Method for detecting ctc using near-infrared light-responsive bismuth-based nanocomposite

Abstract

The present application relates to the field of biological detection, in particular to a method for detecting CTC by using near-infrared light responsive bismuth-based nanocomposite.The present application first synthesizes a kind of near-infrared light responsive bismuth-based nanocomposite with high photo-thermal conversion efficiency, the photo-thermal conversion efficiency is 63.4%, and a layer of glucose is coated on the surface of the material to improve the sensitivity and targeting of the material, the high uptake of glucose by tumor cells is used, the cells after taking the material are separated and photo-thermal experiment is carried out to obtain the linear relationship between temperature rise value △T and cell number to detect tumor cells.The present application has high CTC capture accuracy compared with the traditional CTC detection method, avoids false negative;the photo-thermal conversion rate is high, the cell surface is rich in probes, the sensitivity is improved;the instrument equipment is simple, and the detection cost is low.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biological detection, in particular to a method for detecting CTC by using near-infrared light responsive bismuth-based nanocomposites. BACKGROUND

[0002] CTC refers to tumor cells that are detached from tumor lesions and disseminate into peripheral blood circulation, which represents the molecular characteristics of tumor lesions and can develop into tumor metastases under certain conditions, and is the source of tumor hematogenous metastasis. Compared with other free tumor markers in the blood, CTC is visible and more advanced evidence of tumor existence, and is associated with metastasis. Many studies have shown that CTC can be used as an indicator for cancer prognosis and survival prediction, and can be an important basis for early diagnosis of cancer. Cancer metastasis is the main cause of cancer death, and CTC is an intermediate process and necessary condition for cancer metastasis. CTC detection in peripheral blood is a "liquid biopsy" scheme that can be performed multiple times, and the number and molecular information can guide clinical diagnosis and treatment. Therefore, CTC detection has important clinical significance. At present, CTC detection mainly faces two challenges. First, the rarity of CTC, which may contain only a few to dozens of CTC per 10 mL of blood. The small number itself and the interference of a large number of blood cells pose a great technical challenge to CTC detection. The current market and dozens of CTC detection methods in academic literature basically follow the two-step principle of "first enrichment and then identification". However, the enrichment process is usually cumbersome and leads to the loss of CTC, and the current mainstream scheme based on immunofluorescence staining is difficult to effectively identify whether a cell is indeed a tumor cell, resulting in false positives, low detection sensitivity, and low use efficiency. The second challenge of CTC detection is how to reliably identify tumor cells in a free state in the blood.

[0003] Under laser irradiation, the energy level of electrons generated by gold nanoparticles jumps and rearranges, and plasmonic resonance occurs. This photothermal effect can be used for tumor multi-modal imaging and cancer treatment, and is also widely used in photocatalytic physics, sensors, biomedical imaging, and hazard detection. Although bismuth is a heavy metal, bismuth and bismuth compounds are generally considered safe and non-toxic. Bismuth-based nanomaterials have many functions due to their high X-ray attenuation coefficient, strong near-infrared absorption, excellent photothermal conversion performance, and long circulation half-life. Studies have found that the photothermal conversion performance of gold / bismuth bimetallic nanocomposites is significantly better than that of single-metal nanomaterials, and the diagnosis and treatment of tumors can also be significantly enhanced.

[0004] At present, the research on photothermal effect is mostly focused on the fields of cancer photothermal therapy, biomedical imaging, etc., and there are relatively few reports for establishing detection methods. The detection principle of the method is that after laser irradiation for a fixed time, the amount of the detected substance is proportional to the photothermal effect of the nanomaterial, that is, the amount of the detected substance is linearly related to the temperature rise value, and the method has the advantages of simple and rapid operation, high sensitivity and the like.

[0005] Tumor cells overexpress glucose uptake, and glucose analogs (2-DG) are also over-uptaken by tumor cells and do not participate in intracellular metabolism. SUMMARY

[0006] In order to overcome the defects of the prior art, the application discloses a method for detecting CTCs by using near-infrared light responsive bismuth-based nanocomposites.

[0007] The application achieves the application purposes through the following technical solutions.

[0008] The method for detecting CTCs by using near-infrared light responsive bismuth-based nanocomposites is characterized by the following steps:

[0009] S1: Incubate the prepared glucose-coated near-infrared light responsive bismuth-based nanocomposites with the blood sample from which red blood cells are removed;

[0010] S2: Perform photothermal detection on the treated blood sample by using 800nm-1000nm laser;

[0011] S3: Establish the linear relationship between the temperature rise value ΔT and the number of cells,

[0012] S4: Investigate the consistency of the standard curve established in human peripheral blood and the standard curve established in the culture medium.

[0013] The method for detecting CTCs by using near-infrared light responsive bismuth-based nanocomposites is characterized by:

[0014] In step S1, the prepared bismuth-based nanocomposites have a nanometer morphology, and the preparation of the glucose-coated near-infrared light responsive bismuth-based nanocomposites is implemented in the following steps:

[0015] S1.1: Synthesize Au@Bi2S3 nanocomposites by coating bismuth on the surface of gold nanorods,

[0016] S1.2: Connect the amphiphilic PEG and glucose through amidation and adsorb them on the surface of the material through electrostatic adsorption,

[0017] S1.3: Stir the PEG-Glu with the material and then centrifuge and clean.

[0018] The method for detecting CTCs by using the near-infrared light responsive bismuth-based nanocomposite material, characterized in that: the Au@Bi2S3 nanoparticles synthesized in step S1.1 have a particle size of 50 nm to 60 nm.

[0019] The method for detecting CTCs by using the near-infrared light responsive bismuth-based nanocomposite material, characterized in that:

[0020] In step S1.2, the connection of the amphiphilic PEG and glucose is implemented according to the following steps, so that the synthesized glucose-coated bismuth-based nanocomposite material is accurately adsorbed on the surface of the circulating tumor cells or absorbed into the interior:

[0021] S: After the mPEG-COOH is ultrasonically dissolved in DMSO, EDC•HCL and NHS are added and low-speed stirring is performed in the dark.

[0022] S: Glucosamine hydrochloride is added, and low-speed stirring is continuously performed in the dark for 24 h.

[0023] S: After dialysis for 48 h, freeze-drying is performed to obtain a solid.

[0024] The method for detecting CTCs by using the near-infrared light responsive bismuth-based nanocomposite material, characterized in that:

[0025] In step S3, the linear relationship is established according to the following steps:

[0026] After different numbers of tumor cells are incubated with the material in a PBS solution soaked well plate and then transferred into a centrifuge tube, TMB and H2O2 are added, and then 800 mn to 1000 nm laser irradiation is performed for 60 s. The obtained data are analyzed to obtain the linear relationship between the temperature rise value ΔT in the PBS and the number of cells.

[0027] The method for detecting CTCs by using the near-infrared light responsive bismuth-based nanocomposite material, characterized in that:

[0028] In step S3, the linear relationship is established according to the following steps:

[0029] 5 mL of healthy blood is taken, red blood cells are removed by adding a red blood cell lysis solution, different numbers of tumor cells are placed in a well plate and incubated with the material, and then transferred into a centrifuge tube. After TMB and H2O2 are added, 800 nm to 1000 nm laser irradiation is performed for 60 s. The obtained data are analyzed to obtain the linear relationship between the temperature rise value ΔT in the human peripheral blood and the number of cells.

[0030] The application targets tumor cells by synthesizing a glucose-coated near-infrared light-responsive bismuth-based nanocomposite, and detects CTC by the temperature rise value of human peripheral blood through photothermal effect, which has the advantages of convenient operation, low use cost, accurate result, and can realize batch detection.

[0031] The synthesized material has excellent photothermal conversion efficiency, and the photothermal conversion efficiency is 63.4%, and can catalyze H2O2 to further oxidize TMB to enhance the photothermal response.

[0032] The method used in the application can quickly and accurately adsorb on the surface of tumor cells or be absorbed into the inside of cells by using the high uptake of glucose by tumor cells, and has high sensitivity. Subsequently, the linear relationship between the temperature rise value △T in PBS and human peripheral blood and the number of cells is established, and there is no obvious difference between the linear relationships, which can provide accurate detection results for disease diagnosis.

[0033] The application utilizes the high uptake of glucose by tumor cells, and combines the photothermal nanomaterial to establish the linear relationship between the temperature rise value △T in human peripheral blood and the number of cells by the photothermal analysis method to detect CTC. Normal cells have low glucose uptake or even no uptake, which improves the targeting and sensitivity of the material, avoids false negatives, and more accurately detects CTC, which has important clinical guiding significance for clinical application of CTC detection and evaluation of disease condition and treatment.

[0034] The application has the following beneficial effects:

[0035] 1. Non-invasive, highly related to the prognosis of multiple cancers, can monitor the risk of cancer recurrence, has high sensitivity, and the glycosylated glucose is a glucose analog and does not participate in the metabolism of tumor cells, does not affect the uptake of tumor cells, and realizes high-specificity recognition.

[0036] 2. The method provided by the application can more accurately detect CTC, has simple steps, can solve the problem of low expression of tumor factor recognition in traditional CTC detection system, and can solve the problem of high clinical detection cost. DETAILED DESCRIPTION

[0037] Figure 1 is a synthesis diagram of nano Au@Bi2S3 in the application,

[0038] Figure 2 is a schematic diagram of the glucose-coated material targeting tumor cells in the application,

[0039] Figure 3 is a schematic diagram of the process of photothermal detection of CTC in the application,

[0040] Figure 4is a TEM image of Au@Bi2S3 nanoparticles synthesized in the present application,

[0041] Figure 5 is a UV image of Au@Bi2S3 nanoparticles synthesized in the present application,

[0042] Figure 6 is a photothermal performance image of Au@Bi2S3 nanoparticles synthesized in the present application,

[0043] Figure 7 is a FT-IR image of mPEG-Glu synthesized in the present application,

[0044] Figure 8 is a linear relationship between the temperature rise value ΔT and the cell number of PBS and human peripheral blood obtained in the present application,

[0045] Figure 9 is a comparison chart of the quantitative results of the CTC number of tumor patients by the fluorescence detection method and the photothermal detection method of the present application. DETAILED DESCRIPTION

[0046] The present application is further illustrated by specific examples below.

[0047] I. Preparation of nano Au@Bi2S3:

[0048] The specific targeting probe used is Au@Bi2S3 nanoparticles, as shown in the following steps: Figure 1

[0049] First, Au@Bi2S3 nanoparticles are synthesized.

[0050] II. Preparation of seed solution: After ultrasonic dispersion of CTAB in deionized water, HAuCl4 is added under low-speed stirring, and NaBH4 solution is quickly added into the above mixed solution under high-speed stirring. After stopping the rapid stirring, the solution is aged and ready for use.

[0051] III. Preparation of growth solution: CTAB and 5-bromosalicylic acid are dispersed in deionized water, and AgNO3 solution is added after cooling and fully dissolving. After stirring, the solution is aged. Then, HAuCl4 solution is added, and the solution is fully mixed under slow stirring, and the color of the solution changes to orange yellow. Subsequently, AA is added, and the solution becomes colorless after vigorous stirring. Then, the synthesized seed solution is added, and the solution becomes wine red.

[0052] IV. The above obtained solution is added into a three-necked flask, and then AA and hexamethylene tetramine, thioacetamide and bismuth acetate solutions are added. The mixture is placed in a vacuum environment, and finally centrifuged, washed and dispersed in deionized water for standby use.

[0053] ​The core of the embodiment comprises Au@Bi2S3 nanoparticles with a particle size of 50-60 nm and a photo-thermal conversion efficiency of 63.4%, and the mPEG-Glu is coated on the surface of the material.

[0054] The targeting substance mPEG-Glu described in the embodiment can be taken into cells or on the surface of cells in a large amount through high uptake of glucose by tumor cells.

[0055] Tumor cells have overexpression of glucose uptake, while normal cells have low or even no uptake of glucose. The purpose of targeting tumor cells is achieved by coating glucose on the surface of the material.

[0056] The glucose-coated material targets tumor cells as shown in Figure 2 .

[0057] The TEM image of the synthesized Au@Bi2S3 nanoparticles is shown in Figure 4 , the UV image is shown in Figure 5 , and the photo-thermal performance image is shown in Figure 6 .

[0058] The FT-IR image of the synthesized mPEG-Glu is shown in Figure 7 .

[0059] II. Glycosylation of Au@Bi2S3 nanoparticles:

[0060] The connection of amphiphilic PEG and glucose and the coating on the surface of the material are implemented according to the following steps:

[0061] Dissolve mPEG-COOH in DMSO, ultrasonically dissolve, and add EDC•HCL and NHS under stirring in the dark.

[0062] II. Then add glucose amine hydrochloride under stirring in the dark.

[0063] III. The obtained solution is dialyzed in a dialysis bag for 24 h, and finally freeze-dried to obtain mPEG-Glu solid.

[0064] IV. Mix the material and an appropriate amount of mPEG-Glu in DMF under stirring, and finally centrifuge and wash.

[0065] III. Establishment of a CTC detection system based on cell metabolism:

[0066] The linear relationship between the temperature rise value ΔT and the number of cells is established according to the following steps:

[0067] Different numbers of tumor cells are incubated in the well plate, and the material is added after the cells adhere.

[0068] II. After incubation with the material, wash away the excess material and transfer into a centrifuge tube.

[0069] III. Add TMB and H2O2 into the centrifuge tube, add acetate buffer until the solution turns blue.

[0070] IV. Irradiate the material in the centrifuge tube with 800nm-1000nm laser for 60s, get the △T of different cell numbers under laser irradiation, calculate the linear relationship between the temperature rise △T in PBS and cell number.

[0071] V. Collect 5 mL of human healthy blood, remove red blood cells with red blood cell lysis solution.

[0072] VI. Mix different numbers of tumor cells in human healthy blood and incubate in the well plate, add the material after the cells adhere.

[0073] VII. After incubation with the material, wash away the excess material with PBS and transfer into a centrifuge tube.

[0074] VIII. Add TMB and H2O2 into the centrifuge tube, add acetate buffer until the solution turns blue.

[0075] IX. Irradiate the material in the centrifuge tube with 800nm-1000nm laser, get the △T of different cell numbers under laser irradiation, calculate the linear relationship between the temperature rise △T in human peripheral blood and cell number.

[0076] X. Investigate the consistency of the standard curve established in human peripheral blood and the standard curve established in culture medium.

[0077] The process of photo-thermal detection of CTC is as shown in Figure 3 .

[0078] The linear relationship between the temperature rise △T in PBS and cell number is as shown in Figure 8 .

[0079] The comparison of the quantitative results of tumor patient CTC number by fluorescence detection method and photo-thermal detection method is as shown in Figure 9 .

Claims

1. A method for detecting CTCs using near-infrared light-responsive bismuth-based nanocomposite materials, characterized by: Follow these steps in sequence: S1: The near-infrared light-responsive bismuth-based nanocomposite material coated with glucose was co-incubated with a blood sample in which red blood cells were removed; S2: Use an 800nm~1000nm laser to perform photothermal detection on the processed blood sample; S3: Establish a linear relationship between the temperature rise ΔT and the number of cells. S4: Examine the consistency between the standard curve established in human peripheral blood and the standard curve established in culture medium.

2. The method for detecting CTC using near-infrared light-responsive bismuth-based nanocomposite materials as described in claim 1, characterized in that: In step S1, the bismuth-based nanocomposite material has a nano-morphology. The preparation of glucose-coated near-infrared responsive bismuth-based nanocomposite material is carried out sequentially according to the following steps: S1.1: Synthesis of Au@Bi2S3 nanocomposite material by coating bismuth onto the surface of gold nanorods. S1.2: Amphiphilic PEG and glucose are linked by amidation and then electrostatically adsorbed onto the material surface. S1.3: Stir the PEG-Glu with the material and then centrifuge and wash.

3. The method for detecting CTCs using near-infrared light-responsive bismuth-based nanocomposite materials as described in claim 2, characterized in that: In step S1.1, the synthesized Au@Bi2S3 nanoparticles have a particle size of 50 nm to 60 nm.

4. The method for detecting CTC using near-infrared light-responsive bismuth-based nanocomposite materials as described in claim 3, characterized in that: In step S1.2, the connection between the amphiphilic PEG and glucose is performed as follows: S: After ultrasonically dissolving mPEG-COOH in DMSO, add EDC•HCl and NHS and stir at low speed in the dark. S: Add glucosamine hydrochloride and continue stirring at low speed in the dark for 24 hours. S: After dialysis for 48 hours, freeze-drying yielded a solid.

5. The method for detecting CTCs using near-infrared light-responsive bismuth-based nanocomposites as described in any one of claims 1 to 4, characterized in that: In step S3, the linear relationship is established according to the following steps: Different numbers of tumor cells and materials were co-incubated with PBS-soaked well plates and then transferred into centrifuge tubes. TMB and H2O2 were added, and the tubes were irradiated with an 800nm-1000nm laser for 60s. The obtained data were analyzed to obtain the linear relationship between the temperature rise ΔT in PBS and the number of cells.

6. The method for detecting CTCs using near-infrared light-responsive bismuth-based nanocomposite materials as described in claim 5, characterized in that: In step S3, the linear relationship is established according to the following steps: 5 mL of healthy blood was drawn, and erythrocyte lysis buffer was added to remove erythrocytes. Different numbers of tumor cells were placed in well plates and incubated with the materials. The cells were then transferred to centrifuge tubes, TMB and H2O2 were added, and the cells were irradiated with an 800 nm to 1000 nm laser for 60 s. The obtained data were analyzed to obtain the linear relationship between the temperature rise value ΔT in human peripheral blood and the number of cells.

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