Method for detecting CTC by using near-infrared light response bismuth-based nanocomposite material

Abstract

The invention relates to the field of biological detection, in particular to a method for detecting CTC through a near-infrared light response bismuth-based nanocomposite. The near-infrared light response bismuth-based nano composite material with high photothermal conversion efficiency is synthesized firstly, the photothermal conversion efficiency is 63.4%, the surface of the material is coated with a layer of glucose so as to improve the sensitivity and targeting property of the material, and the sensitivity and targeting property of the material are improved by utilizing the high uptake of tumor cells to glucose. And separating the cells after material uptake and carrying out a photo-thermal experiment to obtain a linear relation between a temperature rise value T and the number of the cells so as to detect tumor cells. Compared with a traditional CTC detection method, the CTC capture accuracy is high, and false negative is avoided; the photothermal conversion rate is high, more probes are enriched on the cell surface, and the sensitivity is improved; instruments and equipment are simple, and detection cost is low.

Description

Technical Field

[0001] The invention relates to the field of biological detection, and in particular to a method for detecting CTCs using a near-infrared light-responsive bismuth-based nanocomposite material. Background Art

[0002] CTC refers to tumor cells that fall off from tumor lesions and spread into the peripheral blood circulation. It represents the molecular characteristics of tumor lesions and can develop into tumor metastases under certain conditions. It is the source of tumor hematogenous metastasis. Compared with other free tumor markers in the blood, CTC is a visible evidence of the existence of higher-grade tumors and is related to metastasis. Many studies have shown that CTC can be used as an indicator for cancer prognosis and survival prediction, and can be used as an important basis for early diagnosis of cancer. Cancer metastasis is the main cause of cancer death. CTC is an intermediate process and necessary condition for cancer metastasis. CTC detection in peripheral blood is a "liquid biopsy" program that can be performed multiple times. Its number and molecular information can guide clinical diagnosis and treatment. Therefore, CTC detection has important clinical significance. At present, CTC detection faces two major challenges. The first is the rarity of CTC. There may be only a few to dozens of CTCs in every 10 mL of blood. The extremely small number itself and the interference of massive blood cells bring great technical challenges to the detection of CTC. Currently, dozens of CTC detection methods on the market and in academic literature basically follow the two-step principle of "enrichment first, then identification", but the enrichment process is usually cumbersome and leads to the loss of CTCs. The current mainstream immunofluorescence staining-based schemes are difficult to effectively identify whether a cell is indeed a tumor cell, and there are deficiencies such as false positive test results, low detection sensitivity, and low efficiency. The second challenge of CTC detection is how to reliably identify tumor cells in a free state in the blood.

[0003] Under the irradiation of nano-gold laser, electrons undergo energy level transitions and rearrange themselves, resulting in a plasma resonance effect. This photothermal effect can be used for multimodal tumor imaging and cancer treatment, and is also widely used in photocatalytic physics, sensors, biomedical imaging, and hazardous substance detection. Although bismuth is a heavy metal, bismuth and bismuth compounds are generally considered safe and non-toxic. The advantages of bismuth-based nanomaterials, such as high X-ray attenuation coefficient and strong near-infrared absorption, excellent photothermal conversion performance and long circulation half-life, give them more functions. Studies have found that the photothermal conversion performance of gold / bismuth bimetallic nanocomposites is significantly better than that of their respective single metal nanomaterials, and the diagnosis and treatment of tumors can also be significantly enhanced.

[0004] At present, the research on photothermal effect is mainly focused on cancer photothermal therapy, biomedical imaging and other fields, and there are relatively few reports on the establishment of detection methods. The detection principle of this method is that after a fixed time of laser irradiation, the amount of the substance to be tested is proportional to the photothermal effect of the nanomaterial, that is, the amount of the substance to be tested is linearly related to the temperature increase value. This method has the advantages of simple and fast operation and high sensitivity.

[0005] Tumor cells significantly overexpress glucose uptake, and glucose analogs (2-DG) are also excessively taken up by tumor cells and do not participate in intracellular metabolism. Summary of the invention

[0006] In order to overcome the defects of the prior art and provide a biological detection method with convenient operation, low use cost and accurate results, the present invention discloses a method for detecting CTC using a near-infrared light-responsive bismuth-based nanocomposite material.

[0007] The present invention achieves the purpose of the invention through the following technical solutions: A method for detecting CTC using a near-infrared light-responsive bismuth-based nanocomposite material, characterized by: performing the following steps in sequence: S1: The prepared glucose-coated near-infrared light-responsive bismuth-based nanocomposite was co-incubated with a blood sample depleted of red blood cells; S2: Use 800nm ​​to 1000nm laser to perform photothermal detection on the processed blood sample; S3: Establish a linear relationship between the temperature rise value △T and the number of cells, S4: Investigate the consistency of the standard curve established in human peripheral blood with that established in culture medium.

[0008] The method for detecting CTC using a near-infrared light-responsive bismuth-based nanocomposite material is characterized by: In step S1, the bismuth-based nanocomposite material prepared has a nano-morphology, and the preparation of the glucose-coated near-infrared light-responsive bismuth-based nanocomposite material is carried out in sequence according to the following steps: S1.1: Coating bismuth on the surface of gold nanorods to synthesize Au@Bi2S3 nanocomposite materials. S1.2: The amphiphilic PEG and glucose are connected by amidation and adsorbed on the surface of the material by electrostatic adsorption. S1.3: Stir PEG-Glu and the material and then centrifuge to wash.

[0009] The method for detecting CTC using near-infrared light-responsive bismuth-based nanocomposite materials is characterized in that in step S1.1, the particle size of the synthesized Au@Bi2S3 nanoparticles is 50nm to 60nm.

[0010] The method for detecting CTC using a near-infrared light-responsive bismuth-based nanocomposite material is characterized by: In step S1.2, the connection between the amphiphilic PEG and glucose is carried out according to the following steps, so that the synthesized glucose-coated bismuth-based nanocomposite material is accurately adsorbed on the surface of circulating tumor cells or absorbed into the interior: S: After dissolving mPEG-COOH in DMSO by ultrasonication, add EDC•HCL and NHS and stir at low speed in the dark.

[0011] S: Add glucosamine hydrochloride and continue stirring at low speed in the dark for 24 hours.

[0012] S: Dialyze for 48 hours and freeze-dry to obtain a solid.

[0013] The method for detecting CTC using a near-infrared light-responsive bismuth-based nanocomposite material is characterized by: In step S3, the linear relationship is established according to the following steps: Different numbers of tumor cells were incubated with materials in a well plate soaked in PBS solution and then transferred into a centrifuge tube. After adding TMB and H2O2, the cells were irradiated with 800mn-1000nm laser for 60s. The obtained data were analyzed to obtain a linear relationship between the temperature rise value △T in PBS and the number of cells.

[0014] The method for detecting CTC using a near-infrared light-responsive bismuth-based nanocomposite material is characterized by: In step S3, the linear relationship is established according to the following steps: 5 mL of healthy blood was drawn, and red blood cells were removed by adding red blood cell lysis solution. Different numbers of tumor cells were placed in a well plate and incubated with the material, then transferred into a centrifuge tube. After adding TMB and H2O2, the cells were irradiated with 800nm-1000nm laser for 60s. The obtained data were analyzed to obtain a linear relationship between the temperature increase value △T and the number of cells in human peripheral blood.

[0015] The present invention targets tumor cells by synthesizing a glucose-coated near-infrared light-responsive bismuth-based nanocomposite material, and detects CTCs by increasing the temperature of human peripheral blood through the photothermal effect. The method has the advantages of convenient operation, low cost, accurate results, and batch detection.

[0016] The material synthesized by the present invention has excellent photothermal conversion efficiency of 63.4%, and can catalyze H2O2 to oxidize TMB to enhance photothermal reaction.

[0017] The method used in the present invention uses glycosylated materials and high glucose uptake by tumor cells to quickly and accurately adsorb on the surface of tumor cells or absorb into the interior of cells with high sensitivity. Subsequently, a linear relationship between the temperature increase value △T and the number of cells in PBS and human peripheral blood is established. There is no significant difference in the linear relationship between the two, which can provide accurate detection results for disease diagnosis.

[0018] The present invention utilizes the high uptake of glucose by tumor cells, combines photothermal nanomaterials, and establishes a linear relationship between the temperature rise value △T and the number of cells in human peripheral blood through photothermal analysis to detect CTC. Normal cells have low or even no uptake of glucose, which improves the targeting and sensitivity of the material, avoids false negatives, and detects CTC more accurately, which has important clinical guiding significance for the clinical application of CTC detection, disease assessment and treatment.

[0019] The present invention has the following beneficial effects: 1. It is non-invasive, highly correlated with the prognosis of multiple cancers, and can monitor the risk of cancer recurrence. It has high sensitivity, and glycosylated glucose is a glucose analogue that does not participate in the metabolism of tumor cells and does not affect the uptake of tumor cells, achieving highly specific recognition.

[0020] 2. The method provided by the present invention can detect CTC more accurately, with simple steps, and can solve the problem of identifying low-expressed tumor factors that are difficult to detect by traditional CTC detection systems, as well as the high cost of clinical detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 This is a schematic diagram of the synthesis of nano Au@Bi2S3 in the present invention. Figure 2 This is a schematic diagram of the glucose-encapsulated material targeting tumor cells in the present invention. Figure 3 Schematic diagram of the photothermal detection CTC process in the present invention, Figure 4 This is the TEM image of Au@Bi2S3 nanoparticles synthesized by the present invention. Figure 5 This is the UV image of Au@Bi2S3 nanoparticles synthesized by the present invention. Figure 6 This is the photothermal performance diagram of Au@Bi2S3 nanoparticles synthesized by the present invention. Figure 7 is the FI-IR diagram of the mPEG-Glu synthesized by the present invention, Figure 8 The linear relationship between the temperature increase value △T and the number of cells in PBS and human peripheral blood obtained by the present invention is: Fig. 9It is a comparison chart of the quantitative results of the number of CTCs in tumor patients using the fluorescence detection method of the present invention and the photothermal detection method. DETAILED DESCRIPTION

[0022] The present invention is further illustrated by the following specific examples.

[0023] 1. Preparation of Nano-Au@Bi2S3: The specific targeting probe used is Au@Bi2S3 nanoparticles, such as Figure 1 As shown, prepare according to the following steps: Au@Bi2S3 nanoparticles were first synthesized.

[0024] II. Preparation of seed solution: Dissolve CTAB in deionized water and disperse by ultrasonic, then add HAuCl4 under low-speed stirring, and quickly add NaBH4 solution into the mixed solution under high-speed stirring, stop stirring after rapid stirring, and let it stand for aging before use.

[0025] III. Preparation of growth solution: Disperse CTAB and 5-bromosalicylic acid in deionized water, add AgNO3 solution after fully dissolving and cooling, stir and let stand. Then add HAuCl4 solution, stir slowly to mix thoroughly, the solution color turns orange-yellow. Then add AA, stir vigorously, the solution turns colorless, add the synthesized seed solution, the solution turns wine red.

[0026] IV. Add the above solution into a three-necked flask, then add AA and hexamethylenetetramine, thioacetamide and bismuth acetate solution. Place the mixture in a vacuum environment, and finally centrifuge and wash to disperse in deionized water for later use.

[0027] The core of this embodiment includes Au@Bi2S3 nanoparticles with a particle size of 50nm to 60nm and a photothermal conversion efficiency of 63.4%. The mPEG-Glu is coated on the surface of the material.

[0028] The targeting substance mPEG-Glu described in this example can be taken up in large quantities into cells or on the cell surface due to the high uptake of glucose by tumor cells.

[0029] Tumor cells overexpress glucose uptake, while normal cells have low or even no glucose uptake. The purpose of targeting tumor cells is achieved by coating the surface of the material with glucose.

[0030] Glucose-wrapped materials target tumor cells Figure 2 shown.

[0031] TEM images of synthesized Au@Bi2S3 nanoparticles are shown in Figure 4 As shown, the UV map is Figure 5 As shown in the photothermal performance diagram Figure 6 shown.

[0032] The FI-IR image of synthesized mPEG-Glu is shown in Figure 7 shown.

[0033] 2. Glycosylation of Au@Bi2S3 nanoparticles: The amphiphilic PEG and glucose are connected and coated on the surface of the material according to the following steps: Dissolve mPEG-COOH in DMSO, sonicate to dissolve, add EDC•HCL and NHS and stir in the dark.

[0034] II. Then add glucosamine hydrochloride and stir in the dark.

[0035] III. The obtained solution was dialyzed with a dialysis bag for 24 hours and finally freeze-dried to obtain mPEG-Glu solid.

[0036] IV. Mix the material and an appropriate amount of mPEG-Glu in DMF and stir, and finally centrifuge and wash.

[0037] 3. Establishment of CTC detection system based on cell metabolism: To establish a linear relationship between the temperature rise value △T and the number of cells, follow the steps below: Different numbers of tumor cells were incubated in the well plate, and materials were added after the cells adhered to the wall.

[0038] II. After incubation with the material, wash away the excess material and transfer into a centrifuge tube.

[0039] III. Add TMB and H2O2 into a centrifuge tube and add acetate buffer until the solution turns blue.

[0040] IV. The material in the centrifuge tube was irradiated with 800nm-1000nm laser for 60s, and the △T of different cell numbers under laser irradiation was obtained. The linear relationship between the temperature rise value △T in PBS and the cell number was calculated.

[0041] V. Collect 5 mL of healthy human blood and add red blood cell lysis buffer to remove red blood cells.

[0042] VI. Mix different numbers of tumor cells with healthy human blood and incubate them in a well plate. Add materials after the cells adhere to the wall.

[0043] VII. After incubation with the material, wash away the excess material with PBS and transfer into a centrifuge tube.

[0044] VIII. Add TMB and H2O2 to a centrifuge tube, add acetate buffer and wait for the solution to turn blue.

[0045] IX. The material in the centrifuge tube was irradiated with 800nm-1000nm laser to obtain the △T of different cell numbers under laser irradiation, and the linear relationship between the temperature rise value △T and the cell number in human peripheral blood was calculated.

[0046] X. Examine the consistency of the standard curve established in human peripheral blood with that established in culture medium.

[0047] Photothermal detection of CTC process Figure 3 shown.

[0048] The linear relationship between the temperature increase △T and the number of cells in PBS and human peripheral blood is as follows Figure 8 shown.

[0049] Comparison of the quantitative results of CTC number in tumor patients by fluorescence detection method and photothermal detection method Fig. 9 shown.

Claims

1. A method for detecting CTCs using a near-infrared light-responsive bismuth-based nanocomposite material, characterized in that: Follow the steps below to implement: S1: The prepared glucose-coated near-infrared light-responsive bismuth-based nanocomposite was co-incubated with a blood sample depleted of red blood cells; S2: Use 800nm ​​to 1000nm laser to perform photothermal detection on the processed blood sample; S3: Establish a linear relationship between the temperature rise value △T and the number of cells, S4: Investigate the consistency of the standard curve established in human peripheral blood with that established in culture medium.

2. The method for detecting CTC using a near-infrared light-responsive bismuth-based nanocomposite material as claimed in claim 1, characterized in that: In step S1, the bismuth-based nanocomposite material prepared has a nano-morphology, and the preparation of the glucose-coated near-infrared light-responsive bismuth-based nanocomposite material is carried out in sequence according to the following steps: S1.1: Coating bismuth on the surface of gold nanorods to synthesize Au@Bi2S3 nanocomposite materials. S1.2: The amphiphilic PEG and glucose are connected by amidation and adsorbed on the surface of the material by electrostatic adsorption. S1.3: Stir PEG-Glu and the material and then centrifuge to wash.

3. The method for detecting CTC using a near-infrared light-responsive bismuth-based nanocomposite material as claimed in claim 2, characterized in that: At S1.1, the particle size of the synthesized Au@Bi2S3 nanoparticles is 50mn~60nm.

4. The method for detecting CTC using a near-infrared light-responsive bismuth-based nanocomposite material as claimed in claim 3, characterized in that: In step S1.2, the connection between amphiphilic PEG and glucose is carried out as follows: S: After dissolving mPEG-COOH in DMSO by ultrasonication, add EDC•HCL and NHS and stir at low speed in the dark. 5.S: Add glucosamine hydrochloride and continue stirring at low speed in the dark for 24 hours. 6.S: After 48 hours of dialysis, freeze-drying was performed to obtain a solid.

7. The method for detecting CTC using a near-infrared light-responsive bismuth-based nanocomposite material according to 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 were incubated with materials in a well plate soaked in PBS solution and then transferred into a centrifuge tube. After adding TMB and H2O2, the cells were irradiated with 800mn-1000nm laser for 60s. The obtained data were analyzed to obtain a linear relationship between the temperature rise value △T in PBS and the number of cells.

8. The method for detecting CTC using a near-infrared light-responsive bismuth-based nanocomposite material as claimed 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 red blood cells were removed by adding red blood cell lysis solution. Different numbers of tumor cells were placed in a well plate and incubated with the material, then transferred into a centrifuge tube. After adding TMB and H2O2, the cells were irradiated with 800nm-1000nm laser for 60s. The obtained data were analyzed to obtain a linear relationship between the temperature increase value △T and the number of cells in human peripheral blood.

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