A method for fabricating a high-throughput photothermal biosensor for detecting tumor cells and its detection application.

Abstract

This invention discloses a method for preparing a photothermal biosensor for high-throughput detection of tumor cells and its detection application. The method includes the following steps: synthesizing a MnO2@CuS nanocomposite material with excellent photothermal properties and coupling it with aptamer Apt2 to construct a signal probe; immobilizing aptamer Apt1 on an aminated glass slide using a microarray to prepare a high-throughput capture substrate; adding a sample solution containing different concentrations of tumor cells to the capture substrate, followed by the addition of a signal probe dispersion to obtain the photothermal biosensor for high-throughput detection of tumor cells; and using an 808 nm laser for irradiation and recording the temperature rise with an infrared thermal imager to achieve quantitative analysis of tumor cells. The advantages are high sensitivity, high selectivity, simple and rapid operation, and high-throughput detection, making it suitable for early non-invasive screening of tumors.

Description

Technical Field

[0001] This invention relates to a method for high-throughput detection of tumor cells, and more particularly to a method for preparing a photothermal biosensor for high-throughput detection of tumor cells and its detection application. Background Technology

[0002] Malignant tumors are a major disease threatening human life and health, and early diagnosis is crucial for improving patient survival rates. Tumor cells, as cells directly derived from tumor tissue, are closely related to the occurrence, development, and metastasis of tumors. Therefore, sensitive and rapid detection of tumor cells has significant clinical value for the auxiliary diagnosis, treatment evaluation, and prognostic monitoring of tumors.

[0003] Current methods for detecting tumor cells mainly include flow cytometry, immunofluorescence imaging, microfluidic chips, bioimaging, and biosensing technologies. Among these, traditional detection methods mostly rely on complex instruments and equipment, have cumbersome operating procedures, high detection costs, and require a high level of expertise from operators, making it difficult to achieve high-throughput, on-site, and low-cost detection.

[0004] Photothermal (PT) detection technology, as an emerging signal output method, has received widespread attention in the field of bioanalysis in recent years. Based on the thermal effect generated by photosensitive materials under specific wavelength excitation, it detects target analytes through temperature change signals, offering advantages such as low background noise, high sensitivity, strong anti-interference capability, and compatibility with various reading terminals.

[0005] High-throughput detection technology can process large numbers of samples simultaneously, meeting the key requirements for disease screening and large-scale clinical testing. In recent years, with the development of nanomaterials, functional interfaces, and miniaturized devices, the combination of photothermal signals and high-throughput platforms has become possible. Developing a novel biosensor that uses photothermal as the output signal and achieves rapid response and high-throughput detection holds significant research and application potential for highly sensitive detection of tumor cells. Currently, there are no publicly reported research reports on the fabrication methods of photothermal biosensors for high-throughput detection of tumor cells. Summary of the Invention

[0006] The technical problem to be solved by the present invention is to provide a method for preparing a high-throughput photothermal biosensor for detecting tumor cells with high sensitivity, high selectivity, simple and rapid operation and high throughput, and its detection application.

[0007] The technical solution adopted by this invention to solve the above-mentioned technical problems is as follows: a method for preparing a high-throughput photothermal biosensor for detecting tumor cells, comprising the following steps:

[0008] Step 1: Preparation of photothermal signal probe MnO2@CuS-Apt2

[0009] a. Mix 3-5 mL of tetramethylammonium hydroxide, 1-3 mL of 30 wt% hydrogen peroxide, and 10-20 mL of ultrapure water, and sonicate at room temperature for 20-40 min; then add 8-12 mL of 0.2-0.5 mol / L manganese chloride tetrahydrate MnCl2·4H2O solution, and stir the reaction at room temperature for 10-15 h; after the reaction is completed, centrifuge to collect the product, wash with water and ethanol, and dry in an oven at 50-70 ℃ to obtain manganese dioxide MnO2 nanosheets;

[0010] b. Disperse 20-30 mg of MnO2 nanosheets and 500-700 mg of copper nitrate trihydrate Cu(NO3)2·3H2O in 20-30 mL of ultrapure water, add 5-15 mg of hexadecyltrimethylammonium bromide, sonicate for 20-40 min, let stand for 10-14 h, and collect the precipitate by centrifugation; redisperse the precipitate in 40-60 mL of 0.01-0.1 mol / L thioacetamide solution, and react in a water bath at 70-90 ℃ for 2-4 h for in-situ sulfidation; after the reaction is completed, centrifuge, wash and dry to obtain manganese dioxide@copper sulfide (MnO2@CuS) composite material;

[0011] c. Incubate the MnO2@CuS aqueous dispersion with the aptamer Apt2 to obtain the MnO2@CuS-Apt2 signal probe;

[0012] Step 2: Construction of a high-throughput photothermal sensing platform

[0013] a. Clean the glass slides and perform amino-functionalization treatment to obtain amino-functionalized glass slides;

[0014] b. Divide the aminated glass slide obtained in step a into multiple sensing regions and modify the aptor Apt1 to obtain the high-throughput capture substrate Apt1 / slide;

[0015] c. Add 100–300 μL of the sample solution containing different concentrations of tumor cells to the capture substrate Apt1 / slide and incubate at room temperature for 0.5–1.5 h to allow the target cells to be specifically captured by the surface-fixed Apt1. Wash with PBS buffer to remove unbound cells. Then add 10–30 μL of MnO2@CuS-Apt2 signal probe dispersion and incubate at room temperature for 0.5–1.5 h to allow the probe to bind to the captured cells via Apt2. After washing, a high-throughput photothermal biosensor for tumor cell detection is obtained.

[0016] The preparation method of the MnO2@CuS-Apt2 signal probe described in step 1c is as follows: Take 0.5~2 mL of 1~3 mg / mL MnO2@CuS aqueous dispersion, add 50~200 μL of 10~30 μmol / L thiol-modified aptamer Apt2, and shake at room temperature for 4~8 h. Remove unbound aptamers by centrifugation. Then add 0.5~2 mL of 0.5~2 mmol / L 6-mercapto-1-hexanol solution and incubate at room temperature for 0.5~1.5 h to block non-specific active sites on the surface of the nanomaterial. Finally, after centrifugation and washing, redisperse the obtained product in 0.5~2 mL of pH 7.0~8.0 phosphate buffer to obtain the MnO2@CuS-Apt2 signal probe dispersion.

[0017] Further, the preparation method of the aminated glass slide described in step 2a is as follows: the glass slide is placed in acetone, an ethanol solution of 0.5-2 mol / L sodium hydroxide (NaOH), and a water / ethanol mixture of 0.5-2 mol / L NaOH in sequence and ultrasonically cleaned for 10-30 min respectively. It is then rinsed with ultrapure water and dried with nitrogen. The clean glass slide is then immersed in a 1-3 wt% ethanol solution of 3-aminopropyltriethoxysilane (APTES) and reacted for 0.5-2 h to introduce amino functional groups onto the surface of the glass slide, thereby obtaining the aminated glass slide.

[0018] Further, the preparation method of the high-throughput capture substrate Apt1 / slide described in step 2b includes the following steps: 8-24 independent circular detection regions, each with a diameter of 4-8 mm, are divided on the surface of an aminated glass slide using insulating tape; within each circular region, the following modifications are performed sequentially: 10-30 μL of 3-7 wt% glutaraldehyde solution is added, and the reaction is carried out at room temperature for 0.5-1.5 h; 10-30 μL of 0.5-2 μmol / L amino-modified aptamer Apt1 is added, and the reaction is carried out at room temperature for 0.5-1.5 h, allowing Apt1 to crosslink with the amino groups on the glass slide; subsequently, 10-30 μL of 0.5-2 wt% bovine serum albumin (BSA) solution is added, and the mixture is incubated at room temperature to block non-specific binding sites; after each modification step, the slide is washed 2-4 times with ultrapure water, ultimately yielding the high-throughput capture substrate Apt1 / slide with multiple independent detection units. The aptamer is a selective recognition aptamer for epithelial cell adhesion molecule (EpCAM), a protein overexpressed on the cell membrane surface, and is a nucleic acid aptamer that can specifically bind to EpCAM.

[0019] Furthermore, the sequence of aptamer Apt1 is: 5'-NH2-CACTACAGAGGTTGCGTCTGTCCCACGTTGTCATGGGGGGTTGGCCTG-3'; the sequence of aptamer Apt2 is: 5'-SH-CACTACAGAGGTTGCGTCTGTCCCACGTTGTCATGGGGGGTTGGCCTG-3'.

[0020] Furthermore, the tumor cells are breast cancer cells MCF-7, colorectal cancer cells HT-29, prostate cancer cells LNCaP, and bladder cancer cells HT-1376.

[0021] This invention also provides the detection application of a high-throughput photothermal biosensor for detecting tumor cells prepared by the above method. This method is not for diagnosis or treatment purposes and includes the following steps: irradiating the detection area of ​​the photothermal biosensor with a laser, acquiring photothermal images and recording temperature changes using an infrared thermal imager, and achieving qualitative and quantitative analysis of the target tumor cells by analyzing the temperature rise value ΔT.

[0022] Furthermore, the laser is an 808 nm near-infrared laser with a power of 1 W.

[0023] The detection principle of this invention is as follows: Figure 1 As shown, taking bladder cancer cells HT-1376 as an example, the capture unit immobilizes the EpCAM aptamer Apt1 onto the surface of an APTES-modified glass slide via glutaraldehyde crosslinking. The signaling unit MnO2@CuS-Apt2 generates a significant photothermal effect under 808 nm laser excitation. After the target cells are captured by Apt1, they bind to MnO2@CuS-Apt2 to form a sandwich structure, allowing the photothermal probe to bind to the EpCAM protein on the surface of the captured cells through Apt2, thereby fixing the photothermal material in the sensing area. After washing, the temperature rise (ΔT) after laser irradiation is detected using an infrared thermal imager, which is proportional to the logarithm of the cell concentration, and quantitative analysis is achieved through a standard curve. This platform utilizes a multi-unit microarray design, combining high-throughput multi-sample processing with rapid visualization detection capabilities.

[0024] Compared with the prior art, the advantages of the present invention are as follows:

[0025] 1. High throughput: For the first time, photothermal sensing is combined with multi-channel microarray technology, enabling the simultaneous detection of multiple samples on a single substrate, which greatly improves detection efficiency and meets the throughput requirements of clinical screening;

[0026] 2. High sensitivity: The use of MnO2@CuS nanocomposite material with high efficiency photothermal conversion performance as a signal tag achieves significant amplification of the target cell detection signal;

[0027] 3. High specificity: It utilizes the EpCAM protein-specific aptamers Apt1 and Apt2 for dual recognition, effectively avoiding interference from non-target cells (such as blood cells and normal epithelial cells) in complex biological samples, and has strong anti-interference ability.

[0028] 4. Simple and fast operation: The detection process does not require complicated and expensive instruments. The results are displayed intuitively through infrared thermal imaging, which can realize semi-quantitative judgment with naked eyes. The process from sample addition to result output is completed quickly.

[0029] 5. Low cost and easy to promote: The sensor manufacturing process is simple, the required reagents are inexpensive, and it can be mass-produced, which is conducive to the transformation of technology and the popularization of clinical applications.

[0030] In summary, this invention successfully achieved highly sensitive, selective, and rapidly visualized detection of various tumor cells by constructing a high-throughput photothermal aptamer sensing platform based on MnO2@CuS nanoprobes. The platform exhibits stable signal output, fast response speed, and high throughput. This platform organically combines efficient photothermal materials with multi-channel microarray technology, offering advantages such as strong signal amplification, ease of operation, low cost, and easy deployment, effectively overcoming the limitations of traditional detection methods in terms of throughput, equipment dependence, and operational complexity. The photothermal biosensing platform constructed in this invention not only provides a novel and reliable tool for early screening of tumor cells, liquid biopsy, and clinical diagnosis, but also lays the technical foundation for the further application of photothermal biosensing technology in life analysis and precision medicine. Attached Figure Description

[0031] Figure 1 A schematic diagram illustrating the principle of a photothermal biosensor for high-throughput detection of tumor cells;

[0032] Figure 2 (A) is a scanning electron microscope image of MnO2, (B) is a scanning electron microscope image of MnO2@CuS, (C) is an X-ray diffraction pattern of MnO2 and MnO2@CuS, and (D) is an ultraviolet-visible absorption spectrum of MnO2@CuS and MnO2@CuS-Apt2.

[0033] Figure 3 (A) is the photothermal response curve of MnO2 and MnO2@CuS, and (B) is the feasibility analysis diagram of the photothermal biosensor.

[0034] Figure 4 (A) is the photothermal response diagram of the photothermal biosensor to different concentrations of tumor cells, (B) is the thermogram of the photothermal biosensor to different concentrations of tumor cells, and (C) is the sensitivity curve of the photothermal biosensor to different concentrations of tumor cells.

[0035] Figure 5For the performance analysis of photothermal biosensors, the following are included: (A) signal stability, (B) repeatability, (C) selectivity analysis, and (D) storage stability plot. Detailed Implementation

[0036] The present invention will be further described in detail below with reference to the accompanying drawings and embodiments. I. Specific Implementation Methods

[0038] Example 1: A method for preparing a high-throughput photothermal biosensor for detecting tumor cells and its detection applications, such as... Figure 1 As shown, it includes the following steps:

[0039] Step 1: Preparation of photothermal signal probe MnO2@CuS-Apt2

[0040] a. Mix 4.3 mL of tetramethylammonium hydroxide, 2 mL of 30 wt% hydrogen peroxide and 15 mL of ultrapure water, and sonicate at room temperature for 30 min; then add 10 mL of 0.3 mol / L manganese chloride tetrahydrate (MnCl2·4H2O) solution, and stir at room temperature for 12 h; after the reaction is complete, collect the product by centrifugation, wash with water and ethanol, and dry in an oven at 60 ℃ to obtain manganese dioxide (MnO2) nanosheets;

[0041] b. Disperse 25 mg of MnO2 nanosheets obtained in step 1a with 600 mg of copper nitrate trihydrate (Cu(NO3)2·3H2O) in 25 mL of ultrapure water, add 9 mg of hexadecyltrimethylammonium bromide, sonicate for 30 min, let stand for 12 h, and collect the precipitate by centrifugation; redisperse the precipitate in 50 mL of 0.05 mol / L thioacetamide solution, and react in an 80 ℃ water bath for 3 h for in-situ sulfidation; after the reaction is completed, centrifuge, wash and dry to obtain manganese dioxide@copper sulfide (MnO2@CuS) composite material;

[0042] c. Take 1 mL of 2 mg / mL MnO2@CuS aqueous dispersion, add 100 μL of 20 μmol / L thiol-modified aptamer Apt2 (sequence: 5'-SH-CACTACAGAGGTTGCGTCTGTCCCACGTTGTCATGGGGGGTTGGCCTG-3'), and shake at room temperature for 6 h. Remove unbound aptamers by centrifugation. Then add 1 mL of 1 mmol / L 6-mercapto-1-hexanol solution and incubate at room temperature for 1 h to block non-specific active sites on the nanomaterial surface. Finally, after centrifugation and washing, redisperse the obtained product in 1 mL of pH 7.4 phosphate buffer (PBS) to obtain the MnO2@CuS-Apt2 signal probe dispersion.

[0043] Step 2: Construction of a high-throughput photothermal sensing platform

[0044] a. Place the glass slides sequentially in acetone, an ethanol solution of 1 mol / L sodium hydroxide (NaOH), and a water / ethanol mixture of 1 mol / L NaOH (water to ethanol volume ratio 1:1) for ultrasonic cleaning for 20 min each. Rinse thoroughly with ultrapure water and dry with nitrogen. Then immerse the clean slides in a 2 wt% ethanol solution of 3-aminopropyltriethoxysilane (APTES) and react for 1 h to introduce amino functional groups onto the surface of the slides, thus obtaining aminated slides.

[0045] b. Sixteen independent circular detection regions, each with a diameter of 6 mm, were delineated on the surface of an aminated glass slide using insulating tape. Within each circular region, the following modifications were performed sequentially: 20 μL of 5 wt% glutaraldehyde solution was added, and the mixture was reacted at room temperature for 1 h; 20 μL of 1 μmol / L amino-modified aptamer Apt1 (sequence: 5'-NH2-CACTACAGAGGTTGCGTCTGTCCCACGTTGTCATGGGGGGTTGGCCTG-3') was added, and the mixture was reacted at room temperature for 1 h, allowing Apt1 to crosslink with the amino groups on the slide surface via glutaraldehyde; subsequently, 20 μL of 1 wt% bovine serum albumin (BSA) solution was added, and the mixture was incubated at room temperature for 1 h to block non-specific binding sites; after each modification step, the slide was washed three times with ultrapure water, ultimately yielding a high-throughput capture substrate Apt1 / slide with 16 independent detection units.

[0046] c. 200 μL of the test sample solution containing different concentrations of bladder cancer cells HT-1376 was dropped onto the capture substrate Apt1 / slide and incubated at room temperature for 1 h to allow the target cells to be specifically captured by the surface-fixed Apt1; unbound cells were removed by washing with PBS buffer; then 20 μL of MnO2@CuS-Apt2 signal probe dispersion was added and incubated at room temperature for 1 h to allow the probe to bind to the captured cells via Apt2; after washing with PBS buffer, a high-throughput photothermal biosensor for tumor cell detection was obtained.

[0047] Step 3, Detection method: The detection area was irradiated with an 808 nm near-infrared laser (1 W power), and photothermal images were acquired and temperature changes were recorded using an infrared thermal imager. The quantitative analysis of bladder cancer cells HT-1376 was achieved by analyzing the temperature rise value ΔT.

[0048] Example 2 is the same as Example 1 above, except that:

[0049] Step 1: Preparation of photothermal signal probe MnO2@CuS-Apt2

[0050] a. Mix 3.5 mL tetramethylammonium hydroxide, 1.5 mL 30 wt% hydrogen peroxide and 12 mL ultrapure water, and sonicate at room temperature for 25 min; then add 9 mL 0.4 mol / L MnCl2·4H2O solution, and stir the reaction at room temperature for 13 h; after the reaction is complete, centrifuge to collect the product, wash with water and ethanol, and dry in an oven at 55 ℃ to obtain MnO2 nanosheets;

[0051] b. 22 mg of MnO2 nanosheets prepared in step a and 550 mg of Cu(NO3)2·3H2O were dispersed in 22 mL of ultrapure water, and 7 mg of hexadecyltrimethylammonium bromide was added. After ultrasonic dispersion for 25 min, the mixture was allowed to stand for 11 h, and the precipitate was collected by centrifugation. The precipitate was redispersed in 45 mL of 0.08 mol / L thioacetamide solution and reacted in a 75 ℃ water bath for 2.5 h for in-situ sulfidation. After the reaction was completed, the mixture was centrifuged, washed, and dried to obtain the manganese dioxide@copper sulfide (MnO2@CuS) composite material.

[0052] c. Take 0.8 mL of 2.5 mg / mL MnO2@CuS aqueous dispersion, add 80 μL of 25 μmol / L thiol-modified aptamer Apt2 (sequence same as in Example 1), shake at room temperature for 5 h, and remove unbound aptamers by centrifugation; then add 0.8 mL of 1.5 mmol / L 6-mercapto-1-hexanol solution, incubate at room temperature for 45 min to block non-specific active sites on the surface of the nanomaterial; finally, after centrifugation and washing, redisperse the obtained product in 0.8 mL of pH 7.4 phosphate buffer (PBS) to obtain the MnO2@CuS-Apt2 signal probe dispersion;

[0053] Step 2: Construction of a high-throughput photothermal sensing platform

[0054] a. Place the glass slides in acetone, 1.5 mol / L NaOH in ethanol solution, and 1.5 mol / L NaOH in water / ethanol mixture for ultrasonic cleaning for 15 min each, rinse with ultrapure water and dry with nitrogen gas; then immerse the clean slides in 2.5 wt% APTES in ethanol solution and react for 45 min to introduce amino functional groups on the surface of the slides, thus obtaining aminated slides.

[0055] b. Twelve independent circular detection regions, each with a diameter of 5 mm, were divided on the surface of an aminated glass slide using insulating tape. Within each circular region, the following modifications were performed sequentially: 15 μL of 6 wt% glutaraldehyde solution was added and reacted at room temperature for 45 min; 15 μL of 1.5 μmol / L amino-modified aptamer Apt1 (sequence as in Example 1) was added and reacted at room temperature for 45 min, allowing Apt1 to crosslink with the amino groups on the slide surface via glutaraldehyde; subsequently, 15 μL of 1.5 wt% bovine serum albumin solution was added and incubated at room temperature for 45 min to block non-specific binding sites; after each modification step, the slide was washed three times with ultrapure water, ultimately yielding a high-throughput capture substrate Apt1 / slide with 12 independent detection units.

[0056] c. 150 μL of the test sample solution containing different concentrations of breast cancer cells MCF-7 was dropped onto the capture substrate Apt1 / slide and incubated at room temperature for 45 min to allow the target cells to be specifically captured by the surface-fixed Apt1; unbound cells were removed by washing with PBS buffer; then 15 μL of MnO2@CuS-Apt2 signal probe dispersion was added and incubated at room temperature for 45 min to allow the probe to bind to the captured cells via Apt2; after washing with PBS buffer, a high-throughput photothermal biosensor for tumor cell detection was obtained.

[0057] Step 3, Detection method: The detection area is irradiated with an 808 nm near-infrared laser (1 W power), and photothermal images are acquired and temperature changes are recorded using an infrared thermal imager. The quantitative analysis of breast cancer cells MCF-7 is achieved by analyzing the temperature rise value ΔT.

[0058] Example 3 is the same as Example 1 above, except that:

[0059] Step 1: Preparation of photothermal signal probe MnO2@CuS-Apt2

[0060] a. Mix 4.8 mL tetramethylammonium hydroxide, 2.5 mL 30 wt% hydrogen peroxide and 18 mL ultrapure water, and sonicate at room temperature for 35 min; then add 11 mL 0.25 mol / L MnCl2·4H2O solution, and stir the reaction at room temperature for 14 h; after the reaction is complete, centrifuge to collect the product, wash with water and ethanol, and dry in an oven at 65 ℃ to obtain MnO2 nanosheets;

[0061] b. Disperse 28 mg of MnO2 nanosheets obtained in step a and 650 mg of Cu(NO3)2·3H2O in 28 mL of ultrapure water, add 12 mg of hexadecyltrimethylammonium bromide, sonicate for 35 min, let stand for 13 h, and collect the precipitate by centrifugation; redisperse the precipitate in 55 mL of 0.03 mol / L thioacetamide solution, and react in an 85 ℃ water bath for 3.5 h for in-situ sulfidation; after the reaction is completed, centrifuge, wash and dry to obtain manganese dioxide@copper sulfide (MnO2@CuS) composite material;

[0062] c. Take 1.5 mL of 1.5 mg / mL MnO2@CuS aqueous dispersion, add 150 μL of 15 μmol / L thiol-modified aptamer Apt2 (sequence same as in Example 1), shake at room temperature for 7 h, and remove unbound aptamers by centrifugation; then add 1.5 mL of 0.8 mmol / L 6-mercapto-1-hexanol solution and incubate at room temperature for 75 min to block non-specific active sites on the surface of the nanomaterial; finally, after centrifugation and washing, redisperse the obtained product in 1.5 mL of pH 7.4 phosphate buffer (PBS) to obtain the MnO2@CuS-Apt2 signal probe dispersion;

[0063] Step 2: Construction of a high-throughput photothermal sensing platform

[0064] a. Place the glass slides sequentially in acetone, an ethanol solution of 0.8 mol / L sodium hydroxide (NaOH), and a water / ethanol mixture of 0.8 mol / L NaOH (volume ratio 1:2) for ultrasonic cleaning for 25 min each. Rinse thoroughly with ultrapure water and dry with nitrogen. Then immerse the clean slides in a 1.5 wt% ethanol solution of 3-aminopropyltriethoxysilane (APTES) and react for 80 min to introduce amino functional groups onto the slide surface, thus obtaining an aminated slide.

[0065] b. Twenty independent circular detection regions, each with a diameter of 7 mm, were divided on the surface of an aminated glass slide using insulating tape. Within each circular region, the following modifications were performed sequentially: 25 μL of 4 wt% glutaraldehyde solution was added and reacted at room temperature for 75 min; 25 μL of 0.8 μmol / L amino-modified aptamer Apt1 (sequence as in Example 1) was added and reacted at room temperature for 75 min, allowing Apt1 to crosslink with the amino groups on the slide surface via glutaraldehyde; subsequently, 25 μL of 0.8 wt% bovine serum albumin (BSA) solution was added and incubated at room temperature for 75 min to block non-specific binding sites; after each modification step, the slide was washed three times with ultrapure water, ultimately yielding a high-throughput capture substrate Apt1 / slide with 20 independent detection units.

[0066] c. 250 μL of the test sample solution containing different concentrations of colorectal cancer cells HT-29 was dropped onto the capture substrate Apt1 / slide and incubated at room temperature for 75 min to allow the target cells to be specifically captured by the surface-fixed Apt1; unbound cells were removed by washing with PBS buffer; then 25 μL of MnO2@CuS-Apt2 signal probe dispersion was added and incubated at room temperature for 75 min to allow the probe to bind to the captured cells via Apt2; after washing with PBS buffer, a high-throughput photothermal biosensor for tumor cell detection was obtained.

[0067] Step 3, Detection method: The detection area was irradiated with an 808 nm near-infrared laser (1 W power), and photothermal images were acquired and temperature changes were recorded using an infrared thermal imager. The quantitative analysis of colorectal cancer cells HT-29 was achieved by analyzing the temperature rise value ΔT.

[0068] II. Analysis of Experimental Results.

[0069] The morphology, crystal structure, and optical properties of the materials were characterized using a Nova NanoSEM 450 scanning electron microscope (FEI, USA), a D8 Focus X-ray diffractometer (Bruck, Germany), and a UV-1800 UV-Vis spectrophotometer (Shimadzu, Japan). Photothermal response measurements were recorded using a Fotric 246M infrared thermal imager (Feichuke, China), with an 808 nm near-infrared laser (1 W power) as the excitation source. The sensing area consisted of 16 independent circular detection units (6 mm in diameter) divided on an aminated glass slide.

[0070] 1. Material Characterization

[0071] like Figure 2 As shown in Figure (A), the scanning electron microscope (SEM) image reveals that pure MnO2 exhibits a typical lamellar and wrinkled structure with a large specific surface area, which is beneficial for subsequent recombination. Figure 2 As shown in (B), after Cu 2+ After adsorption and in-situ sulfidation, a large number of nanoparticles were deposited on the surface of the resulting MnO2@CuS composite material, indicating that CuS had been successfully loaded onto the MnO2 substrate. Figure 2 As shown in (C), the X-ray diffraction (XRD) pattern further confirms that MnO2@CuS retains the crystal characteristics of MnO2 while exhibiting characteristic diffraction peaks consistent with the CuS standard card (PDF#06-0464). Furthermore, as... Figure 2As shown in Figure (D), the aptamer modification effect was verified by UV-Vis absorption spectroscopy. Compared with unmodified MnO2@CuS, MnO2@CuS-Apt2 showed a nucleic acid characteristic absorption peak near 260 nm, indicating that Apt2 had been successfully coupled to the nanomaterial surface, providing a molecular recognition basis for the sensing platform. Subsequent experiments used bladder cancer cells HT-1376 as a model, employing the high-throughput photothermal biosensor for detecting tumor cells prepared by the method described in Example 1.

[0072] 2. Sensor photothermal performance and sensing feasibility analysis

[0073] The photothermal conversion properties of the material, such as Figure 3 As shown in (A), under 808 nm laser irradiation, the temperature of the bare glass slide did not change significantly, while the temperature of the MnO2@CuS modified glass slide rose rapidly and stabilized after about 45 s, demonstrating excellent photothermal effect. Therefore, 45 s (marked by the dashed line) was selected as the standard reading time for subsequent temperature quantification and thermal imaging.

[0074] Figure 3 Image (B) shows infrared thermograms and temperature values ​​of different modified interfaces. The images include a bare slide, an Apt1 modified slide, and a slide with a temperature of 1.0 × 10⁻⁶. 3 The interface temperatures (HT-1376 / Apt1 / slide) of cells / mL HT-1376 were 28.3℃, 28.4℃, and 28.6℃, respectively, all close to room temperature, indicating that the HT-1376 itself has almost no photothermal response. However, after introducing the MnO2@CuS-Apt2 probe and binding it to the captured cells, the temperature of the resulting MnO2@CuS / HT-1376 / Apt1 / slide composite structure significantly increased to 60.5℃. This result confirms that the MnO2@CuS nanoprobe has good photothermal properties and can generate a stable and significant thermal signal after specifically binding to the target cells, verifying the feasibility of this photothermal sensing strategy.

[0075] 3. Sensor detection sensitivity analysis

[0076] To evaluate the sensor sensitivity, different concentrations of HT-1376 cells (1.0 × 10⁻⁶) were tested. 1 ~1.0×10 6 The photothermal response (cells / mL). For example... Figure 4 As shown in Figure (A), the temperature rise curve changes significantly with increasing cell concentration; the higher the concentration, the greater the temperature rise. Figure 4 As shown in (B), after 45 s of irradiation, the color of the infrared thermogram transitions from low-concentration light blue and green to high-concentration yellow and orange-red, thus realizing the visualization and discrimination of the detection results.

[0077] like Figure 4 As shown in Figure (C), quantitative analysis indicates that the logarithm of temperature change (y, ℃) and cell concentration (x, cells / mL) is within 1.0 × 10⁻⁶. 1 ~1.0×10 6 The relationship showed good linearity within the range of cells / mL, and the linear regression equation was y = 10.7lgx + 0.66 (R²). 2 = 0.993). Based on the signal-to-noise ratio (S / N = 3), the limit of detection (LOD) of this sensor is calculated to be 2 cells / mL. These results demonstrate that this sensing platform possesses a wide linear range, high sensitivity, and excellent visualization capabilities, showing application potential in bladder cancer liquid biopsy.

[0078] 4. Sensor Performance Analysis

[0079] A systematic evaluation of the overall performance of the sensor is conducted. For example... Figure 5 As shown in (A), at 1.0 × 10 4 At a concentration of HT-1376 per cell / mL, the platform underwent three laser-switched cycles, and the temperature response curves showed good stability and reversibility, indicating that it possesses reliable thermal response recovery capabilities and supports multi-cycle detection. Figure 5 As shown in Figure (B), the results of the six parallel experiments were highly consistent, with a very small relative standard deviation (RSD), demonstrating that the sensor has excellent repeatability.

[0080] like Figure 5 As shown in (C), the selective assay results indicate that only HT-1376 cells caused a significant temperature rise, while the response signals of other non-target cells were close to the background level. This is attributed to the high affinity and specificity of Apt1 for the EpCAM protein, ensuring the sensor's anti-interference capability in complex samples. Figure 5 As shown in (D), the storage stability test shows that after the sensor was stored at 4℃ for 14 days, the photothermal signal did not decrease significantly, indicating that its performance is stable and suitable for batch preparation and storage.

[0081] 5. Sensor precision and accuracy analysis

[0082] To verify the reliability of the sensor in actual sample detection, a spiked recovery experiment was conducted on urine samples from healthy individuals, and the results are shown in Table 1.

[0083] Table 1. Sensor detection of HT-1376 in urine samples ( x ± s , n = 5)

[0084]

[0085] As shown in Table 1, the recoveries ranged from 98.2% to 102.0% at different spiking concentrations, with relative standard deviations (RSDs) ranging from 1.7% to 3.2%. These results indicate that the sensor possesses good detection accuracy and precision, enabling reliable analysis of tumor cells in actual biological samples.

[0086] 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 high-throughput photothermal biosensor for detecting tumor cells, characterized in that... Includes the following steps: Step 1: Preparation of photothermal signal probe MnO2@CuS-Apt2 a. Mix 3-5 mL tetramethylammonium hydroxide, 1-3 mL 30 wt% hydrogen peroxide, and 10-20 mL ultrapure water, and sonicate at room temperature for 20-40 min; then add 8-12 mL 0.2-0.5 mol / L MnCl2·4H2O solution, and stir the reaction at room temperature for 10-15 h; after the reaction is complete, centrifuge to collect the product, wash with water and ethanol, and dry in an oven at 50-70 ℃ to obtain MnO2 nanosheets; b. Disperse 20-30 mg of MnO2 nanosheets and 500-700 mg of Cu(NO3)2·3H2O in 20-30 mL of ultrapure water, add 5-15 mg of cetyltrimethylammonium bromide, sonicate for 20-40 min, let stand for 10-14 h, and collect the precipitate by centrifugation; redisperse the precipitate in 40-60 mL of 0.01-0.1 mol / L thioacetamide solution, and react in a water bath at 70-90 ℃ for 2-4 h for in-situ sulfidation; after the reaction is completed, centrifuge, wash and dry to obtain the MnO2@CuS composite material; c. The MnO2@CuS aqueous dispersion was incubated with the aptamer Apt2 to obtain the MnO2@CuS-Apt2 signal probe. The specific steps are as follows: Take 0.5~2 mL of 1~3 mg / mL MnO2@CuS aqueous dispersion, add 50~200 μL of 10~30 μmol / L thiol-modified aptamer Apt2, and shake at room temperature for 4~8 h. Remove unbound aptamers by centrifugation. Then add 0.5~2 mL of 0.5~2 mmol / L 6-mercapto-1-hexanol solution and incubate at room temperature for 0.5~1.5 h to block non-specific active sites on the surface of the nanomaterial. Finally, after centrifugation and washing, the obtained product is redispersed in 0.5~2 mL of phosphate buffer at pH 7.0~8.0 to obtain the MnO2@CuS-Apt2 signal probe dispersion. Step 2: Construction of a high-throughput photothermal sensing platform a. Clean the glass slides and perform amino-functionalization treatment to obtain amino-functionalized glass slides; b. Divide the aminated slide obtained in step a into multiple sensing regions and modify it with aptamer Apt1 to obtain a high-throughput capture substrate Apt1 / slide. The specific steps are as follows: Divide the surface of the aminated slide into 8-24 independent circular detection regions with a diameter of 4-8 mm using insulating tape; modify each circular region sequentially as follows: add 10-30 μL of 3-7 wt% glutaraldehyde solution and react at room temperature for 0.5-1.5 h; add 10-30 μL of 0.5-2 μmol / L amino-modified aptamer Apt1 and react at room temperature for 0.5-1.5 h to crosslink Apt1 with the amino groups on the slide; then add 10-30 μL of 0.5-2 wt% bovine serum albumin (BSA) solution and incubate at room temperature to block non-specific binding sites; After each modification step, the substrate was washed with ultrapure water 2-4 times to finally obtain a high-throughput capture substrate Apt1 / slide with multiple independent detection units. c. Add 100–300 μL of the sample solution containing different concentrations of tumor cells to the capture substrate Apt1 / slide, incubate at room temperature for 0.5–1.5 h, and then wash with PBS buffer to remove unbound cells; then add 10–30 μL of MnO2@CuS-Apt2 signal probe dispersion, and incubate at room temperature for 0.5–1.5 h to obtain a high-throughput photothermal biosensor for detecting tumor cells.

2. The method for preparing a high-throughput photothermal biosensor for detecting tumor cells according to claim 1, characterized in that... The preparation method of the aminated glass slide described in step 2a is as follows: The glass slide is placed in acetone, 0.5~2 mol / L sodium hydroxide (NaOH) in ethanol solution, and 0.5~2 mol / L NaOH in water / ethanol mixture for ultrasonic cleaning for 10~30 min respectively, rinsed with ultrapure water and dried with nitrogen gas. Then, the clean glass slide is immersed in a 1-3 wt% ethanol solution of 3-aminopropyltriethoxysilane (APTES) and reacted for 0.5-2 h to introduce amino functional groups onto the surface of the glass slide, thus obtaining an aminated glass slide.

3. The method for preparing a high-throughput photothermal biosensor for detecting tumor cells according to claim 1, characterized in that: The sequence of aptamer Apt1 is: 5'-NH2-CACTACAGAGGTTGCGTCTGTCCCACGTTGTCATGGGGGGTTGGCCTG-3'; the sequence of aptamer Apt2 is: 5'-SH-CACTACAGAGGTTGCGTCTGTCCCACGTTGTCATGGGGGGTTGGCCTG-3'.

4. A method for preparing a photothermal biosensor for high-throughput detection of tumor cells according to any one of claims 1-3, characterized in that: The tumor cells mentioned are breast cancer cells MCF-7, colorectal cancer cells HT-29, prostate cancer cells LNCaP, or bladder cancer cells HT-1376.

5. A detection application of a high-throughput photothermal biosensor for detecting tumor cells prepared by the method of any one of claims 1-3, wherein the application is not for diagnostic or therapeutic purposes, characterized in that... Includes the following steps: The detection area of ​​the photothermal biosensor is illuminated with a laser, and photothermal images are acquired and temperature changes are recorded using an infrared thermal imager. Qualitative and quantitative analysis of the target tumor cells is achieved by analyzing the temperature rise value ΔT.

6. The detection application according to claim 5, characterized in that: The laser is an 808 nm near-infrared laser with a power of 1 W.

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