution, incubate at room temperature for 2-3 h to block non-specific active sites, centrifuge at 4400-5000 × g and redisperse in 2 mL of water to obtain signal units GO@PTCA-APTs;

[0041] (3) Construction of Faraday cage-type electrochemiluminescence biosensor

[0042] Add 25 μL of the capture unit and 25 μL of the signal unit to 0.9 mL of cell standard solution, incubate at 37 °C for 1–1.5 h, and then magnetically separate. The resulting complex of capture unit-circulating tumor cell-signal unit is redispersed in 30 μL of PBS solution and dropped onto the surface of a magnetic glassy carbon electrode (MGCE). This completes the one-step preparation of a Faraday cage electrochemical sensor for detecting circulating tumor cells.

[0043] Example 2

[0044] The difference is the same as in Embodiment 1 above:

[0045] (1) Synthesis of the trapping unit Fe3O4-APTs

[0046] a. First, dissolve 0.5g of anhydrous FeCl3 and 0.2g of sodium citrate in 15mL of ethylene glycol, then add 0.9g of anhydrous sodium acetate. After stirring for 30min, transfer the mixed solution to a stainless steel high-pressure reactor lined with polytetrafluoroethylene and place it in a constant temperature drying oven at 200℃ for 10h. After cooling to room temperature, wash twice with ultrapure water and ethanol in sequence, and disperse in 15mL of water after magnetic separation to obtain a carboxylated Fe3O4 (Fe3O4-COOH) dispersion.

[0047] b. Add 1 mL of EDC / NHS mixed solution to 1 mL of Fe3O4-COOH dispersion obtained in step (1)a. Stir at room temperature for 6 h, then add 0.6 mL of 7 μmol / L aptamer MUC1 (Apt1) and 0.6 mL of 7 μmol / L nucleolin aptamer AS1411 (Apt2). Incubate at 4 °C for 6–8 h, then add 0.2 mL of 2 wt% bovine serum albumin (BSA) solution. Incubate at room temperature for 2–3 h to block non-specific active sites. Finally, magnetically separate and redisperse in 1 mL of water to obtain the Fe3O4-APTs dispersion of the capture unit.

[0048] (2) Synthesis of signal unit GO@PTCA-APTs

[0049] a. Add 10 mg of 3,4,9,10-perylenetetracarboxylic acid dianhydride (PTCDA) to 2 mL of freshly prepared 4 mol / L NaOH solution. Heat and stir at 70 °C until PTCDA is completely dissolved. After the solution turns yellow-green, continue to add 1 mol / L HCl dropwise until the mixture turns completely red. Centrifuge at 4400–5000 × g and wash the precipitate three times alternately with anhydrous ethanol and ultrapure water. Redisperse the precipitate in 3 mL of water to obtain a 3,4,9,10-perylenetetracarboxylic acid (PTCA) dispersion.

[0050] b. Add 0.1 mL of 2 mg / mL PTCA dispersion obtained in step (2)a to 2 mL of 0.5 mg / mL graphene oxide (GO), stir at room temperature in the dark for 12 h to obtain GO@PTCA dispersion; add 1 mL of EDC / NHS mixed solution to 1 mL of GO@PTCA dispersion, stir at room temperature in the dark for 6 h, add 0.4 mL of 7 μmol / L aptamer MUC1 (Apt1) and 0.4 mL of 7 μmol / L nucleolin aptamer AS1411 (Apt2), incubate at 4℃ for 6-8 h, add 0.2 mL of 2 wt% bovine serum albumin (BSA) solution, incubate at room temperature for 2-3 h to block non-specific active sites, centrifuge at 4400-5000×g and redisperse in 1 mL of water to obtain signaling units GO@PTCA-APTs;

[0051] (3) Construction of Faraday cage-type electrochemiluminescence biosensor

[0052] Add 20 μL of the capture unit and 20 μL of the signal unit to 0.8 mL of cell standard solution, incubate at 37 °C for 1–1.5 h, and then magnetically separate. The resulting complex of capture unit-circulating tumor cell-signal unit is redispersed in 20 μL of PBS solution and dropped onto the surface of a magnetic glassy carbon electrode (MGCE). This completes the one-step preparation of a Faraday cage electrochemical sensor for detecting circulating tumor cells.

[0053] Example 3

[0054] The difference is the same as in Embodiment 1 above:

[0055] (1) Synthesis of the trapping unit Fe3O4-APTs

[0056] a. First, dissolve 0.7g of anhydrous FeCl3 and 0.3g of sodium citrate in 20mL of ethylene glycol, then add 1.2g of anhydrous sodium acetate. After stirring for 30min, transfer the mixed solution to a stainless steel high-pressure reactor lined with polytetrafluoroethylene and place it in a constant temperature drying oven at 220℃ for 10h. After cooling to room temperature, wash twice with ultrapure water and ethanol in sequence, and disperse in 20mL of water after magnetic separation to obtain a carboxylated Fe3O4 (Fe3O4-COOH) dispersion.

[0057] b. Add 2 mL of EDC / NHS mixed solution to 2 mL of Fe3O4-COOH dispersion obtained in step (1)a. Stir at room temperature for 6 h, then add 0.8 mL of 5 μmol / L aptamer MUC1 (Apt1) and 0.8 mL of 5 μmol / L nucleolin aptamer AS1411 (Apt2). Incubate at 4 °C for 6–8 h, then add 0.4 mL of 2 wt% bovine serum albumin (BSA) solution. Incubate at room temperature for 2–3 h to block non-specific active sites. Finally, magnetically separate and redisperse in 3 mL of water to obtain the Fe3O4-APTs dispersion of the capture unit.

[0058] (2) Synthesis of signal unit GO@PTCA-APTs

[0059] a. Add 20 mg of 3,4,9,10-perylenetetracarboxylic acid dianhydride (PTCDA) to 5 mL of freshly prepared 5 mol / L NaOH solution. Heat and stir at 80 °C until PTCDA is completely dissolved. After the solution turns yellow-green, continue to add 2 mol / L HCl dropwise until the mixture turns completely red. Centrifuge at 4400–5000 × g and wash the precipitate 5 times alternately with anhydrous ethanol and ultrapure water. Redisperse the precipitate in 8 mL of water to obtain a 3,4,9,10-perylenetetracarboxylic acid (PTCA) dispersion.

[0060] b. Add 0.3 mL of 2-4 mg / mL PTCA dispersion obtained in step (2)a to 5 mL of 0.5 mg / mL graphene oxide (GO), and stir at room temperature in the dark for 16 h to obtain GO@PTCA dispersion; add 2 mL of EDC / NHS mixed solution to 3 mL of GO@PTCA dispersion, stir at room temperature in the dark for 10 h, then add 0.7 mL of 5 μmol / L aptamer MUC1 (Apt1) and 0.4 mL of 5 μmol / L nucleolin aptamer AS1411 (Apt2), incubate at 4℃ for 6-8 h, add 0.4 mL of 2 wt% bovine serum albumin (BSA) solution, incubate at room temperature for 2-3 h to block non-specific active sites, centrifuge at 4400-5000×g and redisperse in 3 mL of water to obtain signal units GO@PTCA-APTs;

[0061] (3) Construction of Faraday cage-type electrochemiluminescence biosensor

[0062] Add 30 μL of the capture unit and 30 μL of the signal unit to 1 mL of cell standard solution, incubate at 37 °C for 1–1.5 h, and then magnetically separate. The resulting complex of capture unit-circulating tumor cell-signal unit is redispersed in 40 μL of PBS solution and dropped onto the surface of a magnetic glassy carbon electrode (MGCE). This completes the one-step preparation of a Faraday cage electrochemical sensor for detecting circulating tumor cells.

[0063] In specific embodiment two, the Faraday cage-type electrochemiluminescence biosensor for detecting circulating tumor cells prepared in specific embodiment one above is used as the working electrode, a platinum electrode as the auxiliary electrode, and an Ag / AgCl electrode (the reference electrode contains a 3 mol / L KCl solution) as the reference electrode. It is placed in a 0.1 mol / L Tris-HCl buffer solution containing 0.1 mol / L co-reaction reagent K₂S₂O₈ at pH 8.7. Electrochemiluminescence (ECL) is used, with a potential range of -1.9 V to 0.0 V, a potential scan rate of 0.1 V / s, and a photomultiplier tube voltage of 800 V. The ECL intensity corresponding to different concentrations of circulating tumor cells (MCF-7) is measured. The concentration of circulating tumor cells in the test solution is obtained based on the ECL signal value. The detection principle is as follows: Figure 1 As shown.

[0064] like Figure 2 As shown, the electrochemiluminescence intensity increases with increasing MCF-7 concentration.

[0065] like Figure 3 As shown, in 10 to 10 6 Within the cell / mL range, the electrochemiluminescence intensity (y) showed a good linear relationship with the logarithm of the MCF-7 concentration (x). The linear regression equation was y = 1857.17 * logx - 1013.29, and the correlation coefficient R² was [value missing]. 2 =0.997. Since the electrochemiluminescence intensity of the blank control is approximately 150 a.u., based on a signal-to-noise ratio (S / N) of 3, the limit of detection (LOD) is approximately 3 cells / mL. Specific Implementation Example 3

[0067] like Figure 4 As shown, the sensor prepared using Specific Embodiment 1 was used to detect blank and 10 samples respectively. 5 Cervical cancer cells / mL (HeLa), 10 4 Electrochemiluminescence (ECL) signal maps of the target MCF-7 (cells / mL) and a mixture of two circulating tumor cells. The signal detection results of HeLa, at a concentration 10 times higher than that of the target MCF-7, were similar to the blank group, with almost no ECL signal generated. The signal intensity of MCF-7 was close to that of the cell mixture sample containing MCF-7, indicating that this ECL biosensor has good selectivity for the detection of the target MCF-7. Specific Implementation Example 4

[0069] The accuracy and precision of the sensor were evaluated using the standard addition method. As shown in Table 1,

[0070] Table 1. Detection results of MCF-7 in the treated blood ( n=5)

[0071]

[0072] As shown in Table 1, the standard addition method was used to detect different concentrations of MCF-7 in the treated blood, and the recovery rate was 93%–107%, with an RSD of 2%–7%, indicating that the ECL biosensor has good accuracy and precision. Specific Implementation Example 5

[0074] Real blood sample analysis

[0075] like Figure 5 As shown, the sensor prepared in Specific Example 1 was used to detect clinical samples from a blank sample, two healthy individuals, and two breast cancer patients. The ECL signal detection results in the blood of the two healthy individuals were similar to those in the blank sample. According to the linear equation in Specific Example 2, the measured MCF-7 concentrations in the two breast cancer patients were approximately 7 cells / mL and 8 cells / mL, respectively, indicating that the ECL biosensor is practical for real-world sample detection.

[0076] The foregoing description is not intended to limit the invention, nor is the invention limited to the examples given. Any changes, modifications, additions, or substitutions made by those skilled in the art within the scope of the invention should also be considered within the protection scope of the invention.

Claims

1. A method for preparing a Faraday cage electrochemical sensor for detecting circulating tumor cells, characterized in that... Includes the following steps: (1) Synthesis of the trapping unit Fe3O4-APTs a. First, dissolve 0.5–0.7 g of anhydrous FeCl3 and 0.2–0.3 g of sodium citrate in 15–20 mL of ethylene glycol, then add 0.9–1.2 g of anhydrous sodium acetate. After stirring for 30 min, transfer the mixed solution to a stainless steel high-pressure reactor lined with polytetrafluoroethylene and place it in a constant temperature drying oven at 200–220 °C for 10 h. After cooling to room temperature, wash twice with ultrapure water and ethanol, and disperse in 15–20 mL of water after magnetic separation to obtain a carboxylated Fe3O4 dispersion. b. Add 1-2 mL of EDC / NHS mixed solution to 1-2 mL of carboxylated Fe3O4 dispersion obtained in step (1)a. Stir at room temperature for 6 h, then add 0.6-0.8 mL of 5-7 μmol / L aptamer MUC1Apt1 and 0.6-0.8 mL of 5-7 μmol / L nucleolin aptamer AS1411Apt2. Incubate at 4 °C for 6-8 h, then add 0.2-0.4 mL of 2 wt% bovine serum albumin solution. Incubate at room temperature for 2-3 h, then magnetically separate and redisperse in 1-3 mL of water to obtain the Fe3O4-APTs dispersion of the capture unit. (2) Synthesis of signal unit GO@PTCA-APTs a. Add 10-20 mg of 3,4,9,10-perylenetetracarboxylic acid dianhydride (PTCDA) to 2-5 mL of freshly prepared 4-5 mol / L NaOH solution. Heat and stir at 70-80 °C until PTCDA is completely dissolved. After the solution turns yellow-green, continue to add 1-2 mol / L HCl dropwise until the mixture turns completely red. Centrifuge at 4400-5000 × g and wash the precipitate 3-5 times alternately with anhydrous ethanol and ultrapure water. Redisperse the precipitate in 3-8 mL of water to obtain the PTCA dispersion. b. Add 0.1-0.3 mL of 2-4 mg / mL PTCA dispersion obtained in step (2)a to 2-5 mL of 0.5 mg / mL graphene oxide (GO), and stir at room temperature in the dark for 12-16 h to obtain GO@PTCA dispersion; add 1-2 mL of EDC / NHS mixed solution to 1-3 mL of GO@PTCA dispersion, and stir at room temperature in the dark for 6-10 h, then add 0.4-0.7 mL of 5-7 μmol / L aptamer MUC1Apt1 and 0.4 mL of 5-7 μmol / L nucleolin aptamer AS1411Apt2, incubate at 4℃ for 6-8 h, add 0.2-0.4 mL of 2 wt% bovine serum albumin solution, incubate at room temperature for 2-3 h, centrifuge at 4400-5000×g and redisperse in 1-3 mL of water to obtain signal unit GO@PTCA-APTs dispersion; (3) Construction of Faraday cage-type electrochemiluminescence biosensor Add 20–30 μL of capture unit dispersion and 20–30 μL of signal unit dispersion to 0.8–1 mL of cell standard solution, incubate at 37 °C for 1–1.5 h, and then magnetically separate. Redisperse the resulting complex capture unit-circulating tumor cell-signal unit in 20–40 μL of PBS solution and drop it onto the surface of a magnetic glassy carbon electrode to prepare a Faraday cage electrochemical sensor for detecting circulating tumor cells in one step.

2. The method for preparing the Faraday cage electrochemical sensor for detecting circulating tumor cells according to claim 1, characterized in that: The concentration of EDC in the EDC / NHS mixed solution is 0.4 mol / L, and the concentration of NHS is 0.1 mol / L.

3. The method for preparing the Faraday cage electrochemical sensor for detecting circulating tumor cells according to claim 1, characterized in that: The sequence of the aptamer MUC1 Apt1 is: 5′-NH2-GCAGTTGATCCTTTGGATACCCTGG-3′; the sequence of the nucleolin aptamer AS1411 Apt2 is: 5′-NH2-TTTGGTGGTGGTGGTTGTGGTGGTGGTG-3′.

4. The method for preparing the Faraday cage electrochemical sensor for detecting circulating tumor cells according to claim 1, characterized in that: The circulating tumor cells mentioned are MCF-7.

5. A method for detecting circulating tumor cells using a Faraday cage-type electrochemiluminescence biosensor prepared by any one of claims 1-4, wherein the method is not for diagnostic or therapeutic purposes, characterized in that... Includes the following steps: Using the prepared Faraday cage-type electrochemiluminescence biosensor for detecting circulating tumor cells as the working electrode, a platinum electrode as the auxiliary electrode, and an Ag / AgCl electrode as the reference electrode, the biosensor was placed in a 0.1 mol / L Tris-HCl buffer solution containing 0.1 mol / L co-reaction reagent K2S2O8 at pH 8.

7. The ECL intensity corresponding to different concentrations of circulating tumor cells was determined by electrochemiluminescence method, and the concentration of circulating tumor cells in the test solution was obtained based on the ECL signal value.

6. The method for detecting circulating tumor cells using a Faraday cage-type electrochemiluminescence biosensor according to claim 5, characterized in that... The electrochemiluminescence method described herein has a potential range of -1.9V to 0.0V, a potential scan rate of 0.1V / s, and a photomultiplier tube voltage of 800V.

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