Preparation method and application of photo-thermal biosensor for tumor cell quantification and vitality double-index liquid biopsy

Abstract

The invention discloses a preparation method and application of a photo-thermal biosensor for tumor cell quantification and vitality dual-index liquid biopsy. Phosphatidylserine (PS) is exposed by enriching and cracking tumor cells, is identified and combined by an aptamer Apt2, and is driven to be separated from Apt2-Cu2-xTe NSs; after magnetic separation, separated supernatant is subjected to photo-thermal detection; the photo-thermal signal intensity is positively correlated with the number of cells, so that quantitative detection is realized; during cell apoptosis, PS is turned out of the membrane, Apt2-Cu2-xTe NSs is combined with the surface of the membrane, separated supernatant is subjected to photo-thermal detection after magnetic separation, and the photo-thermal signal enhancement amplitude is in negative correlation with cell activity. The platform has the advantages of POCT characteristics, low cost and portability, and a new tool is provided for precise tumor diagnosis and treatment.

Description

Technical Field

[0001] The present invention relates to a photothermal biosensor, and in particular to a preparation method and application of a photothermal biosensor for liquid biopsy of tumor cell quantification and vitality dual indicators. Background Art

[0002] The progression assessment and efficacy monitoring of malignant tumors are highly dependent on the survival status and functional characteristics of tumor cells. Tumor metastasis caused by tumor cells is the main factor leading to clinical treatment failure. Liquid biopsy technology provides a new non-invasive detection strategy for precise tumor diagnosis and treatment by analyzing tumor cells and their related biomarkers in biological samples such as blood and urine. Although the dynamic changes in tumor cell number and activity are of great guiding value for early diagnosis, treatment optimization and efficacy evaluation, due to their low abundance distribution and heterogeneity in body fluid samples, existing detection technologies still face severe challenges in terms of sensitivity, specificity and clinical applicability.

[0003] Current tumor cell detection technologies primarily focus on quantitative analysis. Traditional methods such as flow cytometry and the Cell-Search system offer high accuracy, but they are expensive, require specialized personnel, and lack sensitivity. Emerging technologies such as colorimetry, fluorescence, surface plasmon resonance, photoelectrochemistry, and electrochemiluminescence sensors have been developed in recent years, demonstrating superior sensitivity and quantitative capabilities. However, these technologies often involve complex procedures and are susceptible to interference from complex sample backgrounds. Tumor cell viability, a key indicator of cell proliferation potential, metabolic activity, and metastatic capacity, is still largely limited to traditional techniques such as trypan blue staining and Calcein-AM / PI double staining. These techniques suffer from high error rates, time-consuming procedures, stain toxicity, and cumbersome procedures. While a deep learning model developed by our research group previously enabled the detection of tumor cell viability in complex blood samples, its construction required extensive data training, which was time-consuming and limited the ability to enrich and sensitively quantify tumor cells in large blood samples. Clearly, simultaneous detection of tumor cell number and viability can achieve a paradigm shift from static counting to dynamic functional analysis, redefining the role of liquid biopsy in the overall management of cancer—elevating it from an auxiliary diagnostic tool to a core engine guiding treatment decisions, thereby promoting precision medicine for cancer. Notably, to date, no studies have reported simultaneous detection of tumor cell number and viability.

[0004] In response to the above technical bottlenecks, the present invention proposes a scientific proposition to construct a multi-dimensional tumor cell detection system: How to achieve simultaneous and accurate detection of tumor cell number and vitality? A breakthrough in this problem will be directly related to key clinical links such as tumor metastasis risk assessment and personalized medication guidance. Based on this, the present invention innovatively developed a dual-index sensing platform based on photothermal effect. Its technical principle is: using epithelial cell adhesion molecule (EpCAM) aptamers to efficiently enrich tumor cells, and constructing a dual detection channel through the extracellular exposure characteristics of phosphatidylserine (PS) membrane - PS on the surface of the intact cell membrane reflects cell vitality, and the total PS in the cell lysate characterizes the number of cells. The experiment uses Cu 2-x Te nanosheets, used as signal conversion elements, enabled simultaneous detection of both cell count and viability in MCF-7 breast cancer and HT-1376 bladder cancer models. This platform combines the advantages of point-of-care (POCT) technology with low cost and portability, providing a novel tool for precision cancer diagnosis and treatment. Currently, there are no published reports, either domestically or internationally, on photothermal sensors that can simultaneously and accurately detect both cell count and viability. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide a method for preparing a photothermal sensor for dual-indicator detection of tumor cell number and vitality with high sensitivity, instant detection, low cost, simple and rapid operation, and its application.

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

[0007] 1. A method for preparing and applying a photothermal biosensor for dual-indicator liquid biopsy of tumor cell quantification and viability, which is not intended for diagnosis or treatment, and comprises the following steps:

[0008] (1) Preparation of Fe3O4-Apt1

[0009] 0.5-3.0 mg of Fe₃O₄-NH₂ was added to 1-5 mL of a 2.5 wt% aqueous glutaraldehyde solution at room temperature. After stirring for 1-4 hours and washing with water, the solution was redispersed in 1-5 mL of PBS. 50-500 μL of a 20 μmol / L Apt1 solution was then added and incubated at room temperature with shaking for 1-5 hours. Unbound Apt1 was then collected and washed using magnetic adsorption. 0.5-3.0 mL of 1 wt% BSA was then added and incubated at room temperature for 1 hour to block nonspecific binding sites. Finally, the solution was collected and washed using magnetic adsorption and redispersed in 0.5-3.0 mL of PBS to obtain a Fe₃O₄-Apt1 dispersion.

[0010] (2) Preparation of GO@Fe3O4-Apt2

[0011] 5–20 mg of EDC and 1–10 mg of NHS were added to 1–5 mL of a 1 mg / mL GO dispersion and sonicated for 10–60 min to activate the carboxyl groups of GO. Subsequently, 0.5–3.0 mg of Fe₃O₄-NH₂ was added and stirred at 37–100°C for 1–5 h. After washing, the mixture was redispersed in 0.5–3.0 mL of PBS to obtain a GO@Fe₃O₄ dispersion. 50–500 μL of 20 μmol / L Apt₂ was then added and incubated at room temperature with shaking for 1–5 h. Finally, the mixture was collected by magnetic adsorption, washed, and redispersed in 1–5 mL of PBS to obtain a GO@Fe₃O₄-Apt₂ dispersion.

[0012] (3)GO@Fe3O4-Apt2-Cu 2-x Preparation of Te NSs

[0013] Add 0.5-0.7g CuCl2·2H2O, 0.4-1.0g NaOH, 0.2-0.8g Na2TeO3, and 0.4-0.8g PVP to 20-40mL EG and stir at room temperature for 1-3h. Then transfer the mixture to a 50mL Teflon-lined stainless steel reactor and heat to 160-200℃ and keep it for 24-48h. After that, centrifuge and wash and dry at 50-70℃ to obtain CuCl2·2H2O. 2-x TeNSs powder. Take 1.0~3.0mg Cu 2-x Te NSs were dispersed in 0.5-2.0 mL PBS, and 0.5-2.0 mL GO@Fe3O4-Apt2 dispersion was added. The mixture was shaken and incubated overnight at 2-10 °C. The mixture was collected by magnetic adsorption and redispersed in 0.5-2 mL PBS to obtain GO@Fe3O4-Apt2-Cu 2-x Te NSs dispersion.

[0014] (4) Actual sample pretreatment

[0015] The number of tumor cells was counted using a cell counting plate, and the solution was serially diluted using culture medium to obtain a series of tumor cell standard solutions.

[0016] For MCF-7 cells, MCF-7 cells were added to 10-fold diluted healthy human whole blood samples to a final concentration of 5.0 × 10 6 , 5.0×10 5 , 5.0×10 4 , 5.0×10 3 , 5.0×10 2 and 50 cells / mL.

[0017] For HT-1376 cells, HT-1376 cells were added to healthy human urine samples to a final concentration of 5.0 × 10 6 , 5.0×10 5 , 5.0×10 4 , 5.0×10 3 , 5.0×10 2 and 50 cells / mL.

[0018] Add 100-500 μL of Fe₃O₄-Apt1 dispersion to 100-500 μL of tumor cell sample solution and incubate at 25-45°C for 10-60 minutes. Collect the cells by magnetic adsorption and redisperse them in 100-500 μL of PBS. Add 10-100 μL of 1 kU / mL DNase I to cleave Apt1 and release the enriched tumor cells. Then, collect the supernatant by magnetic separation and centrifuge at 800 × g to collect the captured tumor cells. Redisperse them in 100-500 μL of culture medium.

[0019] (5) Tumor cell concentration detection

[0020] Take the culture medium containing tumor cells in (4), lyse the cells and then detect the concentration of tumor cells.

[0021] (6) Photothermal signal-tumor cell viability standard curve

[0022] a. Trypsinize well-adherent MCF-7 cells to obtain a tumor cell suspension and count them. Inoculate 100 μL of tumor cell suspension into each well of a 96-well culture plate to ensure that the number of tumor cells is approximately 1.0 × 10 3 indivual.

[0023] b. MCF-7 cells were cultured at 37°C in 5% CO2 for 3-5 hours to allow the tumor cells to adhere to the wall.

[0024] c. Remove the supernatant and add PBS containing a certain concentration of H2O2 for incubation.

[0025] d. Transfer the suspension in the wells to a 2 mL filtered centrifuge tube, add PBS to the wells to wash the tumor cells, and transfer the PBS used to wash the tumor cells to a 2 mL filtered centrifuge tube to collect the suspended tumor cells.

[0026] e. Add 10-50 μL of 0.25% trypsin digestion solution to the wells and treat for 1-3 minutes. Discard the trypsin digestion solution and place the wells in a 5% CO2, 37°C culture environment for 1-2 minutes. Add 50-100 μL of new culture medium and use a pipette to repeatedly pipette to suspend the tumor cells.

[0027] f. Subsequently, the tumor cell suspension was centrifuged at 800 × g for 3–10 min to separate the tumor cells from the culture medium and dispersed in 10–50 μL of cell culture medium along with the tumor cells on the filter membrane of a 2 mL centrifuge tube.

[0028] g. Finally, add 50-100 μL GO@Fe3O4-Apt2-Cu 2-x The Te NSs dispersion was incubated at 37°C for 10–60 min, magnetically separated, and 10–50 μL of the upper layer was transferred to a 100 μL EP tube for photothermal testing.

[0029] As for HT-1376 cells, except for adding a certain concentration of cisplatin in PBS for incubation in step c, adding 10-50 μL of 0.25% trypsin digestion solution for treatment for 5-10 minutes in step e, discarding the trypsin digestion solution, and placing the cells in a 5% CO2, 37°C culture environment for 1-5 minutes, all other aspects are the same as those of MCF-7 cells.

[0030] The CCK-8 method was used to calibrate the cell viability of MCF-7 cells treated with different concentrations of H2O2 and HT-1376 cells treated with different concentrations of cisplatin.

[0031] (7) Photothermal determination of tumor cell viability in actual samples

[0032] Take the culture medium containing tumor cells in (4) and put it on the cell counting plate. Use a microscope to count the tumor cells to calculate the tumor cell concentration. After adjusting the concentration, add GO@Fe3O4-Apt2-Cu 2-x Te NSs dispersion, followed by photothermal testing.

[0033] Furthermore, the sequence of the epithelial cell adhesion molecule EpCAM aptamer Apt1 is as follows: 5′-NH2-CACTACAGAGGTTGCGTCTGTCCCACGTTGTCATGGGGGGTTGGCCTG-3′; the sequence of the phosphatidylserine PS aptamer Apt2 is as follows: 5′-SH-TTAAAGACTT-3′.

[0034] Furthermore, the tumor cells are breast cancer cells MCF-7 and bladder cancer cells HT-1376.

[0035] Furthermore, the tumor cell concentration test described in step (5) is as follows: 10-30 μL of the culture medium containing tumor cells in step (4) is taken, 10-30 μL of RIPA cell lysis buffer is added, and the tumor cells are fully lysed by ultrasonication for 5-10 minutes. Then 10-100 μL of GO@Fe3O4-Apt2-Cu is added. 2-xThe Te NSs dispersion was incubated at 25–45°C for 10–60 min, magnetically separated, and 10–30 μL of the upper layer was transferred to a 100 μL EP tube for photothermal testing.

[0036] Furthermore, the H2O2 treatment of MCF-7 cells was performed as described in step (6) c, specifically as follows: 50-100 μL of the upper culture medium was removed, 20-100 μL of PBS containing a certain concentration of H2O2 (0, 3, 6, 9, 12, 15, 18, 21, 24, 27, 30 mmol / L) was added, and the cells were incubated for 20-60 min. As for HT-1376 cells, PBS containing a certain concentration of cisplatin (0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 mmol / L) was added in step (6) c and the cells were incubated for 1-2 h.

[0037] Furthermore, the actual sample activity photothermal assay described in step (7) is specifically performed as follows: 50 to 200 μL of the culture medium containing tumor cells in step (4) is placed on a cell counting plate, and tumor cells are counted using a microscope to calculate the tumor cell concentration. The concentration is adjusted to 5.0×10 4 cells / mL, take 10-50 μL tumor cell suspension and add 20-100 μL GO@Fe3O4-Apt2-Cu 2-x The Te NSs dispersion was incubated at 25–45 °C for 10–60 min, magnetically separated, and 10–50 μL of the upper layer was transferred to a 100 μL EP tube for photothermal testing.

[0038] 2. A method for using a photothermal biosensor for dual-indicator liquid biopsy of tumor cell quantification and viability. This method is not intended for diagnosis or treatment and comprises the following steps: 1. Exciting a 100 μL EP tube containing 20 μL of a test solution using a near-infrared laser (1 W) with a wavelength of 808 nm. The temperature response and thermal imaging are recorded using an infrared thermal imager and its accompanying AnalyzIR software. The tumor cell concentration in the test solution is calculated based on the temperature signal.

[0039] Principle of invention: PS is a component present in all human cells, usually located on the inner side of the cell membrane. However, when cell vitality weakens, PS will flip from the inner side of the cell membrane to the outer side. Based on this characteristic, Figure 1 As shown, by enriching and lysing tumor cells, PS is fully exposed and specifically recognized and bound by Apt2, making GO@Fe3O4 and Apt2-Cu 2-x After magnetic separation, the upper layer of the separated liquid was photothermally detected. The more cells there were, the more PS there was. Therefore, the Apt2-Cu 2-xThe more Te NSs there are, the more obvious the photothermal signal response is, and thus quantitative detection of cells can be achieved. For cell viability detection, when the number of cells remains unchanged but apoptosis occurs, that is, the viability is weakened, PS is externalized to the cell membrane and is covered by GO@Fe3O4-Apt2-Cu 2-x Apt2 recognition on Te NSs enables Apt2-Cu 2-x Te NSs separated from GO@Fe3O4 and bound to the intact cell membrane. After magnetic separation, the separated upper layer was photothermally detected. The lower the cell viability, the more PS on the cell membrane was turned outward, and the more Apt2-Cu bound to the cell membrane was detected. 2-x The more Te NSs there are, the more pronounced the photothermal signal response becomes, enabling cell viability detection. Based on this method, a photothermal sensing platform for dual-indicator detection of tumor cell quantification and viability based on photothermal response was constructed.

[0040] Compared with the prior art, the advantages of the present invention are:

[0041] 1. Point-of-care testing: This method uses aptamer-mediated magnetic enrichment and photothermal signal conversion technology, eliminating the need for complex pretreatment or large instruments. The procedure is simplified (incubation time ≤ 30 minutes), and the test results are directly read using a portable infrared thermal imager. It is suitable for bedside or resource-limited scenarios.

[0042] 2. Dual-indicator simultaneous detection

[0043] A dual detection channel for tumor cell number and viability based on the extracellular exposure characteristics of phosphatidylserine (PS) is proposed. The total PS in the cell lysate characterizes the number, and the PS on the intact cell membrane surface reflects the viability. This breaks through the limitation of traditional methods that can only detect a single indicator (number or viability), and realizes a paradigm shift from static counting to dynamic functional analysis.

[0044] 3. Universality: It can detect tumor cells with high EpCAM expression without changing the aptamer. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 Flowchart of the photothermal sensor for simultaneous detection of tumor cell number and viability;

[0046] Figure 2 SEM images of (A) Fe3O4 and (B) GO@Fe3O4; (C) XRD patterns of Fe3O4 and GO@Fe3O4; (D) Zeta potential diagram of Fe3O4-NH2, GO and GO@Fe3O4;

[0047] Figure 3 Cu 2-x(A) TEM image and (B) XRD pattern of Te; (C) Te 3d and (D) Cu 2p high-resolution XPS spectra;

[0048] Figure 4 The biosensor platform is sensitive to different concentrations (5.0×10 1 ~5.0×10 6 cells / mL) (A) Photothermal response curves of MCF-7 and (D) HT-1376 cells; Thermal imaging of the sensing platform at different concentrations of (B) MCF-7 and (E) HT-1376; Linear relationship between temperature difference (ΔT) and the logarithm of (C) MCF-7 and (F) HT-1376 cell concentrations;

[0049] Figure 5 (A) Repeatability and (B) stability of quantitative detection of MCF-7 cells; (C) Repeatability and (D) stability of quantitative detection of HT-1376 cells;

[0050] Figure 6 (A) Cell viability values ​​of MCF-7 cells after treatment with different concentrations of H2O2 (0-30 mmol / L) measured by CCK-8; (B) Cell viability values ​​of HT-1376 cells after treatment with different concentrations of cisplatin (0-10 mmol / L) measured by CCK-8

[0051] Figure 7 Figure 3. Photothermal response of the biosensor platform to (A) MCF-7 and (D) HT-1376 cells with different viabilities; (B) Thermal imaging of the sensing platform under different viabilities of MCF-7 and (E) HT-1376 cells; Linear relationship between temperature difference (ΔT) and viability values ​​of (C) MCF-7 and (F) HT-1376 cells. DETAILED DESCRIPTION

[0052] The present invention will be described in further detail below with reference to the accompanying drawings and embodiments. 1. Specific embodiments

[0054] Example 1

[0055] A method for preparing and applying a photothermal biosensor for dual-indicator liquid biopsy of tumor cell quantification and viability, which is not intended for diagnosis or treatment, comprises the following steps:

[0056] (1) Preparation of Fe3O4-Apt1

[0057] 2 mg of Fe₃O₄-NH₂ was added to 2 mL of 2.5 wt% glutaraldehyde solution, stirred at room temperature for 2 hours, washed with water, and then redispersed in 2 mL of PBS. 100 μL of 20 μmol / L Apt1 solution was added, and the mixture was incubated at room temperature with shaking for 2 hours. Unbound Apt1 was then collected and washed by magnetic adsorption. 1 mL of 1 wt% BSA was added, and the mixture was incubated at room temperature for 1 hour to block nonspecific binding sites. Finally, the mixture was collected and washed by magnetic adsorption and redispersed in 1 mL of PBS to obtain a Fe₃O₄-Apt1 dispersion.

[0058] (2) Preparation of GO@Fe3O4-Apt2

[0059] 10 mg of EDC and 5 mg of NHS were added to 2 mL of a 1 mg / mL GO dispersion and sonicated for 30 minutes to activate the carboxyl groups of GO. Subsequently, 2 mg of Fe₃O₄-NH₂ was added, and the mixture was stirred at 80°C for 2 hours. After washing, the mixture was redispersed in 2 mL of PBS to obtain a GO@Fe₃O₄ dispersion. 100 μL of 20 μmol / L Apt₂ was then added, and the mixture was incubated at room temperature with shaking for 2 hours. Finally, the mixture was collected by magnetic adsorption, washed, and redispersed in 1 mL of PBS to obtain a GO@Fe₃O₄-Apt₂ dispersion.

[0060] Figure 2 The synthesized Fe3O4 and GO@Fe3O4 were characterized by scanning electron microscopy (SEM) and X-ray diffraction (XRD). The Fe3O4 synthesized by hydrothermal method was regular spherical with a size of about 200nm ( Figure 2 A). After the carboxyl groups of graphene oxide are activated by EDC / NHS, they can couple with Fe3O4-NH2 to form GO@Fe3O4( Figure 2 B). In addition, the XRD characteristic diffraction peaks of Fe3O4 match well with the Fe3O4 standard card (JCPDS No.88-0315), indicating that Fe3O4 was successfully synthesized; GO@Fe3O4 has a broad peak between 20° and 30°, which is attributed to GO. The other characteristic peaks are basically consistent with those of Fe3O4, indicating that Fe3O4 is successfully loaded on GO without affecting the crystal structure of Fe3O4 ( Figure 2 C). Zeta potential analysis ( Figure 2 D) shows that the Zeta potentials of Fe3O4-NH2 (a) and GO (b) are 18.7 mV and -37.6 mV, respectively. The Zeta potential of GO@Fe3O4 (c) is 10.1 mV, further proving that GO@Fe3O4 was successfully synthesized by assembling the two.

[0061] (3)GO@Fe3O4-Apt2-Cu 2-x Preparation of Te NSs

[0062] 0.61g CuCl2·2H2O, 0.96g NaOH, 0.40g Na2TeO3, and 0.53g PVP were added to 36mL EG and stirred at room temperature for 2h. The mixture was then transferred to a 50mL Teflon-lined stainless steel reactor and heated to 180℃ for 36h. After that, it was centrifuged, washed, and dried at 60℃ to obtain Cu 2-x Te NSs powder. Take 2 mg Cu 2-x Te NSs were dispersed in 1 mL PBS, and 1 mL GO@Fe3O4-Apt2 dispersion was added. The mixture was shaken and incubated overnight at 4 °C. The mixture was collected by magnetic adsorption and redispersed in 1 mL PBS to obtain GO@Fe3O4-Apt2-Cu 2-x Te NSs dispersion.

[0063] Figure 3 The synthesized Cu was characterized by SEM and transmission electron microscopy (TEM). 2-x Te was characterized by morphology, and it can be seen that Cu 2-x Te is a thin nanosheet structure ( Figures S1 and Figure 3 A), its XRD diffraction peak is similar to Cu 2-x Te standard card (JCPDS No.10-0421) matches well ( Figure 3 B). The full X-ray photoelectron spectroscopy (XPS) spectrum shows peaks for Cu, Te, C, and O (Figure S2); the Te 3d spectrum fitting yields four peaks, namely, Te at 572.5 and 582.8 eV. 2+ , Te at 575.9 and 586.4 eV 4+ ( Figure 3 C); Cu 2p spectrum fitting yields four peaks, namely Cu at 935.0 and 954.3 eV 2+ , Cu at 932.6 and 952.2 eV + ( Figure 3 D). The above results show that Cu 2-x Te was successfully synthesized.

[0064] (4) Actual sample pretreatment

[0065] The number of tumor cells was counted using a cell counting plate, and the solution was serially diluted using culture medium to obtain a series of tumor cell standard solutions.

[0066] For MCF-7 cells, MCF-7 cells were added to 10-fold diluted healthy human whole blood samples to a final concentration of 5.0 × 10 6 , 5.0×10 5 , 5.0×10 4, 5.0×10 3 , 5.0×10 2 and 50 cells / mL.

[0067] For HT-1376 cells, HT-1376 cells were added to healthy human urine samples to a final concentration of 5.0 × 10 6 , 5.0×10 5 , 5.0×10 4 , 5.0×10 3 , 5.0×10 2 and 50 cells / mL.

[0068] 200 μL of Fe₃O₄-Apt1 dispersion was added to 200 μL of tumor cell sample solution and incubated at 37°C for 30 minutes. The suspension was collected by magnetic adsorption and redispersed in 200 μL of PBS. 50 μL of 1 kU / mL DNase I was added to cleave Apt1 and release the enriched tumor cells. The supernatant was then collected by magnetic separation and centrifuged at 800 × g to collect the captured tumor cells, which were then redispersed in 200 μL of culture medium.

[0069] (5) Tumor cell concentration detection

[0070] Take 20 μL of the culture medium containing tumor cells in (4), add 10 μL of RIPA cell lysis buffer, and sonicate for 5 minutes to fully lyse the tumor cells. Then add 50 μL of GO@Fe3O4-Apt2-Cu 2-x The Te NSs dispersion was incubated at 37 °C for 30 min, magnetically separated, and 20 μL of the upper layer was transferred to a 100 μL EP tube for photothermal testing.

[0071] (6) Photothermal signal-tumor cell viability standard curve

[0072] a. Trypsinize well-adherent MCF-7 cells to obtain a tumor cell suspension and count them. Inoculate 100 μL of tumor cell suspension into each well of a 96-well culture plate to ensure that the number of tumor cells is approximately 1.0 × 10 3 indivual.

[0073] b. MCF-7 cells were cultured at 37°C in 5% CO2 for 4 h to allow tumor cells to adhere to the wall.

[0074] c. Remove 75 μL of the upper culture medium, add 50 μL of PBS containing a certain concentration of H2O2 (0, 3, 6, 9, 12, 15, 18, 21, 24, 27, 30 mmol / L) and incubate for 40 min.

[0075] d. Transfer the suspension in the wells to a 2 mL filtered centrifuge tube, add PBS to the wells to wash the tumor cells, and transfer the PBS used to wash the tumor cells to a 2 mL filtered centrifuge tube to collect the suspended tumor cells.

[0076] e. Add 20 μL of 0.25% trypsin digestion solution to the wells and treat for 1.5 minutes. Discard the trypsin digestion solution and place the wells in a 5% CO2, 37°C culture environment for 1 minute. Add 100 μL of new culture medium and use a pipette to repeatedly pipette to suspend the tumor cells.

[0077] f. Subsequently, the tumor cell suspension was centrifuged at 800 × g for 5 min to separate the tumor cells from the culture medium and dispersed in 20 μL of cell culture medium together with the tumor cells on the filter membrane of a 2 mL centrifuge tube.

[0078] g. Finally, 50 μL GO@Fe3O4-Apt2-Cu was added to the above tumor cell suspension. 2-x The Te NSs dispersion was incubated at 37 °C for 30 min, magnetically separated, and 20 μL of the upper layer was transferred to a 100 μL EP tube for photothermal testing.

[0079] As for HT-1376 cells, except that in step c, PBS containing certain concentrations of cisplatin (0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10 mmol / L) was added and incubated for 1 hour, and in step e, 20 μL of 0.25% trypsin digestion solution was added and treated for 8 minutes, the trypsin digestion solution was discarded, and the cells were placed in a 5% CO2, 37°C culture environment for 2 minutes, all other aspects were the same as those of MCF-7 cells.

[0080] The CCK-8 method was used to calibrate the cell viability of MCF-7 cells treated with different concentrations of H2O2 and HT-1376 cells treated with different concentrations of cisplatin.

[0081] (7) Photothermal determination of tumor cell viability in actual samples

[0082] Take 100 μL of the culture medium containing tumor cells in (4) and place it on a cell counting plate. Count the tumor cells using a microscope to calculate the tumor cell concentration. Adjust the concentration to 5.0×10 4 cells / mL, take 20μL tumor cell suspension and add 50μL GO@Fe3O4-Apt2-Cu 2-x The Te NSs dispersion was incubated at 37 °C for 30 min, magnetically separated, and 20 μL of the upper layer was transferred to a 100 μL EP tube for photothermal testing.

[0083] Example 2

[0084] The same as the above embodiment 1, the difference is:

[0085] A method for preparing and applying a photothermal biosensor for dual-indicator liquid biopsy of tumor cell quantification and viability, which is not intended for diagnosis or treatment, comprises the following steps:

[0086] (1) Preparation of Fe3O4-Apt1

[0087] 1.5 mg of Fe₃O₄-NH₂ was added to 1 mL of 2.5 wt% glutaraldehyde solution, stirred at room temperature for 1 hour, and washed with water. The solution was then redispersed in 1 mL of PBS. 50 μL of 20 μmol / L Apt1 solution was then added, and the mixture was incubated at room temperature with shaking for 1 hour. Unbound Apt1 was then collected and washed by magnetic adsorption. 0.5 mL of 1 wt% BSA was then added, and the mixture was incubated at room temperature for 2 hours to block nonspecific binding sites. Finally, the solution was collected and washed by magnetic adsorption and redispersed in 0.5 mL of PBS to obtain a Fe₃O₄-Apt1 dispersion.

[0088] (2) Preparation of GO@Fe3O4-Apt2

[0089] 5 mg of EDC and 2 mg of NHS were added to 1 mL of a 1 mg / mL GO dispersion and sonicated for 20 minutes to activate the carboxyl groups of GO. Subsequently, 1 mg of Fe₃O₄-NH₂ was added, stirred at 50°C for 1 hour, washed, and redispersed in 1 mL of PBS to obtain a GO@Fe₃O₄ dispersion. 50 μL of 20 μmol / L Apt₂ was then added, and the mixture was incubated with shaking at room temperature for 1 hour. Finally, the mixture was collected by magnetic adsorption, washed, and redispersed in 2 mL of PBS to obtain a GO@Fe₃O₄-Apt₂ dispersion.

[0090] (3)GO@Fe3O4-Apt2-Cu 2-x Preparation of Te NSs

[0091] 0.55g CuCl2·2H2O, 0.75g NaOH, 0.57g Na2TeO3, and 0.62g PVP were added to 30mL EG and stirred at room temperature for 1.5h. The mixture was then transferred to a 50mL Teflon-lined stainless steel reactor and heated to 170℃ for 24h. Afterwards, the mixture was washed by centrifugation and dried at 50℃ to obtain CuCl2·2H2O. 2-x Te NSs powder. Take 1.5 mg Cu 2-x Te NSs were dispersed in 0.5 mL PBS, and 0.5 mL GO@Fe3O4-Apt2 dispersion was added. The mixture was shaken and incubated overnight at 2 °C. The mixture was collected by magnetic adsorption and redispersed in 0.5 mL PBS to obtain GO@Fe3O4-Apt2-Cu 2-xTe NSs dispersion.

[0092] (4) Actual sample pretreatment

[0093] The number of tumor cells was counted using a cell counting plate, and the solution was serially diluted using culture medium to obtain a series of tumor cell standard solutions.

[0094] For MCF-7 cells, MCF-7 cells were added to 10-fold diluted healthy human whole blood samples to a final concentration of 5.0 × 10 6 , 5.0×10 5 , 5.0×10 4 , 5.0×10 3 , 5.0×10 2 and 50 cells / mL.

[0095] For HT-1376 cells, HT-1376 cells were added to healthy human urine samples to a final concentration of 5.0 × 10 6 , 5.0×10 5 , 5.0×10 4 , 5.0×10 3 , 5.0×10 2 and 50 cells / mL.

[0096] 100 μL of Fe₃O₄-Apt1 dispersion was added to 150 μL of tumor cell sample solution and incubated at 25°C for 20 minutes. The suspension was collected by magnetic adsorption and redispersed in 150 μL of PBS. 20 μL of 1 kU / mL DNase I was added to cleave Apt1 and release the enriched tumor cells. The supernatant was then collected by magnetic separation and centrifuged at 800 × g to collect the captured tumor cells. The cells were then redispersed in 150 μL of culture medium.

[0097] (5) Tumor cell concentration detection

[0098] Take 10 μL of the culture medium containing tumor cells in (4), add 15 μL of RIPA cell lysis buffer, and sonicate for 8 minutes to fully lyse the tumor cells. Then add 30 μL of GO@Fe3O4-Apt2-Cu 2-x The Te NSs dispersion was incubated at 25 °C for 15 min, magnetically separated, and 10 μL of the upper layer was transferred to a 100 μL EP tube for photothermal testing.

[0099] (6) Photothermal signal-tumor cell viability standard curve

[0100] a. Trypsinize well-adherent MCF-7 cells to obtain a tumor cell suspension and count them. Inoculate 100 μL of tumor cell suspension into each well of a 96-well culture plate to ensure that the number of tumor cells is approximately 1.0 × 10 3 indivual.

[0101] b. MCF-7 cells were cultured at 37°C in 5% CO2 for 3 h to allow tumor cells to adhere to the wall.

[0102] c. Remove 50 μL of the upper culture medium, add 30 μL of PBS containing a certain concentration of H2O2 (0, 3, 6, 9, 12, 15, 18, 21, 24, 27, 30 mmol / L) and incubate for 20 min.

[0103] d. Transfer the suspension in the wells to a 2 mL filtered centrifuge tube, add PBS to the wells to wash the tumor cells, and transfer the PBS used to wash the tumor cells to a 2 mL filtered centrifuge tube to collect the suspended tumor cells.

[0104] e. Add 10 μL of 0.25% trypsin digestion solution to the wells and treat for 2 minutes. Discard the trypsin digestion solution and place in a 5% CO2, 37°C culture environment for 1.5 minutes. Add 50 μL of new culture medium and use a pipette to repeatedly pipette to suspend the tumor cells.

[0105] f. Subsequently, the tumor cell suspension was centrifuged at 800 × g for 8 min to separate the tumor cells from the culture medium and dispersed in 10 μL of cell culture medium together with the tumor cells on the filter membrane of a 2 mL centrifuge tube.

[0106] g. Finally, 60 μL GO@Fe3O4-Apt2-Cu was added to the above tumor cell suspension. 2-x The Te NSs dispersion was incubated at 25 °C for 20 min, magnetically separated, and 10 μL of the upper layer was transferred to a 100 μL EP tube for photothermal testing.

[0107] As for HT-1376 cells, except that in step c, PBS containing certain concentrations of cisplatin (0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 mmol / L) was added and incubated for 1.5 h, and in step e, 10 μL of 0.25% trypsin digestion solution was added and treated for 5 min, the trypsin digestion solution was discarded, and the cells were placed in a 5% CO2, 37°C culture environment for 3 min, all other aspects were the same as those of MCF-7 cells.

[0108] The CCK-8 method was used to calibrate the cell viability of MCF-7 cells treated with different concentrations of H2O2 and HT-1376 cells treated with different concentrations of cisplatin.

[0109] (7) Photothermal determination of tumor cell viability in actual samples

[0110] Take 80 μL of the culture medium containing tumor cells in (4) and place it on a cell counting plate. Count the tumor cells using a microscope to calculate the tumor cell concentration. Adjust the concentration to 5.0×10 4 cells / mL, 10 μL of tumor cell suspension was added with 30 μL of GO@Fe3O4-Apt2-Cu 2-x The Te NSs dispersion was incubated at 25 °C for 20 min, magnetically separated, and 10 μL of the upper layer was transferred to a 100 μL EP tube for photothermal testing.

[0111] Example 3

[0112] The same as the above embodiment 1, the difference is:

[0113] A method for preparing and applying a photothermal biosensor for dual-indicator liquid biopsy of tumor cell quantification and viability, which is not intended for diagnosis or treatment, comprises the following steps:

[0114] (1) Preparation of Fe3O4-Apt1

[0115] 3 mg of Fe₃O₄-NH₂ was added to 5 mL of 2.5 wt% glutaraldehyde solution, stirred at room temperature for 3 hours, washed with water, and then redispersed in 5 mL of PBS. 200 μL of 20 μmol / L Apt1 solution was added, and the mixture was incubated at room temperature with shaking for 3 hours. Unbound Apt1 was then collected and washed by magnetic adsorption. 5 mL of 1 wt% BSA was added and incubated at room temperature for 3 hours to block nonspecific binding sites. Finally, the mixture was collected and washed by magnetic adsorption and redispersed in 5 mL of PBS to obtain a Fe₃O₄-Apt1 dispersion.

[0116] (2) Preparation of GO@Fe3O4-Apt2

[0117] 20 mg of EDC and 8 mg of NHS were added to 5 mL of a 1 mg / mL GO dispersion and sonicated for 60 minutes to activate the carboxyl groups of GO. Subsequently, 5 mg of Fe₃O₄-NH₂ was added, and the mixture was stirred at 90°C for 3 hours. After washing, the mixture was redispersed in 5 mL of PBS to obtain a GO@Fe₃O₄ dispersion. 200 μL of 20 μmol / L Apt₂ was then added, and the mixture was incubated at room temperature with shaking for 3 hours. Finally, the mixture was collected by magnetic adsorption, washed, and redispersed in 5 mL of PBS to obtain a GO@Fe₃O₄-Apt₂ dispersion.

[0118] (3)GO@Fe3O4-Apt2-Cu 2-x Preparation of Te NSs

[0119] 0.70 g CuCl2·2H2O, 0.98 g NaOH, 0.75 g Na2TeO3, and 0.78 g PVP were added to 40 mL EG and stirred at room temperature for 3 h. The mixture was then transferred to a 50 mL Teflon-lined stainless steel reactor and heated to 200 ° C for 48 h. Afterwards, it was centrifuged and washed and dried at 70 ° C to obtain Cu 2-x Te NSs powder. Take 3mg Cu 2-x Te NSs were dispersed in 2 mL PBS, and 2 mL GO@Fe3O4-Apt2 dispersion was added. The mixture was shaken and incubated overnight at 10 °C. The mixture was collected by magnetic adsorption and redispersed in 2 mL PBS to obtain GO@Fe3O4-Apt2-Cu 2-x Te NSs dispersion.

[0120] (4) Actual sample pretreatment

[0121] The number of tumor cells was counted using a cell counting plate, and the solution was serially diluted using culture medium to obtain a series of tumor cell standard solutions.

[0122] For MCF-7 cells, MCF-7 cells were added to 10-fold diluted healthy human whole blood samples to a final concentration of 5.0 × 10 6 , 5.0×10 5 , 5.0×10 4 , 5.0×10 3 , 5.0×10 2 and 50 cells / mL.

[0123] For HT-1376 cells, HT-1376 cells were added to healthy human urine samples to a final concentration of 5.0 × 10 6 , 5.0×10 5 , 5.0×10 4 , 5.0×10 3 , 5.0×10 2 and 50 cells / mL.

[0124] 500 μL of Fe₃O₄-Apt1 dispersion was added to 500 μL of tumor cell sample solution and incubated at 45°C for 50 minutes. The suspension was collected by magnetic adsorption and redispersed in 500 μL of PBS. 80 μL of 1 kU / mL DNase I was added to cleave Apt1 and release the enriched tumor cells. The supernatant was then collected by magnetic separation and centrifuged at 800 × g to collect the captured tumor cells, which were then redispersed in 500 μL of culture medium.

[0125] (5) Tumor cell concentration detection

[0126] Take 30 μL of the culture medium containing tumor cells in (4), add 30 μL of RIPA cell lysis buffer, and ultrasonicate for 10 minutes to fully lyse the tumor cells. Then add 80 μL of GO@Fe3O4-Apt2-Cu 2-x The Te NSs dispersion was incubated at 45 °C for 50 min, magnetically separated, and 30 μL of the upper layer was transferred to a 100 μL EP tube for photothermal testing.

[0127] (6) Photothermal signal-tumor cell viability standard curve

[0128] a. Trypsinize well-adherent MCF-7 cells to obtain a tumor cell suspension and count them. Inoculate 100 μL of tumor cell suspension into each well of a 96-well culture plate to ensure that the number of tumor cells is approximately 1.0 × 10 3 indivual.

[0129] b. MCF-7 cells were cultured at 37°C in 5% CO2 for 5 h to allow tumor cells to adhere to the wall.

[0130] c. Remove 80 μL of the upper culture medium, add 80 μL of PBS containing a certain concentration of H2O2 (0, 3, 6, 9, 12, 15, 18, 21, 24, 27, 30 mmol / L) and incubate for 50 min.

[0131] d. Transfer the suspension in the wells to a 2 mL filtered centrifuge tube, add PBS to the wells to wash the tumor cells, and transfer the PBS used to wash the tumor cells to a 2 mL filtered centrifuge tube to collect the suspended tumor cells.

[0132] e. Add 50 μL of 0.25% trypsin digestion solution to the wells and treat for 3 minutes. Discard the trypsin digestion solution and place in a 5% CO2, 37°C culture environment for 2 minutes. Add 80 μL of new culture medium and use a pipette to repeatedly pipette to suspend the tumor cells.

[0133] f. Subsequently, the tumor cell suspension was centrifuged at 800 × g for 10 min to separate the tumor cells from the culture medium and dispersed in 80 μL of cell culture medium together with the tumor cells on the filter membrane of a 2 mL centrifuge tube.

[0134] g. Finally, 80 μL GO@Fe3O4-Apt2-Cu was added to the above tumor cell suspension. 2-x The Te NSs dispersion was incubated at 45 °C for 50 min, magnetically separated, and 30 μL of the upper layer was transferred to a 100 μL EP tube for photothermal testing.

[0135] As for HT-1376 cells, except that in step c, PBS containing certain concentrations of cisplatin (0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 mmol / L) was added and incubated for 2 hours, and in step e, 10 μL of 0.25% trypsin digestion solution was added for 8 minutes, the trypsin digestion solution was discarded, and the cells were placed in a 5% CO2, 37°C culture environment for 5 minutes, all other aspects were the same as those of MCF-7 cells.

[0136] The CCK-8 method was used to calibrate the cell viability of MCF-7 cells treated with different concentrations of H2O2 and HT-1376 cells treated with different concentrations of cisplatin.

[0137] (7) Photothermal determination of tumor cell viability in actual samples

[0138] Take 200 μL of the culture medium containing tumor cells in (4) and place it on a cell counting plate. Count the tumor cells using a microscope to calculate the tumor cell concentration. Adjust the concentration to 5.0×10 4 cells / mL, 50 μL of tumor cell suspension was added with 100 μL of GO@Fe3O4-Apt2-Cu 2-x The Te NSs dispersion was incubated at 45 °C for 50 min, magnetically separated, and 30 μL of the upper layer was transferred to a 100 μL EP tube for photothermal testing.

[0139] 2. Experimental Results Analysis

[0140] 1. Photothermal sensing analysis of cell concentration

[0141] Under the optimal experimental conditions, the biosensor platform was studied for MCF-7 ( Figure 4 A, 4B, 4C) and HT-1376( Figure 4 D, 4E, 4F) Cell quantitative analysis performance. Figure 4 As shown in A and 4D, the photothermal response signal (temperature) increases with the increase of illumination time, and the temperature is basically stable at 90s. Therefore, the temperature and thermal imaging at 90s are selected for subsequent analysis. As the cell concentration increases, the temperature of the sensing platform ( Figure 4 A, 4D) and thermal imaging color ( Figure 4 B, 4E) also change accordingly. The temperature change ΔT is related to the cell 2 ~5.0×10 6 The logarithm of the concentration in the range of cells / mL showed a good linear relationship, such as Figure 4 As shown in C and 4F, the linear regression equation of MCF-7 cells is y = 10.58*lg x-23.2, and the square correlation coefficient R 2=0.991, based on the signal-to-noise ratio S / N=3, the detection limit (LOD) was calculated to be 160 cells / mL; the linear regression equation for HT-1376 cells was y=9.28*lg x-19.8, and the square correlation coefficient R 2 =0.993, based on a signal-to-noise ratio (S / N) of 3, the LOD is approximately 145 cells / mL. Where y is the temperature difference ΔT (°C) between the sensing platform temperature response and room temperature (20°C), and x is the cell concentration (cells / mL).

[0142] 2. Repeatability and stability analysis

[0143] Repeatability and stability are important indicators for evaluating the performance of the biosensor platform. 4 The repeatability of quantitative detection of MCF-7 and HT-1376 cells was investigated by 6 parallel tests, and the relative standard deviation (RSD) values ​​were 1.3% and 1.8% ( Figure 5 A, 5C), showing high precision. The prepared nanomaterials were stored at 4 ° C and measured continuously for 10 days to explore the long-term storage stability of the sensor. Figure 5 As shown in B and 5D, after 10 days, the photothermal responses of the sensor to MCF-7 and HT-1376 cells remained at 93.4% and 92.7% of the original values, respectively, indicating good storage stability.

[0144] 3. Photothermal Sensing Analysis of Cell Viability

[0145] The analytical performance of the biosensor platform for MCF-7 and HT-1376 cell viability was studied. Figure 6 As shown in A, after MCF-7 cells were treated with PBS containing different concentrations of H2O2 (0, 5, 10, 15, 20, 25, 30 mmol / L), the cell viability values ​​of MCF-7 cells measured by CCK-8 were 99.4%, 83.2%, 70.3%, 51.0%, 36.9%, 20.0%, and 5.2%, respectively. Figure 6 As shown in Figure B, after HT-1376 cells were treated with PBS containing different concentrations (0, 2, 4, 6, 8, 10, 12 mmol / L) of cisplatin, the HT-1376 cell viability values ​​measured by CCK-8 were 99.5%, 83.4%, 63.0%, 51.8%, 33.6%, 17.2%, 4.4%, 18.0%, and 4.0%, respectively. Photothermal testing was performed on cells with different viability values, and it was found that the photothermal signal increased with the decrease of cell viability ( Figure 7 A, 7D), the color of the thermal image also becomes darker ( Figure 7B, 7E). The cell viability of MCF-7 showed a good linear relationship with the photothermal signal in the range of 99.4% to 5.2% ( Figure 7 C), the linear regression equation is y = -0.25*x + 26.7, and the square of the correlation coefficient R 2 =0.994. The cell viability of HT-1376 showed a good linear relationship with the photothermal signal in the range of 99.5% to 4.4% ( Figure 7 F), the linear regression equation is y = -0.19*x + 24.0, and the square correlation coefficient R 2 =0.991. Where y is the temperature difference ΔT (°C) between the temperature response of the sensing platform and room temperature (20°C), and x is the cell viability value (%).

[0146] 3. Application Examples

[0147] 1. Analysis of actual spiked sample cell concentration

[0148] In order to evaluate whether the developed biosensor platform can perform sensitive and reliable cell quantitative detection on real samples, different concentrations (1.0×10 3 , 1.0×10 4 , 1.0×10 5 The biosensor platform was validated using human blood samples spiked with MCF-7 (100 cells / mL) and human urine samples spiked with HT-1376 (100 cells / mL). The test results are shown in Table 1. The recoveries of the sensor platform for MCF-7 and HT-1376 were 97.9% to 101.4% and 96.9% to 99.3%, respectively, with RSDs of 3.7% to 5.0% and 1.9% to 4.7%, respectively. This demonstrates that the constructed cell quantification sensor has promising potential for application in real-world blood and urine samples.

[0149] Table 1 Detection of MCF-7 and HT-1376 in actual samples by the biosensor platform (x±s, n=5)

[0150]

[0151] 2. Analysis of cell viability of actual spiked samples

[0152] Table 2. Activity of MCF-7 and HT-1376 in actual samples detected by the biosensor platform (x±s, n=5)

[0153]

[0154] In order to evaluate whether the developed biosensor platform can perform sensitive and reliable cell viability detection on real samples, three groups of cells with a cell concentration of 5.0×10 4Cells / mL of MCF-7 cells and HT-1376 cells were used as test objects. After the MCF-7 cells were treated with PBS containing different concentrations (8, 18, 28 mmol / L) of H2O2, the cell viability measured by CCK-8 was 75.2%, 48.0%, and 15.3%, respectively. After the HT-1376 cells were treated with PBS containing different concentrations (3, 7, 11 mmol / L) of cisplatin, the cell viability measured by CCK-8 was 74.5%, 45.1%, and 10.4%, respectively. Through the biosensor platform, combined with the linear relationship between cell viability and temperature difference ( Figure 7 C, 7F), and the results are shown in Table 2. The recovery rates of the sensing platform for MCF-7 and HT-1376 tests were 99.2%-102.0% and 98.7%-102.3%, respectively, with RSDs of 2.8%-4.7% and 2.1%-4.7%, respectively, indicating that the constructed cell viability sensor has good potential application capabilities in the detection of actual blood samples and actual urine samples.

[0155] The above description is not intended to limit the present invention, and the present invention is not limited to the above examples. Any changes, modifications, additions or substitutions made by persons of ordinary skill in the art within the spirit and scope of the present invention shall also fall within the scope of protection of the present invention.

Claims

1. A method for preparing and applying a photothermal biosensor for dual-indicator liquid biopsy of tumor cell quantification and viability, which is not intended for diagnosis or treatment, and comprises the following steps: (1) Preparation of Fe3O4-Apt1 0.5-3.0 mg of Fe₃O₄-NH₂ was added to 1-5 mL of a 2.5 wt% aqueous glutaraldehyde solution at room temperature. After stirring for 1-4 hours and washing with water, the solution was redispersed in 1-5 mL of PBS. 50-500 μL of a 20 μmol / L Apt1 solution was then added and incubated at room temperature with shaking for 1-5 hours. Unbound Apt1 was then collected and washed using magnetic adsorption. 0.5-3.0 mL of 1 wt% BSA was then added and incubated at room temperature for 1 hour to block nonspecific binding sites. Finally, the solution was collected and washed using magnetic adsorption and redispersed in 0.5-3.0 mL of PBS to obtain a Fe₃O₄-Apt1 dispersion. (2) Preparation of GO@Fe3O4-Apt2 5–20 mg of EDC and 1–10 mg of NHS were added to 1–5 mL of a 1 mg / mL GO dispersion and sonicated for 10–60 min to activate the carboxyl groups of GO. Subsequently, 0.5–3.0 mg of Fe₃O₄-NH₂ was added and stirred at 37–100°C for 1–5 h. After washing, the mixture was redispersed in 0.5–3.0 mL of PBS to obtain a GO@Fe₃O₄ dispersion. 50–500 μL of 20 μmol / L Apt₂ was then added and incubated at room temperature with shaking for 1–5 h. Finally, the mixture was collected by magnetic adsorption, washed, and redispersed in 1–5 mL of PBS to obtain a GO@Fe₃O₄-Apt₂ dispersion. (3)GO@Fe3O4-Apt2-Cu 2-x Preparation of Te NSs Add 0.5-0.7g CuCl2·2H2O, 0.4-1.0g NaOH, 0.2-0.8g Na2TeO3, and 0.4-0.8g PVP to 20-40mL EG and stir at room temperature for 1-3h. Then transfer the mixture to a 50mL Teflon-lined stainless steel reactor and heat to 160-200℃ and keep it for 24-48h. After that, centrifuge and wash and dry at 50-70℃ to obtain CuCl2·2H2O. 2-x TeNSs powder. Take 1.0~3.0mg Cu 2-x Te NSs were dispersed in 0.5-2.0 mL PBS, and 0.5-2.0 mL GO@Fe3O4-Apt2 dispersion was added. The mixture was shaken and incubated overnight at 2-10 °C. The mixture was collected by magnetic adsorption and redispersed in 0.5-2 mL PBS to obtain GO@Fe3O4-Apt2-Cu 2-x Te NSs dispersion. (4) Actual sample pretreatment The number of tumor cells was counted using a cell counting plate, and the solution was serially diluted using culture medium to obtain a series of tumor cell standard solutions. For MCF-7 cells, MCF-7 cells were added to 10-fold diluted healthy human whole blood samples to a final concentration of 5.0 × 10 6 , 5.0×10 5 , 5.0×10 4 , 5.0×10 3 , 5.0×10 2 and 50 cells / mL. For HT-1376 cells, HT-1376 cells were added to healthy human urine samples to a final concentration of 5.0 × 10 6 , 5.0×10 5 , 5.0×10 4 , 5.0×10 3 , 5.0×10 2 and 50 cells / mL. Add 100-500 μL of Fe₃O₄-Apt1 dispersion to 100-500 μL of tumor cell sample solution and incubate at 25-45°C for 10-60 minutes. Collect the cells by magnetic adsorption and redisperse them in 100-500 μL of PBS. Add 10-100 μL of 1 kU / mL DNase I to cleave Apt1 and release the enriched tumor cells. Then, collect the supernatant by magnetic separation and centrifuge at 800 × g to collect the captured tumor cells. Redisperse them in 100-500 μL of culture medium. (5) Tumor cell concentration detection Take 10-30 μL of the culture medium containing tumor cells in (4), add 10-30 μL of RIPA cell lysis buffer, and sonicate for 5-10 minutes to fully lyse the tumor cells. Then add 10-100 μL of GO@Fe3O4-Apt2-Cu 2-x The Te NSs dispersion was incubated at 25–45°C for 10–60 min, magnetically separated, and 10–30 μL of the upper layer was transferred to a 100 μL EP tube for photothermal testing. (6) Photothermal signal-tumor cell viability standard curve a. Trypsinize well-adherent MCF-7 cells to obtain a tumor cell suspension and count them. Inoculate 100 μL of tumor cell suspension into each well of a 96-well culture plate to ensure that the number of tumor cells is approximately 1.0 × 10 3 indivual. b. MCF-7 cells were cultured at 37°C in 5% CO2 for 3-5 h to allow the tumor cells to adhere to the wall. c. Remove 50-100 μL of the upper culture medium, add 20-100 μL of PBS containing a certain concentration of H2O2 (0, 3, 6, 9, 12, 15, 18, 21, 24, 27, 30 mmol / L) and incubate for 20-60 min. d. Transfer the suspension in the wells to a 2 mL filtered centrifuge tube, add PBS to the wells to wash the tumor cells, and transfer the PBS used to wash the tumor cells to a 2 mL filtered centrifuge tube to collect the suspended tumor cells. e. Add 10-50 μL of 0.25% trypsin digestion solution to the wells and treat for 1-3 minutes. Discard the trypsin digestion solution and place the wells in a 5% CO2, 37°C culture environment for 1-2 minutes. Add 50-100 μL of new culture medium and use a pipette to repeatedly pipette to suspend the tumor cells. f. Subsequently, the tumor cell suspension was centrifuged at 800 × g for 3–10 min to separate the tumor cells from the culture medium and dispersed in 10–50 μL of cell culture medium along with the tumor cells on the filter membrane of a 2 mL centrifuge tube. g. Finally, add 50-100 μL GO@Fe3O4-Apt2-Cu 2-x The Te NSs dispersion was incubated at 37°C for 10–60 min, magnetically separated, and 10–50 μL of the upper layer was transferred to a 100 μL EP tube for photothermal testing. As for HT-1376 cells, except that in step c, PBS containing a certain concentration of cisplatin (0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 mmol / L) was added and incubated for 1 to 2 hours, and in step e, 10 to 50 μL of 0.25% trypsin digestion solution was added for 5 to 10 minutes, the trypsin digestion solution was discarded, and the cells were placed in a 5% CO2, 37°C culture environment for 1 to 5 minutes, all other aspects were the same as those of MCF-7 cells. The CCK-8 method was used to calibrate the cell viability of MCF-7 cells treated with different concentrations of H2O2 and HT-1376 cells treated with different concentrations of cisplatin. (7) Photothermal determination of tumor cell viability in actual samples Take 50-200 μL of the culture medium containing tumor cells in (4) and place it on a cell counting plate. Count the tumor cells using a microscope to calculate the tumor cell concentration. Adjust the concentration to 5.0×10 4 cells / mL, take 10-50 μL of tumor cell suspension and add 20-100 μL of GO@Fe3O4-Apt2-Cu 2-x The Te NSs dispersion was incubated at 25–45 °C for 10–60 min, magnetically separated, and 10–50 μL of the upper layer was transferred to a 100 μL EP tube for photothermal testing.

2. The preparation method and application of a photothermal biosensor for dual-indicator liquid biopsy of tumor cell quantification and viability according to claim 1, characterized in that: The sequence of the epithelial cell adhesion molecule EpCAM aptamer Apt1 is as follows: 5′-NH2-CACTACAGAGGTTGCGTCTGTCCCACGTTGTCATGGGGGGTTGGCCT G-3′; the sequence of the phosphatidylserine PS aptamer Apt2 is as follows: 5′-SH-TTAAAGACTT-3′.

3. The preparation method and application of the photothermal biosensor for dual-indicator liquid biopsy of tumor cell quantification and vitality according to claim 1, characterized in that: The tumor cells are breast cancer cells MCF-7 and bladder cancer cells HT-1376.

4. The preparation method and application of the photothermal biosensor for dual-indicator liquid biopsy of tumor cell quantification and vitality according to claim 1, characterized in that: The tumor cell concentration test described in step (5) is as follows: take 10-30 μL of the culture medium containing tumor cells in step (4), add 10-30 μL of RIPA cell lysis buffer, and sonicate for 5-10 minutes to fully lyse the tumor cells. Then add 10-100 μL of GO@Fe3O4-Apt2-Cu 2-x The Te NSs dispersion was incubated at 25–45°C for 10–60 min, magnetically separated, and 10–30 μL of the upper layer was transferred to a 100 μL EP tube for photothermal testing.

5. The preparation method and application of the photothermal biosensor for dual-indicator liquid biopsy of tumor cell quantification and vitality according to claim 1, characterized in that: MCF-7 and HT-137 cells were treated with H2O2 and cisplatin as described in step (6) c, specifically as follows: 50-100 μL of the upper culture medium was removed, 20-100 μL of PBS containing a certain concentration of H2O2 (0, 3, 6, 9, 12, 15, 18, 21, 24, 27, 30 mmol / L) was added, and the cells were incubated for 20-60 minutes. For HT-1376 cells, PBS containing a certain concentration of cisplatin (0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 mmol / L) was added in step (6) c and the cells were incubated for 1-2 hours.

6. The preparation method and application of the photothermal biosensor for dual-indicator liquid biopsy of tumor cell quantification and vitality according to claim 1, characterized in that: The actual sample activity photothermal assay described in step (7) is as follows: 50-200 μL of the culture medium containing tumor cells in step (3) is placed on a cell counting plate, and tumor cells are counted using a microscope to calculate the tumor cell concentration. The concentration is adjusted to 5.0×10 4 cells / mL, take 10-50 μL of tumor cell suspension and add 20-100 μL of GO@Fe3O4-Apt2-Cu 2-x The Te NSs dispersion was incubated at 25–45 °C for 10–60 min, magnetically separated, and 10–50 μL of the upper layer was transferred to a 100 μL EP tube for photothermal testing.

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