A two-dimensional material biosensor based on CEA detection and a preparation method and application thereof

Abstract

The application discloses a two-dimensional material biosensor based on CEA detection and a preparation method and application thereof, and belongs to the technical field of two-dimensional material biosensing.The two-dimensional material biosensor based on CEA detection is prepared by the following steps: growing a single-layer WS2 crystal on a Si / SiO2 substrate by using a chemical vapor deposition method, transferring the single-layer WS2 crystal on an ITO substrate by using a wet transfer method, and treating the single-layer WS2 crystal by using bis(trifluoromethane)sulfonimide, so that Anti-CEA probe proteins are fixed by surface interfacial chemical modification, and the biosensor is obtained.The single-layer WS2 crystal prepared by the application is easy to grow, has uniform fluorescence, and has good performance; the biosensor has the characteristics of simple process, high sensitivity, low detection limit, biomolecule label-free and the like in the detection of carcinoembryonic antigen.

Description

Technical Field

[0001] This invention belongs to the field of two-dimensional material biosensing technology, specifically relating to a method for detecting carcinoembryonic antigen using biosensors. Background Technology

[0002] Carcinoembryonic antigen (CEA) is an acidic glycoprotein with characteristics of human embryonic antigens. It exists on the surface of cancer cells differentiated from endoderm cells and is a structural protein of the cell membrane. Formed in the cytoplasm, it is secreted through the cell membrane into the extracellular space and then enters surrounding body fluids. Therefore, it can be detected in various body fluids and excretions, including serum, cerebrospinal fluid, breast milk, gastric juice, pleural and peritoneal fluid, urine, and feces. CEA is an embryonic antigen primarily isolated from adenocarcinoma. Its elevation in body tissues is directly proportional to the number of cancer cells; if CEA levels are elevated, the risk of adenocarcinoma increases. As a tumor marker, CEA expression levels accurately reflect the number, proliferation, and mortality of cancer cells, and its levels are extremely low in healthy individuals. When tumor cells appear in a patient, the levels of tumor markers in the body rise abnormally within a short period, which can be used to assess disease progression. Elevated CEA levels are commonly seen in colorectal cancer, pancreatic cancer, gastric cancer, breast cancer, liver cancer, lung cancer, ovarian cancer, and urinary tract tumors.

[0003] Monolayer WS2 crystals possess many unique physical and chemical properties, attracting widespread attention and research from the academic and scientific communities. Monolayer WS2 crystals exhibit a wide, tunable bandgap, strong photoluminescence efficiency, and excellent carrier mobility, making them promising candidates for applications in fields such as biosensing. Currently, the main methods for preparing monolayer WS2 crystals include mechanical exfoliation, liquid-phase exfoliation, and chemical vapor deposition. The bandgap of monolayer WS2 crystals is approximately 1.97 eV, exhibiting strong photoluminescence efficiency and light absorption performance. However, structural defects such as sulfur atom vacancies in monolayer WS2 crystals lead to problems such as poor fluorescence and chemical stability.

[0004] In existing technologies, the paper "A DNAzyme-catalyzed label-free aptasensor based on multifunctional dendrimer-like DNA assembly for sensitive detection of carcinoembryonic antigen" discloses a simple, label-free, and antibody-free aptasensor sensor based on a multifunctional dendrimer-like DNA nanoassembly for detecting CEA. However, the modification process in this paper is relatively complex, the operation process is even more complicated, and the detection limit is relatively high, which is not conducive to industrialization.

[0005] The paper "Magnetic antifouling material-based ratiometric electrochemical biosensor for the accurate detection of CEA in clinical serum" discloses a novel electrochemical biosensor based on multifunctional nanocomposite materials. However, the sample synthesis process in this paper is relatively complex, time-consuming, costly, and has a high detection limit, which is not conducive to industrialization.

[0006] In summary, the main drawbacks of current CEA biosensors are: First, they have a high detection limit and low sensitivity. Second, the synthesis process of the substrate sample is relatively complex. Third, the preparation process is complex, requires sophisticated equipment, and is costly, making them unsuitable for large-scale production. Summary of the Invention

[0007] To address the problems existing in the prior art, the present invention aims to design and provide a CEA biosensor with low detection limit, high detection sensitivity, and rapid and efficient detection, as well as its preparation method. The monolayer WS2 crystal prepared by this invention is easy to grow, exhibits uniform fluorescence, and demonstrates good performance. This biosensor offers advantages such as simple process, high sensitivity, low detection limit, and label-free biomolecule detection of carcinoembryonic antigen. The binding of the monolayer WS2 crystal to carcinoembryonic antigen causes a change in the fluorescence signal of the monolayer WS2 crystal, thereby detecting the concentration of carcinoembryonic antigen.

[0008] This invention primarily utilizes CVD to grow high-quality monolayer WS2 crystals. The monolayer WS2 crystals are treated with the super-strong organic acid TFSI to repair or eliminate structural defects such as sulfur atom vacancies, resulting in poor fluorescence and chemical stability, thereby improving the fluorescence and chemical stability of the monolayer WS2 crystals. Combining fluorescence confocal imaging and electrochemical techniques, the effects of different voltages on the fluorescence of the monolayer WS2 crystals are systematically studied, revealing the charge-sensitive properties of the monolayer WS2 crystals. Further surface / interface chemical modification of the monolayer WS2 crystals enables highly sensitive and specific detection of CEA molecules.

[0009] To achieve the above objectives, the present invention adopts the following technical solution:

[0010] A two-dimensional material biosensor based on CEA detection is obtained by growing a monolayer WS2 crystal onto a Si / SiO2 substrate using chemical vapor deposition, transferring it to an ITO substrate using a wet transfer method, treating the monolayer WS2 crystal with bis(trifluoromethane)sulfonylimide (TFSI), and immobilizing the Anti-CEA probe protein through surface and interface chemical modification to obtain the biosensor.

[0011] The method for fabricating a two-dimensional material biosensor based on CEA detection includes the following steps:

[0012] (1) Transfer the monolayer WS2 crystal grown on the Si / SiO2 substrate by chemical vapor deposition to the ITO substrate, remove the glue on the monolayer WS2 crystal, place it in bis(trifluoromethane)sulfonylimide solution, immerse it on a hot plate, take it out, blow it dry with nitrogen, and then anneal it under the hot plate.

[0013] (2) Soak in 11-mercaptoundecanoic acid (MUA), then treat with carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS), and modify the monolayer WS2 crystal after the above treatment with Anti-CEA to obtain a biosensor.

[0014] In the preparation method described above, the concentration of the bis(trifluoromethane)sulfonylimide solution in step (1) is 0.2 mg / mL; the immersion conditions on the hot plate are: temperature 100-120℃, time 10-12 min; the annealing conditions under the hot plate are: temperature 100-120℃, time 5-6 min.

[0015] The preparation method described above, specifically the method for removing the adhesive on the monolayer WS2 crystal in step (1) is as follows: the monolayer WS2 crystal on the ITO substrate is immersed in acetone.

[0016] The preparation method described herein, wherein the soaking conditions are: temperature 65-70℃, time 15-20min.

[0017] In the preparation method described above, the concentration of 11-mercaptoundecanoic acid in step (2) is 50 mM; the soaking time of 11-mercaptoundecanoic acid is 24 h; and the soaking treatment time of carbodiimide hydrochloride and N-hydroxysuccinimide is 4 to 6 h.

[0018] In the preparation method described above, the concentration ratio of carbodiimide hydrochloride to N-hydroxysuccinimide in step (2) is 4:1; preferably, the concentration of carbodiimide hydrochloride is 400 mM / L and the concentration of N-hydroxysuccinimide is 100 mM / L.

[0019] In the preparation method described above, the Anti-CEA modification time in step (2) is 12 hours.

[0020] A kit for detecting carcinoembryonic antigen concentration, comprising the aforementioned biosensor.

[0021] The application of the two-dimensional material biosensor based on CEA detection in detecting carcinoembryonic antigen concentration.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] 1. The biosensor of this invention utilizes a novel two-dimensional material;

[0024] 2. The biosensor of this invention has a low detection limit and high detection sensitivity;

[0025] 3. Label-free detection of biological samples using the biosensor of this invention;

[0026] 4. The biosensor of this invention has a fast detection time, a simple process, and is easy to operate. Attached Figure Description

[0027] Figure 1 A biological detection diagram of the experimental setup and monolayer WS2 crystal used in this invention;

[0028] Figure 2 Raman spectrum and PL spectrum of the monolayer WS2 crystal prepared for the present invention;

[0029] Figure 3 This is a diagram illustrating the enhanced fluorescence and chemical stability of a single-layer WS2 crystal as described in this invention.

[0030] Figure 4 The graph shows the results of CEA detection using the biosensor of this invention. Detailed Implementation

[0031] Example 1:

[0032] (1) Place 1.5g of S powder and 2.0g of WO3 powder into two corundum boats, respectively. Then place the S boat and the WO3 boat in the low-temperature (240℃) and high-temperature (910℃) zones of a tube furnace, respectively. The heating time and holding time in the low-temperature zone and the high-temperature zone are 30min and 20min, respectively. Place the Si / SiO2 substrate upside down in the WO3 boat and pass 30sccmAr (pressure 160~170Pa) through it to complete the preparation of high-quality monolayer WS2 crystals.

[0033] (2) The monolayer WS2 crystal on the Si / SiO2 substrate was transferred to a conductive ITO substrate using a wet transfer method. The first spin coating was performed (low speed 700 rpm for 10 s, high speed 4000 rpm for 60 s), followed by drying at 170℃ for 10 min. A second spin coating was then performed, followed by drying at 170℃ for another 10 min. The edges were scraped off, and the sample was placed in water at 120℃ for 30 min until it floated. It was then retrieved using the ITO substrate and dried on a drying rack (120℃) for 2–3 h. Next, it was immersed in acetone at 65℃ for 15 min to remove the adhesive from the surface of the monolayer WS2 crystal. Protonation treatment was then performed on the surface of the monolayer WS2 crystal. The monolayer WS2 crystal was placed in a pre-prepared 0.2 mg / mL TFSI solution and immersed on a hot plate (120℃) for 10 min. The sample was then removed, dried with nitrogen, and subsequently annealed at 100℃ for 5 min to improve the fluorescence and chemical stability of the monolayer WS2 crystal.

[0034] (3) The detection instrument used was a confocal fluorescence microscope. The sample was irradiated with a 561nm laser at an appropriate laser power. Then, the fluorescence performance of the TFSI-treated monolayer WS2 crystal was modulated using an electrochemical workstation, and imaging was performed using a photomultiplier tube (PMT) detector. A three-electrode working mode was used to modulate the voltage of the TFSI-treated monolayer WS2 crystal. First, the sample was tested in linear voltammetry mode (-1V to 0.2V). It was found that the fluorescence signal change rate was the largest at -0.7V. Then, the sample was tested in cyclic voltammetry mode (amplitude 50, 100, 200, 300mV; frequency 1, 3, 5, 10Hz). It was found that the fluorescence signal change was the strongest at 300mV and 1Hz.

[0035] (4) The monolayer WS2 crystals treated with TFSI were then soaked in 50 mM 11-mercaptoundecanoic acid (MUA) for 24 h, followed by soaking in 400 mM carbodiimide hydrochloride (EDC) and 100 mM N-hydroxysuccinimide (NHS) at a concentration ratio of 4:1 for 4–6 h, and finally treated with Anti-CEA (10 -4 The sample was modified with g / L for 12 hours to prepare a CEA biosensor.

[0036] like Figure 1This image shows the experimental setup and biodetection of a monolayer WS2 crystal used in this invention. Combining confocal fluorescence imaging and electrochemical techniques, tests were performed using a confocal fluorescence microscope and an electrochemical workstation. A 561nm laser was selected, with an excitation range of 550nm–680nm, allowing for fluorescence imaging of the monolayer WS2 crystal. Testing was conducted using the electrochemical workstation (DC voltage -0.7V, amplitude 300mV, frequency 1Hz). Carcinoembryonic antigen (CEA) and its antibody were linked to the monolayer WS2 crystal using a two-dimensional material interface modification method to complete the detection.

[0037] like Figure 2 Raman spectrum and PL plot of the monolayer WS2 crystal prepared for this invention. Raman characterization was performed using a 532 nm laser, and the Raman spectrum can be observed up to 175 cm⁻¹. -1 192cm -1 212cm -1 230cm -1 297cm -1 323cm -1 350cm -1 418cm -1 Interlaminar vibration A1′ (418.6cm) -1 ) and the intralayer vibration E′ (356.5cm) -1 The two peaks near the [missing information] are consistent with the Raman spectral peaks of monolayer WS2 crystal reported in the literature. PL plots revealed that the monolayer WS2 crystal is a direct bandgap semiconductor with a bandgap of 1.97 eV.

[0038] Example 2:

[0039] Based on relevant literature review, the isoelectric point of CEA protein was determined to be pH 4.5–5.5. CEA was prepared into solutions of different concentrations (10... -15 g / L~10 -9 Different concentrations of CEA solutions (g / L) were detected using a biosensor. Cyclic voltammetry was performed using an electrochemical workstation with parameters of -0.7V, 1Hz, and 300mV. The fluorescence intensity changes of monolayer WS2 crystals at each concentration were then observed.

[0040] like Figure 3 As can be seen, when the photoluminescence (PL) spectra of monolayer WS2 crystals treated with and without TFSI organic acid were tested using a 532nm laser, it was found that the PL of monolayer WS2 crystals treated with TFSI organic acid was significantly enhanced.

[0041] The fluorescence stability of monolayer WS2 crystals treated with and without TFSI organic acid was tested using confocal fluorescence microscopy. A 561 nm laser was used, with an excitation range of 550 nm to 680 nm and a power of 0.5% (0.09 mW). The fluorescence stability of the monolayer WS2 crystals treated with TFSI organic acid was found to be significantly enhanced.

[0042] Example 3:

[0043] The concentration of CEA protein was detected using a combination of confocal fluorescence microscopy and an electrochemical workstation. The confocal fluorescence microscope used a 561 nm laser with an excitation range of 550 nm–680 nm and a power of 0.5% (0.09 mW). The electrochemical workstation was configured with a DC voltage of -0.7 V, an amplitude of 300 mV, and a frequency of 1 Hz. When testing different CEA protein concentrations, a biosensor was used to detect concentrations from low to high. The CEA protein solution was added to the biosensor and allowed to stand for approximately 15–20 minutes to allow for sufficient and specific binding of carcinoembryonic antigen (CEA) and its antibody. The tests were then performed according to the electrochemical workstation parameters. The obtained data were processed and extracted using ImageJ software. Figure 4 The graph shown.

Claims

1. A two-dimensional material biosensor based on CEA detection, characterized in that, Monolayer WS2 crystals were grown on a Si / SiO2 substrate using chemical vapor deposition, then transferred to an ITO substrate using a wet transfer method. The monolayer WS2 crystals were then treated with bis(trifluoromethane)sulfonylimide. Through surface and interface chemical modification, the Anti-CEA probe protein was immobilized to obtain a biosensor.

2. The method for fabricating a two-dimensional material biosensor based on CEA detection as described in claim 1, characterized in that, Includes the following steps: (1) Transfer the monolayer WS2 crystal grown on the Si / SiO2 substrate by chemical vapor deposition to the ITO substrate, remove the glue on the monolayer WS2 crystal, place it in bis(trifluoromethane)sulfonylimide solution, immerse it on a hot plate, take it out, blow it dry with nitrogen, and then anneal it under the hot plate. (2) Soak in 11-mercaptoundecanoic acid, then soak in carbodiimide hydrochloride and N-hydroxysuccinimide, and modify the monolayer WS2 crystal after the above treatment with Anti-CEA to obtain a biosensor.

3. The preparation method according to claim 2, characterized in that, The concentration of the bis(trifluoromethane)sulfonylimide solution in step (1) is 0.2 mg / mL; the immersion conditions on the hot plate are: temperature 100~120℃, time 10~12 min; the annealing conditions under the hot plate are: temperature 100~120℃, time 5~6 min.

4. The preparation method according to claim 2, characterized in that, The specific method for removing the adhesive on the monolayer WS2 crystal in step (1) is as follows: immerse the monolayer WS2 crystal on the ITO substrate in acetone.

5. The preparation method according to claim 4, characterized in that, The soaking conditions are: temperature 65~70℃, time 15~20 min.

6. The preparation method according to claim 2, characterized in that, The concentration of 11-mercaptoundecanoic acid in step (2) is 50 mM; the soaking time of 11-mercaptoundecanoic acid is 24 h; and the soaking time of carbodiimide hydrochloride and N-hydroxysuccinimide is 4-6 h.

7. The preparation method according to claim 2, characterized in that, The concentration ratio of carbodiimide hydrochloride to N-hydroxysuccinimide in step (2) is 4:1; preferably, the concentration of carbodiimide hydrochloride is 400 mM / L and the concentration of N-hydroxysuccinimide is 100 mM / L.

8. The preparation method according to claim 2, characterized in that, The Anti-CEA modification time in step (2) is 12 h.

9. A kit for detecting carcinoembryonic antigen concentration, characterized in that, It includes the biosensor as described in claim 1.

10. The use of the two-dimensional material biosensor based on CEA detection as described in claim 1 in the preparation of a kit for detecting carcinoembryonic antigen concentration.

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