A two-dimensional red phosphorus nanosheet / molybdenum disulfide flower immunogenic structure, its preparation method, and its application.

Abstract

This invention provides a two-dimensional red phosphorus nanosheet / molybdenum disulfide flower immunoassay structure, its preparation method, and its application. The immunoassay substrate, using two-dimensional red phosphorus nanosheets as a carrier, and the immunoassay probe, using 3D MoS2NFs as a carrier, are applied to the detection of SERS tumor markers. The two-dimensional red phosphorus nanosheets are prepared using a hydrothermal method and an ice-bath ultrasonic method. A Raman laser is used to screen for the substrate with the strongest Raman signal. 3D MoS2NFs are prepared using a hydrothermal method. Both red phosphorus and molybdenum disulfide have good biocompatibility and are simple and low-cost to prepare. Using two-dimensional red phosphorus nanosheets as a substrate to construct a sandwich structure and preparing the 3D molybdenum disulfide flower immunoassay probe avoids molecular aggregation on the two-dimensional layered material and improves the sensitivity of SERS detection.

Description

Technical Field

[0001] This invention relates to the field of biodetection technology, and in particular to a two-dimensional red phosphorus nanosheet / molybdenum disulfide flower immunoassay structure, its preparation method, and its application. Background Technology

[0002] Surface-enhanced Raman scattering (SERS) is a highly sensitive and fast-response molecular sensing technique. Most SERS substrates are dominated by noble metals (such as gold and silver). Achieving high-density "hot spots" by controlling the spacing between metal nanoparticles is a key factor in realizing the significant enhancement effect of noble metal SERS; however, this usually requires sophisticated procedures. Furthermore, the poor reproducibility and molecular selectivity of noble metals limit their development in practical applications. In contrast, semiconductors offer a wide variety of types and more tunable parameters. Designing semiconductor substrates holds promise for overcoming many application limitations of noble metal substrates and improving the enhancement effect of semiconductor SERS. Two-dimensional materials possess rich physical properties, such as their inherent van der Waals forces, giving them bandgap-dependent layered structures. Molybdenum disulfide (MoD) exhibits a layered structure, with layers fixed by van der Waals forces. Furthermore, nanostructured MoD has a large specific surface area and high surface activity. Meanwhile, the novel two-dimensional material red phosphorus (RP) possesses a non-toxic, highly stable layered structure, and its broad visible light absorption range gives it the potential for tunable bandgap, potentially leading to continuously tunable SERS performance. However, research on two-dimensional red phosphorus in this area is somewhat lacking. In addition, red phosphorus materials are currently mostly used in the field of photocatalysis, and their application in tumor marker detection urgently needs further exploration.

[0003] According to current SERS-based immunoassay research, the immunoassay process based on two-dimensional layered materials is prone to intermolecular aggregation. Therefore, it is hoped that by combining the above ideas, a reasonable experimental scheme can be designed to solve the problems of error and malignant molecular aggregation in actual detection. By studying non-noble metal nanoparticles as substrates, new avenues will be opened for the application of SERS technology in the field of biomedical detection. Summary of the Invention

[0004] One advantage of this invention is that it provides a two-dimensional red phosphorus nanosheet / molybdenum disulfide flower immunoassay structure, its preparation method, and its application. The prepared two-dimensional red phosphorus nanosheet / molybdenum disulfide flower immunoassay structure is suitable for the detection of SERS tumor markers, exhibiting high sensitivity and accuracy.

[0005] Another advantage of this invention is that it provides a two-dimensional red phosphorus nanosheet / molybdenum disulfide flower immune structure, preparation method and application. The two-dimensional red phosphorus nanosheet is used as an immune substrate to construct a "sandwich" structure and prepare 3D molybdenum disulfide flowers as immune probes. This avoids the aggregation of molecules on two-dimensional layered materials, overcomes the defects of metal substrates, and improves the sensitivity of SERS detection.

[0006] Another advantage of this invention is that it provides a two-dimensional red phosphorus nanosheet / molybdenum disulfide flower immune structure, its preparation method, and its application. By using a simple and efficient method, bulk red phosphorus is transformed into nanosheets with a larger surface area, which significantly improves the molecular adsorption capacity.

[0007] Another advantage of this invention is that it provides a two-dimensional red phosphorus nanosheet / molybdenum disulfide flower immune structure, preparation method and application. The change in the size of the two-dimensional red phosphorus nanosheet adjusts its visible light absorption range and band gap, and the two-dimensional red phosphorus nanosheet with the best SERS performance is obtained by Raman characterization screening, that is, the substrate with the strongest Raman signal is screened.

[0008] Another advantage of this invention is that it provides a two-dimensional red phosphorus nanosheet / molybdenum disulfide flower immune structure, its preparation method and application. Both red phosphorus and molybdenum disulfide have good biocompatibility, and the invention is simple to prepare, low in cost, and easy to use, making it suitable for clinical application.

[0009] Another advantage of this invention is that it provides a two-dimensional red phosphorus nanosheet / molybdenum disulfide flower immunoassay structure, preparation method and application, which overcomes many defects of noble metal substrates that limit their application range due to poor reproducibility and molecular selectivity. The two-dimensional red phosphorus / molybdenum disulfide flower immunoassay structure provided by this invention has a wide range of applications, is more stable and accurate in detection.

[0010] Another advantage of this invention is that it provides a two-dimensional red phosphorus nanosheet / molybdenum disulfide flower immune structure, its preparation method, and its application. Red phosphorus nanosheets and black phosphorus nanosheets are prepared simultaneously through the same experiment. By comparison, it can be found that two-dimensional red phosphorus nanosheets are more suitable as an immune substrate to construct sandwich structures for the detection of tumor markers, with higher detection sensitivity.

[0011] Another advantage of this invention is that it provides a two-dimensional red phosphorus nanosheet / molybdenum disulfide flower immune structure, its preparation method and application. It uses non-precious metal two-dimensional red phosphorus nanosheets as a substrate, which does not require the use of biotoxic surfactants, making it more friendly to the human body and the environment, and thus beneficial to environmental protection.

[0012] Another advantage of this invention is that it provides a two-dimensional red phosphorus nanosheet / molybdenum disulfide flower immunoassay structure, its preparation method, and its application. This overcomes the various defects of using gold or silver nanomaterials as substrates. By using non-precious metal nanoparticles as substrates, it will open up new avenues for the application of SERS technology in the field of biomedical detection and has great significance for the development of the biomedical detection field.

[0013] According to one aspect of the present invention, a method for preparing a two-dimensional red phosphorus nanosheet / molybdenum disulfide flower immune structure is provided, the method comprising the following steps:

[0014] (1) Preparation of two-dimensional red phosphorus nanosheets;

[0015] (2) Screening two-dimensional red phosphorus nanosheets as the optimal substrate;

[0016] (3) Preparation of 3D MoS2 NFs;

[0017] (4) Preparation of immune base;

[0018] (5) Preparation of immune probes; and

[0019] (6) Preparation of sandwich structure.

[0020] In step (1), two-dimensional red phosphorus nanosheets are prepared by hydrothermal method and ice bath ultrasonic method. In step (2), the substrate with the strongest Raman signal is screened by Raman laser to prepare the immune substrate in step (4). In step (3), 3D MoS2 NFs are prepared by hydrothermal method.

[0021] In step (1), the preparation of two-dimensional red phosphorus nanosheets includes the following steps: (101) take red phosphorus, grind and sieve to obtain a sample; (102) add deionized water to the sample to obtain a red phosphorus solution; (103) sonicate the red phosphorus solution and transfer it into a high-pressure autoclave lined with polytetrafluoroethylene, and treat it in a water bath at 200°C for 12 hours; (104) after the hydrothermal treatment is completed, centrifuge and disperse, wash three times with anhydrous ethanol and deionized water respectively, and take the lower precipitate; (105) dissolve the obtained red phosphorus again in deionized water, and sonicate it with a probe-type ultrasonic instrument, with working and intermittent phases of 5s and 3s respectively, and sonicate 99 times as a working cycle; and (106) centrifuge the taken red phosphorus suspension at 4000rpm for 10min, take out the supernatant, and store it in a refrigerator.

[0022] Depending on the working cycle, 0, 4, 8, 12, and 16 working cycles were selected to prepare RP-0, RP-4, RP-8, RP-12, and RP-16 samples, respectively. Among them, the size of RP-0, RP-4, RP-8, RP-12, and RP-16 samples gradually decreased, and the Raman signal of RP-4 sample was the strongest.

[0023] In step (2), red phosphorus supernatant is added dropwise to a silicon wafer, dried, crystal violet molecules are added dropwise, and a Raman laser is used as the excitation source to draw a Raman spectrum.

[0024] The Raman laser has a wavelength of 532 nm, a laser spot diameter of 12.5 μm, a laser power of 1 mW, and an integration time of 10 s.

[0025] The concentration of the red phosphorus supernatant was 1 mg / mL, and the concentration of the crystal violet was 1 × 10⁻⁶. -2 mg / mL.

[0026] Step (3) includes the following steps: Na2MoO4·2H2O, CH3CSNH2 and H4[Si(W3O 10 [4]·xH2O was dissolved in deionized water and stirred. The resulting solution was transferred to a stainless steel autoclave lined with polytetrafluoroethylene and hydrothermally treated at 220°C for 24 hours. After cooling to room temperature, the product was washed and filtered with NaOH, anhydrous ethanol and deionized water in sequence to obtain MoS2 deposits. The washed product was dried in a vacuum drying oven at 60°C for 6 hours to obtain 3D MoS2 NFs, which were then packaged and stored at room temperature.

[0027] The MoS2 NFs prepared by the above steps are three-dimensional irregular spheres with a diameter of approximately 200-400 nm.

[0028] The substrate RP-4 with the best SERS performance selected in step (2) was dropped onto a silicon wafer, dried at room temperature, and then cancer antibody solution was added. The substrate was incubated at 4°C for at least 12 hours, rinsed to remove residual antibodies, and then BSA solution was added and placed at room temperature for 3 hours. Antigen solutions of different concentrations were added to the substrate and incubated at 37°C for 2 hours. The substrate was then washed to obtain the immune substrate.

[0029] The BSA solution uses PBS solvent and bovine serum albumin powder. In this embodiment, the mass of the bovine serum albumin powder is 3%.

[0030] Excess antigen was removed by rinsing with TBS, PBS and deionized water, with the ratio of PBS to deionized water being 1:4 and the ratio of TBS to deionized water being 1:4.

[0031] According to another aspect of the present invention, the present invention also provides a two-dimensional red phosphorus nanosheet / molybdenum disulfide flower immune structure, the two-dimensional red phosphorus nanosheet / molybdenum disulfide flower immune structure comprising: an immune substrate with two-dimensional red phosphorus nanosheets as a carrier and an immune probe with 3D MoS2 NFs as a carrier, wherein the MoS2 NFs are irregular spheres with a diameter of 200-400 nm, and the immune probe and the immune substrate are prepared as a sandwich structure.

[0032] The two-dimensional red phosphorus nanosheet / molybdenum disulfide flower immune structure described herein is prepared using the method provided in this invention.

[0033] According to another aspect of the present invention, the present invention also provides an application of a two-dimensional red phosphorus nanosheet / molybdenum disulfide flower immunostructure, wherein the two-dimensional red phosphorus nanosheet / molybdenum disulfide flower immunostructure is suitable for use in the detection of SERS tumor markers. Attached Figure Description

[0034] Figure 1 This is a scanning electron microscope image of RP-0 prepared according to a preferred embodiment of the present invention.

[0035] Figure 2 These are scanning electron microscope images of RP-4 prepared according to the preferred embodiments described above in this invention.

[0036] Figure 3 These are scanning electron microscope images of RP-8 prepared according to the preferred embodiments described above in this invention.

[0037] Figure 4 These are scanning electron microscope images of RP-12 prepared according to the preferred embodiments described above in this invention.

[0038] Figure 5 These are scanning electron microscope images of RP-16 prepared according to the preferred embodiments described above in this invention.

[0039] Figure 6 The Raman spectra are drawn based on the Raman detection results of crystal violet by RP-0, RP-4, RP-8, RP-12, and RP-16 in the preferred embodiments of the present invention.

[0040] Figure 7 This is a scanning electron microscope image of MoS2NFs prepared according to the preferred embodiment of the present invention.

[0041] Figure 8 This is a scanning electron microscope image of the two-dimensional red phosphorus nanosheets / MoS2NFs immunostructure prepared according to the above-described preferred embodiment of the present invention.

[0042] Figure 9 The results of Raman detection of CEA tumor markers are based on the two-dimensional red phosphorus nanosheets / MoS2NFs immune structure prepared according to the above-described preferred embodiment of the present invention.

[0043] Figure 10 These are scanning electron microscope images of BP NSs prepared according to the preferred embodiments described above in this invention.

[0044] Figure 11 The Raman spectra are plotted based on the Raman detection results of crystal violet by RP-0, RP-16 and BP NSs prepared according to the above embodiments of the present invention. Detailed Implementation

[0045] The following description is intended to disclose the present invention and enable those skilled in the art to implement it. The preferred embodiments described below are merely examples, and other obvious variations will occur to those skilled in the art. The basic principles of the invention defined in the following description can be applied to other embodiments, modifications, improvements, equivalents, and other technical solutions that do not depart from the spirit and scope of the invention.

[0046] Those skilled in the art should understand that, in the disclosure of this invention, the terms "top," "bottom," "inner," "outer," etc., indicate the orientation or positional relationship based on the orientation or positional relationship shown in the accompanying drawings. They are only for the convenience of describing this invention and simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation. Therefore, the above terms should not be construed as limiting this invention.

[0047] It is understood that the term "a" should be understood as "at least one" or "one or more", that is, in one embodiment, the number of an element can be one, while in another embodiment, the number of the element can be multiple, and the term "a" should not be understood as a limitation on the number.

[0048] Reference Appendix Figure 1 To be continued Figure 9 As shown, a preferred embodiment of the present invention discloses a method for preparing a two-dimensional red phosphorus nanosheet / molybdenum disulfide flower (MoS2 NFs) immunostructure based on SERS detection of tumor markers, wherein the method for preparing the two-dimensional red phosphorus nanosheet / molybdenum disulfide flower (MoS2 NFs) immunostructure includes the following steps:

[0049] (1) Preparation of two-dimensional red phosphorus nanosheets;

[0050] (2) Screening two-dimensional red phosphorus nanosheets as the optimal substrate;

[0051] (3) Preparation of 3D MoS2 NFs;

[0052] (4) Preparation of immune base;

[0053] (5) Preparation of immune probes; and

[0054] (6) Preparation of sandwich structure.

[0055] In step (S101), two-dimensional red phosphorus nanosheets are efficiently prepared using a hydrothermal method and an ice bath ultrasonic method. The specific preparation method of the two-dimensional red phosphorus nanosheets includes the following steps:

[0056] (S101) Grind the blocky red phosphorus and sieve it;

[0057] (S102) Prepare a red phosphorus solution using the above-mentioned red phosphorus, and then sonicate it.

[0058] (S103) Transfer the sample to an autoclave and treat it in a water bath;

[0059] (S104) Centrifuge to disperse, wash, and remove the lower layer precipitate;

[0060] (S105) Ultrasound, sampling; and

[0061] (S106) Centrifuge.

[0062] A small amount of commercial red phosphorus was ground in a mortar and then sieved through a 300-mesh sieve. 1 g of the sample was added to 60 ml of deionized water to prepare a red phosphorus solution with a concentration of 1 / 60 g / mL. The solution was sonicated for 30 min, then transferred to a 100 ml stainless steel autoclave lined with polytetrafluoroethylene (PTFE) and hydrothermally treated at 200 °C for 12 h. After hydrothermal treatment, the resulting sample was centrifuged and dispersed. The sample was then washed three times with anhydrous ethanol and deionized water, and the lower precipitate was collected. The red phosphorus was dissolved again in deionized water and transferred to a probe-type ultrasonic instrument. The ultrasonic power was 100 W, the work / interval time was 5 / 3 s, and a total of 99 cycles were performed, which is called one work cycle. Different samples were prepared according to the different work cycles.

[0063] First, a portion of the RP-0 samples that were not ultrasonically tested were taken. Then, samples RP-4, RP-8, RP-12, and RP-16 were taken out every four cycles. That is, samples RP-4, RP-8, RP-12, and RP-16 were taken out after the ultrasonic instrument had worked for 4, 8, 12, and 16 cycles, respectively.

[0064] Finally, the above red phosphorus (RP) suspension was centrifuged at 4000 rpm for 10 min, and the supernatant was collected.

[0065] Specifically, the following samples were prepared:

[0066] Preparation and electron microscopy analysis of samples RP-0 and RP-4:

[0067] In step (S101), a small amount of commercial red phosphorus (RP) is ground in a mortar and then sieved through a 300-mesh sieve.

[0068] In step (S102), 1g of sample is added to 60ml of deionized water to prepare a red phosphorus solution with a concentration of 1 / 60g / ml, and then sonicated for 30min.

[0069] In step (S103), the sample from step (S102) is transferred to a 100ml polytetrafluoroethylene-lined stainless steel autoclave and hydrothermally treated at 200°C for 12 hours.

[0070] In step (S104), after the hydrothermal treatment is completed, the obtained sample is centrifuged and dispersed, and then washed three times with anhydrous ethanol and deionized water respectively before the lower layer precipitate is taken out.

[0071] The step (S105) includes the following two steps:

[0072] (S10501) The red phosphorus obtained in the above step (S104) is dissolved again in deionized water and transferred to a probe-type ultrasonic instrument.

[0073] The probe-type ultrasonic instrument has an ultrasonic power of 100W, a working time of 5s, an interval of 3s, and the working time and interval are spaced apart for a total of 99 times, which is called one working cycle.

[0074] (S10502) First, take a portion of the unultrasonicated red phosphorus sample as RP-0, and then take the red phosphorus sample after 4 working cycles of ultrasound as RP-4.

[0075] In step (S106), the above RP-0 and RP-4 suspensions are centrifuged at 4000 rpm for 10 min, the supernatant is taken out, placed in a refrigerator, and packaged for storage for later use.

[0076] Figure 1 Here is a scanning electron microscope image of the RP-0 prepared in Embodiment 1 of this disclosure; from Figure 1 It can be seen that RP-0 is a red phosphorus sample that has not been sonicated. The wrinkles on its side indicate that the blocky red phosphorus is composed of multiple tightly packed sheets, with a size of about 1 micrometer.

[0077] Figure 2 Here is a scanning electron microscope image of RP-4 prepared in Embodiment 1 of this disclosure; from Figure 2 It can be seen that PR-4 is a layered material. That is to say, after four working cycles of ultrasonic treatment, RP-0 is transformed from a blocky material into a layered material, and the size of RP-4 is approximately 600-800 nanometers.

[0078] Preparation and electron microscopy analysis of sample RP-8:

[0079] In step (S101), a small amount of commercial red phosphorus (RP) is ground in a mortar and then sieved through a 300-mesh sieve.

[0080] In step (S102), 1g of sample is added to 60ml of deionized water to prepare a red phosphorus solution with a concentration of 1 / 60g / ml, and then sonicated for 30min.

[0081] In step (S103), the sample from step (S102) is transferred to a 100ml polytetrafluoroethylene-lined stainless steel autoclave and hydrothermally treated at 200°C for 12 hours.

[0082] In step (S104), after the hydrothermal treatment is completed, the obtained sample is centrifuged and dispersed, and then washed three times with anhydrous ethanol and deionized water respectively before the lower layer precipitate is taken out.

[0083] The step (S105) includes the following two steps:

[0084] (S10501) The red phosphorus obtained in the above step (S104) is dissolved again in deionized water and transferred to a probe-type ultrasonic instrument.

[0085] The probe-type ultrasonic instrument has an ultrasonic power of 100W, a working time of 5s, an interval of 3s, and the working time and interval are spaced apart for a total of 99 times, which is called one working cycle.

[0086] (S10502) Take the red phosphorus sample after 8 working cycles of ultrasound as RP-8.

[0087] In step (S106), the above RP-8 suspension is centrifuged at 4000 rpm for 10 min, the supernatant is taken out, placed in a refrigerator, and packaged for storage for later use.

[0088] Figure 3 Here is a scanning electron microscope image of the RP-8 prepared in Example 2 of this disclosure; from Figure 3 It can be seen that the size of RP-8 is approximately 400-600 nanometers.

[0089] Preparation and electron microscopy analysis of sample RP-12:

[0090] In step (S101), a small amount of commercial red phosphorus (RP) is ground in a mortar and then sieved through a 300-mesh sieve.

[0091] In step (S102), 1g of sample is added to 60ml of deionized water to prepare a red phosphorus solution with a concentration of 1 / 60g / ml, and then sonicated for 30min.

[0092] In step (S103), the sample from step (S102) is transferred to a 100ml polytetrafluoroethylene-lined stainless steel autoclave and hydrothermally treated at 200°C for 12 hours.

[0093] In step (S104), after the hydrothermal treatment is completed, the obtained sample is centrifuged and dispersed, and then washed three times with anhydrous ethanol and deionized water respectively before the lower layer precipitate is taken out.

[0094] The step (S105) includes the following two steps:

[0095] (S10501) The red phosphorus obtained in the above step (S104) is dissolved again in deionized water and transferred to a probe-type ultrasonic instrument.

[0096] The probe-type ultrasonic instrument has an ultrasonic power of 100W, a working time of 5s, an interval of 3s, and the working time and interval are spaced apart for a total of 99 times, which is called one working cycle.

[0097] (S10502) Take the red phosphorus sample after 12 working cycles of ultrasound as RP-12.

[0098] In step (S106), the above RP-12 suspension is centrifuged at 4000 rpm for 10 min, the supernatant is taken out, placed in a refrigerator, and packaged for storage for later use.

[0099] Figure 4 A scanning electron microscope image of RP-12 prepared in Example 3 of this disclosure; from Figure 4 It can be seen that the size of RP-12 is approximately 200-400 nanometers.

[0100] Preparation and electron microscopy analysis of sample RP-16:

[0101] In step (S101), a small amount of commercial red phosphorus (RP) is ground in a mortar and then sieved through a 300-mesh sieve.

[0102] In step (S102), 1g of sample is added to 60ml of deionized water to prepare a red phosphorus solution with a concentration of 1 / 60g / ml, and then sonicated for 30min.

[0103] In step (S103), the sample from step (S102) is transferred to a 100ml polytetrafluoroethylene-lined stainless steel autoclave and hydrothermally treated at 200°C for 12 hours.

[0104] In step (S104), after the hydrothermal treatment is completed, the obtained sample is centrifuged and dispersed, and then washed three times with anhydrous ethanol and deionized water respectively before the lower layer precipitate is taken out.

[0105] The step (S105) includes the following two steps:

[0106] (S10501) The red phosphorus obtained in the above step (S104) is dissolved again in deionized water and transferred to a probe-type ultrasonic instrument.

[0107] The probe-type ultrasonic instrument has an ultrasonic power of 100W, a working time of 5s, an interval of 3s, and the working time and interval are spaced apart for a total of 99 times, which is called one working cycle.

[0108] (S10502) Take the red phosphorus sample after 16 working cycles of ultrasound as RP-16.

[0109] In step (S106), the above RP-16 suspension is centrifuged at 4000 rpm for 10 min, the supernatant is taken out, placed in a refrigerator, and packaged for later use.

[0110] Figure 5 Here is a scanning electron microscope image of RP-16 prepared in Example 4 of this disclosure; from Figure 5 It can be seen that the size of RP-16 is approximately 100-200 nanometers.

[0111] Preparation and electron microscopy analysis of BP NSs samples:

[0112] In addition, based on the above-mentioned preparation of two-dimensional red phosphorus nanosheets, this invention also provides an embodiment of a method for preparing black phosphorus nanosheets.

[0113] The specific steps for preparing black phosphorus nanosheets are as follows:

[0114] Take a small amount of lumpy black phosphorus, grind it into black phosphorus powder, dissolve 1g in deionized water, and transfer it to a probe-type ultrasonic instrument. The ultrasonic power is 100W, the working / interval time is 5 / 3s, and the peeling is carried out for 4 hours. After ultrasonication, the lumpy black phosphorus that has not been super-opened is removed by centrifugation at 2000pm for 10min. The upper dispersion is centrifuged at 10000pm for 10min to obtain black phosphorus nanosheets, which are stored for later use and compared with red phosphorus nanosheets.

[0115] Figure 10 These are scanning electron microscope images of the black phosphorus nanosheets (BP NSs) that underwent ultrasonic treatment in the above steps of this invention. Figure 10 As can be seen, the size of the black phosphorus nanosheets is approximately 1500-2000 nanometers.

[0116] The present invention also provides a method for screening two-dimensional red phosphorus nanosheets, wherein the screening method includes the following steps:

[0117] (A) The supernatant of the prepared two-dimensional red phosphorus nanosheets was dropped onto a 0.5×0.5 silicon wafer;

[0118] (B) Dry at 60℃;

[0119] (C) Add crystal violet molecules dropwise and dry again;

[0120] (D) The red phosphorus sample was tested using a Raman laser;

[0121] (E) Draw Raman spectra and select the substrate with the strongest Raman signal.

[0122] In step (A), 20 microliters of the supernatant of the two-dimensional red phosphorus nanosheets (RP-0, RP-4, RP-8, RP-12, RP-16) prepared in Examples 1, 2, 3, and 4 are taken and dropped onto a 0.5×0.5 silicon wafer.

[0123] In steps (B) and (C), the product is dried at 60°C, then 20 μL of crystal violet (CV) is added dropwise, and the product is dried again.

[0124] In step (D), a Raman laser with a wavelength of 532 nm was used as the excitation source, with a laser spot diameter of 12.5 μm, a numerical aperture of 0.55, a laser power of 1 mW, and an integration time of 10 s. Raman spectra were plotted and compared on five samples to select the substrate with the strongest Raman signal for subsequent immunoassay.

[0125] Figure 6 The above-described SERS spectra of RP-0, RP-4, RP-8, RP-12, and RP-16 against crystal violet, prepared using the method of this invention, are shown at 1616 cm⁻¹. -1 The Raman signals at the locations are 1991, 7010, 5423, 4652, and 3488, respectively. As can be seen from the figure, RP-4 has the best SERS enhancement effect and the strongest Raman signal, making it suitable as the optimal substrate.

[0126] In this invention, firstly, bulk commercial red phosphorus is transformed into two-dimensional red phosphorus nanosheets with a larger surface area through simple hydrothermal treatment and ice bath ultrasonication, thereby improving the molecular adsorption capacity and SERS signal. Secondly, by changing the ultrasonication time, the size of the two-dimensional red phosphorus nanosheets is altered, adjusting their visible light absorption range and band gap. Raman characterization is then used to screen for the red phosphorus nanosheets with the strongest SERS signal. Thirdly, red phosphorus is biodegradable into non-toxic phosphates, possesses biocompatibility, and is stable and low-cost, making it promising for applications in biomedicine.

[0127] To improve the sensitivity of SERS detection of tumor markers, a sandwich structure was constructed based on an optimal red phosphorus nanosheet substrate. The introduction of 3D MoS2 NFs as an immune probe avoids the aggregation of molecules on the two-dimensional layered material and also effectively reduces the interference of fluorescence background.

[0128] Figure 1Here is a scanning electron microscope image of the RP-0 prepared in Example 1 of this disclosure; from Figure 1 It can be seen that the wrinkles on the side of RP-0 indicate that the blocky red phosphorus is composed of multiple tightly packed sheets, with a size of approximately 1 micrometer.

[0129] Figure 2 Here is a scanning electron microscope image of RP-4 prepared in Example 1 of this disclosure; from Figure 2 It can be seen that RP-4 transforms from a blocky material into a layered material after ultrasonic treatment, and the size of RP-4 is approximately 600-800 nanometers.

[0130] Figure 3 Here is a scanning electron microscope image of the RP-8 prepared in Example 1 of this disclosure; from Figure 3 It can be seen that the size of RP-8 is approximately 400-600 nanometers.

[0131] Figure 4 A scanning electron microscope image of RP-12 prepared in Example 1 of this disclosure; from Figure 4 It can be seen that the size of RP-12 is approximately 200-400 nanometers.

[0132] Figure 5 Here is a scanning electron microscope image of the RP16 prepared in Example 1 of this disclosure; from Figure 5 It can be seen that the size of RP-16 is approximately 100-200 nanometers.

[0133] Figure 6 SERS spectra of RP-0, RP-4, RP-8, RP-12, and RP-16 against crystal violet (CV), 1616 cm⁻¹ -1 The Raman signals at the locations were 1991, 7010, 5423, 4652, and 3488, respectively. Among them, RP-4 showed the best SERS enhancement effect and was suitable as the optimal substrate.

[0134] Similar to the above method, a 532nm Raman laser was used to measure the Raman signal of the black phosphorus sample to compare the Raman signals of red phosphorus nanosheets and black phosphorus nanosheets, so as to select the nanosheet with the strongest Raman signal, and then select the nanosheet with the strongest Raman signal as the immune substrate through comparison.

[0135] Take the black phosphorus nanosheets (BP NSs) prepared in the above examples, add 20 μL to a 0.5 × 0.5 mm silicon wafer, dry at 60 °C, add 20 μL of crystal violet molecules (CV), dry again, use a 532 nm Raman laser as the excitation source, the laser spot diameter is 12.5 μm, the numerical aperture is 0.55, the laser power is set to 1 mW, the integration time is 10 s, and a Raman spectrum is plotted.

[0136] In addition, the sample RP-0 from the above embodiment needs to be taken and processed using the same method as above, i.e., 20 μL is dropped onto a 0.5 × 0.5 silicon wafer, dried at 60°C, and then 20 μL of crystal violet (CV) molecules are dropped onto it. After drying again, a Raman laser with a wavelength of 532 nm is used as the excitation source, with a laser spot diameter of 12.5 μm, a numerical aperture of 0.55, a laser power of 1 mW, and an integration time of 10 s. The Raman spectrum is then plotted.

[0137] Take sample RP-16 from the above examples, and use the same method as above to drop 20 μL onto a 0.5 × 0.5 mm silicon wafer. After drying at 60 °C, drop 20 μL of crystal violet (CV) molecules onto the wafer, and dry it again. Use a 532 nm Raman laser as the excitation source, with a laser spot diameter of 12.5 μm, a numerical aperture of 0.55, a laser power of 1 mW, and an integration time of 10 s to plot the Raman spectrum.

[0138] Raman spectra of the three samples (RP-0, RP-16, and BP NSs) were plotted, as shown in the attached diagram. Figure 11 As shown.

[0139] Appendix Figure 11 The figures show the SERS spectra of RP-0, RP-16, and BP NSs against crystal violet (CV). From the figures, it can be seen that at 1616 cm⁻¹... -1 The Raman signals at the locations were 1991, 3488, and 2744, respectively. This indicates that the Raman signal of black phosphorus nanosheets is weaker than that of red phosphorus nanosheets, meaning that the performance of black phosphorus nanosheets is weaker than that of red phosphorus nanosheets. Furthermore, the ultrasonic time required to peel off BP nanosheets is longer.

[0140] Although the SERS performance of red phosphorus (RP) improved from bulk RP-0 to layered RP-16, the enhancement factor only reached 10. 4 Therefore, it is necessary to continue exploring experimental conditions that require less time and have better enhancement effects, as well as the corresponding red phosphorus nanosheets screened under these conditions.

[0141] A comparison between red phosphorus nanosheets and black phosphorus nanosheets shows that red phosphorus nanosheets exhibit significantly superior performance. Furthermore, Raman spectra and electron microscope images of various red phosphorus nanosheets (RP-0, RP-4, RP-8, RP-12, and RP-16) under different experimental conditions reveal that RP-4 exhibits the strongest Raman signal, with an enhancement factor reaching 10. 5 Furthermore, electron microscope images show that RP-4 has the most suitable size. In conclusion, through comparison of various red phosphorus nanosheets and black phosphorus nanosheets, RP-4 is suitable as the optimal substrate.

[0142] Step (3) MoS2 nanoflowers (MoS2 NFs) were prepared using a hydrothermal method:

[0143] Take 3 mmol Na2MoO4·2H2O, 9 mmol CH3CSNH2 and 2.8 mmol H4[Si(W3O 10 [4]·xH2O was dissolved sequentially in 50 mL of deionized water and stirred magnetically. The resulting solution was then transferred to a 100 mL stainless steel autoclave lined with polytetrafluoroethylene and hydrothermally treated at 220 °C for 24 h. After cooling to room temperature, the product was washed sequentially with NaOH, anhydrous ethanol, and deionized water to obtain MoS2 precipitate. Finally, the washed product was dried in a vacuum drying oven at 60 °C for 6 h to obtain 3DMoS2NFs, which were then packaged and stored at room temperature.

[0144] Figure 7 Scanning electron microscope image of MoS2 NFs prepared in Example 3 of this disclosure.

[0145] from Figure 7 It can be seen that MoS2 NFs are 3D irregular spheres with a diameter of approximately 200-400 nanometers.

[0146] Step (4) Preparation of the immune base:

[0147] Take 20 μL of the substrate (RP-4) with the best SERS performance obtained in step (2) and drop it onto a 0.5×0.5 silicon wafer. After drying at room temperature, add 20 μL of 0.2 mg / mL cancer antibody solution and incubate at 4°C for more than 12 h. Wash the substrate three times with TBS, PBS and deionized water to remove residual antibodies. Then add 10 μL of BSA solution (3 wt%) and place at room temperature for 3 h. Add 20 μL of cancer antigen solution of different concentrations onto the substrate and incubate at 37°C for 2 h (using antigen-antibody specific binding). Finally, wash again with TBS, PBS and deionized water to obtain the immune substrate.

[0148] The BSA solution uses PBS solvent and bovine serum albumin powder, with the bovine serum albumin powder accounting for 3% of the total mass. The PBS and TBS are prepared as follows: PBS:deionized water = 1:4, TBS:deionized water = 1:4.

[0149] Step (5) Preparation of immune probes:

[0150] Take 1 ml of the 3D MoS2 NFs obtained in step (3) into a 5 ml centrifuge tube, add 20 μL of crystal violet molecules and shake thoroughly, and incubate at room temperature for 12 h; centrifuge the resulting mixture at 6000 rpm for 10 min and remove the supernatant, then add 1 ml of PBS solution to wash; add 20 μL of 0.2 mg / mL cancer antibody solution to the above sample and incubate at 4 °C for 1.5 h; centrifuge again to remove unlinked antibodies, and add 500 μL of PBS solution to wash again; finally add 10 μL of BSA solution (3 wt%) and place at room temperature for 1 h, centrifuge to remove excess BSA, and add 1 ml of PBS solution to obtain the CV-tagged MoS2 NFs immune probe, which should be stored at 4 °C before use.

[0151] The concentration of 3D MoS2 NFs was 2 mg / mL, and the concentration of crystal violet molecules was 1 × 10⁻⁶. -2 mg / mL.

[0152] Step (6) Preparation of the sandwich structure:

[0153] 20 μL of the immune probe prepared in step (5) was dropped onto the immune substrate obtained in step (4) and incubated at 37°C for 2 h to promote the binding between antigen and antibody; then, excess antigen was removed by rinsing with TBS, PBS and deionized water; finally, the sandwich structure was dried at room temperature and stored at 4°C before use.

[0154] Figure 8 This is a scanning electron microscope image of the immunostructure of two-dimensional red phosphorus nanosheets / MoS2 NFs prepared in Example 4 of this disclosure.

[0155] Figure 9 The image shows the Raman spectroscopy results of the two-dimensional red phosphorus nanosheets / MoS2 NFs immune structure prepared in Example 4 of this disclosure against CEA tumor markers.

[0156] from Figure 9 It can be seen that this immune structure has a good SERS signal enhancement effect, which is evident at 1616 cm⁻¹. -1 The Raman signal strength at that location reached 8344.

[0157] Therefore, through the above experiments and rigorous screening, among the several samples prepared in this invention, RP-4 exhibited the strongest Raman signal and was most suitable as an immune substrate. In the detection of tumor markers, the two-dimensional red phosphorus nanosheets / molybdenum disulfide flower immune structure prepared in this invention demonstrated a good SERS signal enhancement effect when detecting CEA tumor markers, showing a high SERS signal intensity at 1616 cm⁻¹. -1 The Raman signal enhancement at that location reached 8344.

[0158] Those skilled in the art will understand that, following the experimental steps of this invention, they can prepare two-dimensional red phosphorus nanosheets under other experimental conditions without departing from the inventive concept of this invention.

[0159] Therefore, two-dimensional red phosphorus nanosheets / molybdenum disulfide flower immunoassay structures can be used for the detection of tumor markers. This involves a simple and efficient method to convert bulk red phosphorus into nanosheets with larger surfaces, significantly improving molecular adsorption capacity. The size change of the two-dimensional red phosphorus nanosheets adjusts their visible light absorption range and band gap. Raman characterization was used to screen for the two-dimensional red phosphorus nanosheets with the best SERS performance, and comparison with black phosphorus revealed that red phosphorus nanosheets have significant advantages and are suitable as an immunoassay substrate.

[0160] In addition, both red phosphorus and molybdenum disulfide have good biocompatibility and are simple to manufacture and low in cost. By using two-dimensional red phosphorus nanosheets as an immune substrate to construct a sandwich structure and preparing 3D molybdenum disulfide flowers as an immune probe, the aggregation of molecules on two-dimensional layered materials is avoided, while the sensitivity of SERS detection is improved, which can be conveniently used for the detection of tumor markers.

[0161] Furthermore, this study overcomes the problem of deformation of metal nanostructures caused by localized temperature increases in electromagnetic hotspot areas when traditional SERS substrates, such as gold or silver, are used for tumor marker detection. This deformation affects the stability of the assay. Moreover, compared to traditional noble metal substrates, this invention uses two-dimensional red phosphorus nanosheets as the substrate, eliminating the need for biotoxic surfactants, making it more human- and environmentally friendly, and suitable for widespread application in the biomedical field. Therefore, this research will open up new avenues for the application of non-noble metal nanoparticle substrates as SERS substrates in the biomedical detection field.

[0162] Those skilled in the art should understand that the embodiments of the present invention described above and shown in the accompanying drawings are merely examples and do not limit the present invention. The objectives of the present invention have been fully and effectively achieved. The functions and structural principles of the present invention have been demonstrated and explained in the embodiments, and any variations or modifications may be made to the implementation of the present invention without departing from the stated principles.

Claims

1. A method for preparing a two-dimensional red phosphorus nanosheet / molybdenum disulfide flower immune structure, characterized in that, The preparation method includes the following steps: (1) The preparation of two-dimensional red phosphorus nanosheets includes the following steps: (101) Take red phosphorus and grind it to obtain a sample; (102) Add deionized water to the sample to prepare a red phosphorus solution; (103) After sonicating the red phosphorus solution, transfer it into a high-pressure autoclave with a polytetrafluoroethylene liner and treat it in a water bath at 200°C for 12 hours; (104) After the hydrothermal treatment is completed, centrifuge and disperse it, wash it three times with anhydrous ethanol and deionized water respectively, and take the lower precipitate; (105) Dissolve the obtained red phosphorus in deionized water again and sonicate it with a probe ultrasonic instrument, with working and intermittent phases of 5s and 3s respectively, and sonicate 99 times as a working cycle; and (106) Centrifuge the taken red phosphorus suspension at 4000rpm for 10min, take out the supernatant, and store it in a refrigerator; (2) Screening two-dimensional red phosphorus nanosheets as the optimal substrate, wherein the substrate with the strongest Raman signal is screened by Raman laser. According to different working cycles, 0 working cycles, 4 working cycles, 8 working cycles, 12 working cycles and 16 working cycles are selected to prepare RP-0, RP-4, RP-8, RP-12 and RP-16 samples respectively. Among them, the size of RP-0, RP-4, RP-8, RP-12 and RP-16 samples gradually decreases, and the Raman signal of RP-4 sample is the strongest. (3) Preparation of 3D MoS2 NFs; (4) Preparation of immune substrate, wherein the substrate with the best SERS performance selected in step (2) is dropped onto a silicon wafer, dried at room temperature, cancer antibody solution is added, incubated at 4°C for at least 12 h, rinsed to remove residual antibody, BSA solution is added and placed at room temperature for 3 h, antigen solutions of different concentrations are dropped onto the substrate, incubated at 37°C for 2 h, and washed to obtain immune substrate; (5) Preparation of the immunoprobe: Take 1 mL of the 3D MoS2 NFs prepared in step (3) into a 5 mL centrifuge tube, add 20 μL of crystal violet molecules and oscillate, and incubate at room temperature for 12 h; centrifuge the resulting mixture at 6000 rpm for 10 min and remove the supernatant, then add 1 mL of PBS solution to wash; add 20 μL of 0.2 mg / mL cancer antibody solution to the obtained sample and incubate at 4 °C for 1.5 h; centrifuge again to remove unlinked antibodies, and add 500 μL of PBS solution to wash again; finally add 10 μL of BSA solution and place at room temperature for 1 h, centrifuge to remove excess BSA, and add 1 mL of PBS solution to obtain the CV-tagged MoS2 NFs immunoprobe; and (6) Preparation of sandwich structure.

2. The preparation method according to claim 1, wherein in step (3), 3D MoS2NFs are prepared by hydrothermal method.

3. The preparation method according to claim 2, wherein in step (2), red phosphorus supernatant is added dropwise to a silicon wafer, dried, crystal violet molecules are added dropwise, and a Raman laser is used as the excitation source to draw a Raman spectrum.

4. The preparation method according to claim 3, wherein the Raman laser has a laser wavelength of 532 nm, a laser spot diameter of 12.5 μm, a laser power of 1 mW, and an integration time of 10 s.

5. The preparation method according to claim 4, wherein the concentration of the red phosphorus supernatant is 1 mg / mL, and the concentration of the crystal violet in step (2) is 1 × 10⁻⁶. -2 mg / mL.

6. The preparation method according to claim 5, wherein step (3) includes the following steps: Na2MoO4·2H2O, CH3CSNH2 and H4[Si(W3O 10 [4]·xH2O was dissolved in deionized water and stirred. The resulting solution was transferred to a stainless steel autoclave lined with polytetrafluoroethylene and hydrothermally treated at 220 °C for 24 h. After cooling to room temperature, the product was washed and filtered with NaOH, anhydrous ethanol and deionized water in sequence to obtain MoS2 deposits. The washed product was dried in a vacuum drying oven at 60 °C for 6 h to obtain 3D MoS2 NFs, which were then packaged and stored at room temperature.

7. The preparation method according to claim 6, wherein the prepared MoS2 NFs are three-dimensional irregular spheres with a diameter of 200-400 nm.

8. The preparation method according to claim 7, wherein the BSA solution in step (4) and step (5) is prepared using PBS solvent and bovine serum albumin powder, wherein the mass of bovine serum albumin powder is 3%.

9. The preparation method according to claim 8, wherein excess antigen is removed by rinsing with TBS, PBS and deionized water, wherein the PBS and TBS are configured as follows: PBS:deionized water = 1:4, TBS:deionized water = 1:

4.

10. A two-dimensional red phosphorus nanosheet / molybdenum disulfide flower immune structure prepared by the preparation method according to any one of claims 1 to 9, characterized in that, include: An immune substrate using two-dimensional red phosphorus nanosheets as a carrier and an immune probe using 3D MoS2 NFs as a carrier, wherein the MoS2 NFs are irregular spheres with a diameter of 200-400 nm, and the immune probe and immune substrate are prepared into a sandwich structure.

11. The non-diagnostic application of a two-dimensional red phosphorus nanosheet / molybdenum disulfide flower immunoassay structure prepared by the preparation method according to any one of claims 1 to 9, characterized in that, The two-dimensional red phosphorus nanosheet / molybdenum disulfide flower immunostructure is suitable for the detection of SERS tumor markers.

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