Paclitaxel drug resistance SERS substrate and detection method

Abstract

The invention discloses a paclitaxel drug resistance SERS (Surface Enhanced Raman Scattering) substrate and a detection method. Belongs to the technical field of drug resistance detection. The SERS substrate is mixed with ultrapure water after centrifuging a silver nanoparticle-sodium citrate suspension, and the mixture is dropwise added to a silicon wafer and dried to form a coffee ring structure. During detection, cancer tissue crushing liquid is dropwise added to the center of the substrate, SERS signals are collected through a Raman spectrometer and compared with a pre-established drug resistance / sensitivity spectrum database, and drug resistance is judged according to characteristic peaks at 820 cm <-1 >, 1180 cm <-1 >, 1270 cm <-1 > and 1450 cm <-1 >. The method is easy and convenient to operate, short in detection time (within 10 minutes), high in sensitivity and suitable for rapid screening of paclitaxel drug resistance of breast cancer patients.

Description

Technical Field

[0001] The present invention belongs to the technical field of drug resistance detection, and particularly relates to a SERS substrate for paclitaxel drug resistance and a detection method thereof. Background Art

[0002] Breast cancer is one of the most common malignant tumors globally, and its incidence rate ranks first among female cancer patients. Paclitaxel is one of the most common chemotherapy drugs for it and is widely used to treat various types of breast cancer. However, due to the drug resistance of some breast cancers to it, the drug cannot effectively control the disease progression during use, seriously affecting the survival time and quality of life of patients.

[0003] Currently, the identification and evaluation of breast cancer drug resistance mainly rely on the efficacy evaluation after every two cycles of treatment for patients. For patients with drug resistance, the disease often progresses during this ineffective treatment period, thus losing the best treatment opportunity or even life. In addition, there are also some current studies hoping to distinguish whether cancer cells have developed drug resistance through laboratory detection means, such as: gene detection of patients' cancer tissues, in vitro culture and drug sensitivity experiments of patients' cancer cells, etc. The whole process includes tumor sample pretreatment and large instrument detection, which is rather cumbersome, with limited success rate, long detection time and high cost. Another study detects drug resistance by mining drug resistance-related proteins in patients' peripheral blood and constructing corresponding antibody-conjugated nanoparticles. However, this method also has a series of defects, specifically as follows: first, a large amount of blood specimens are required, which is harmful to the patient's body; second, the cost of constructing antibodies is relatively high; third, the cancer focus is not directly detected, resulting in a certain false negative rate; fourth, the peripheral blood specimens require complex pretreatment steps, affecting the rapid output of results.

[0004] Therefore, the development of a rapid detection method for chemotherapy drug resistance in breast cancer patients has very important clinical and social significance, which can more conveniently distinguish the presence or absence of drug resistance and promote the development of the medical and health industry.

[0005] In recent years, the research on the application of SERS (Surface Enhanced Raman Spectroscopy) technology in clinical practice has become more and more extensive. Compared with technologies such as clinical efficacy evaluation and gene detection, it has obvious advantages such as on-site, rapid, high sensitivity and low detection cost. Summary of the Invention

[0006] The object of the present invention is to provide a SERS substrate for paclitaxel resistance and a detection method. At present, there is no relevant technical report on using SERS technology to detect paclitaxel resistance in breast cancer, and the few existing research methods also have defects such as complex pretreatment and low success rate, so they cannot be widely applied clinically. Breast cancer, as the most common malignant tumor among women, seriously threatens the lives of patients. Clinical treatment needs to seize the critical moment and give effective treatment as soon as possible while protecting the stability of the patient's body. There is an urgent need to invent a method that can quickly and accurately obtain the drug sensitivity information of patients.

[0007] Based on these problems, the present invention proposes a SERS substrate and a detection method based on this SERS substrate. SERS technology not only has the characteristics of strong specificity, small sample consumption, and no damage to samples of ordinary Raman spectroscopy, but also has a lower detection limit than ordinary Raman spectroscopy. By detecting cancer tissues from breast cancer patients based on SERS technology, that is, after pretreatment such as ultrasonic fragmentation of tumor tissue samples, the samples are taken and dropped on the coffee ring SERS substrate constructed by silver nanoparticles, and then detection can be carried out, which has the characteristics of accurate detection results, rapid detection, and low cost.

[0008] The present invention first pretreats the cancer tissues of breast cancer patients, constructs a highly efficient and sensitive silver nanoparticle SERS substrate at the same time, and then uses this substrate to perform SERS detection on the cancer tissues. This method can complete the SERS detection of patient tissues within a few minutes and output the drug sensitivity results in real time.

[0009] In order to solve the above technical problems, the specific technical solutions of the present invention are as follows:

[0010] A method for detecting paclitaxel resistance, comprising the following steps:

[0011] 1. Pretreatment of breast cancer patient tissues

[0012] Perform a biopsy on breast cancer patients who are clinically evaluated to be likely to have developed paclitaxel resistance, obtain a cancer tissue strip with a volume of more than 2×2×3 mm and place it in 1 ml of ddH2O, and perform ultrasonic fragmentation on ice to obtain the tissue lysate to be tested.

[0013] 2. Construct a highly efficient coffee ring SERS substrate with silver nanoparticles

[0014] (1) Take a spherical silver nanoparticle-sodium citrate solution with a concentration of 6×10 8 per ml, centrifuge at 8500 r / min for 10 min at room temperature, discard the liquid in the tube, add 80 times its own volume of ddH2O to the precipitate and mix well to form a silver nanoparticle suspension;

[0015] (2) Take 5 μl of the above suspension and gently drop it onto the silica wafer, then transfer it to an oven preheated to 60 °C and bake it dry; after the nanoparticles are baked dry, a coffee ring slightly protruding from the surface of the wafer is formed.

[0016] (3) Take 5 μl of the tissue lysate to be tested and gently drop it in the center of the coffee ring. Due to capillary action, the tissue lysate will move evenly towards the coffee ring without exceeding it.

[0017] (4) Place the silica wafer with the dropped tissue lysate to be tested back into the oven at 60 °C and bake it dry again.

[0018] 3. SERS Detection and Data Output

[0019] (1) After taking out the silica wafer, perform SERS detection using a Raman spectrometer of model RPB-785-1.5-FS. The excitation wavelength of the Raman spectrometer is about 785 nm, the detection power is selected as 200 mW, and the integration time is 5 s to obtain the spectrum of the tissue to be tested.

[0020] (2) Collect the SERS spectra of the patient's tissue and import the spectra into the spectral database of patient tissues that have been identified as paclitaxel-resistant / sensitive for comparison. Use PCA for inter-group analysis, and the analysis data will output the detection result that the tissue to be tested is "paclitaxel-resistant" or "paclitaxel-sensitive".

[0021] In the present invention, the cancer tissues of breast cancer patients are ultrasonically disrupted, so that special small molecules in the cancer cells can be fully released. Using the principle that when the solution evaporates from the inside out, it will drive small particles to move around, a loose and porous coffee ring substrate is constructed, and the SERS detection efficiency of drug-resistant cancer tissues is improved using this substrate.

[0022] Beneficial Effects:

[0023] (1) The method for treating tumor tissues in the present invention is simple to operate, has a short treatment time, and has a low requirement for the professional level of operators.

[0024] (2) The present invention constructs a new type of coffee ring silver nanoparticle substrate, which has high detection sensitivity and short time. It only takes 10 minutes from tissue sampling to the full detection process. When detecting multiple samples, each sample on average takes 3 minutes, and the detection results can be exported in real time, which is suitable for the accurate and rapid detection of drug resistance in breast cancer patients. Description of the Drawings

[0025] Figure 1 It is the SERS spectrogram and comparison of the breast cancer patient tissue and breast cancer paclitaxel-resistant cells to be detected in Example 1. Specific Embodiments

[0026] The present invention will be described in detail below with reference to the accompanying drawings and specific embodiments. However, the following embodiments are only for explaining the present invention, and the protection scope of the present invention should include all the contents of the claims. Moreover, through the description of the following embodiments, those skilled in the art can fully implement all the contents of the claims of the present invention.

[0027] The present invention will be further described below in conjunction with embodiments, but the present invention is not limited to the following embodiments.

[0028] Embodiment 1:

[0029] Patient Li xx, female, 55 years old, was diagnosed with breast cancer in October 2023 and received 4 cycles of paclitaxel chemotherapy. During the review, a suspicious metastatic lesion was found in the liver.

[0030] (1) To understand the sensitivity to paclitaxel drugs, a biopsy was performed on the suspicious metastatic lesion in the liver, and a tissue strip of 2×2×4 mm was obtained.

[0031] (2) The tissue to be tested was placed in 1 ml of ddH2O and ultrasonically broken on ice to obtain a broken solution of the tissue to be tested.

[0032] (3) Take a spherical silver nanoparticle solution with a concentration of 6×10 8 particles / ml, centrifuge at 8500 r / min for 10 min at room temperature, discard the liquid in the tube, add 80 times its own volume of ddH2O to the precipitate and mix well to form a silver nanoparticle suspension;

[0033] (4) Take 5 μl of the above suspension and gently drop it onto a silica wafer, then transfer it to an oven preheated to 60 °C and bake it dry; after the nanoparticles are baked dry, a coffee ring slightly protruding from the surface of the silicon wafer is formed;

[0034] (5) Take 5 μl of the broken solution of the tissue to be tested and gently drop it in the center of the coffee ring, and then place the silicon wafer with the broken solution of the tissue to be tested in the oven at 60 °C again and bake it dry.

[0035] (6) After taking out the silicon wafer, SERS detection was performed using a Raman spectrometer of model RPB-785-1.5-FS. The excitation wavelength of the Raman spectrometer was about 785 nm, the detection power was selected as 200 mW, and the integration time was 5 s to obtain the spectrum of the tissue to be tested.

[0036] (7) Collect the SERS spectra of the patient's tissue and compare them with the spectral data of the tissue of patients who are clearly resistant / sensitive to paclitaxel. If detection peaks appear at Raman shifts of 820 cm -1 , 1180 cm -1 , 1270 cm -1 , 1450 cm -1 etc., the diagnosis is paclitaxel resistance.

[0037] Example 2:

[0038] Patient Wang xx, female, 68 years old, was diagnosed with breast cancer in January 2024 and received 6 cycles of paclitaxel chemotherapy. During the review, suspicious metastatic lesions were found in the lungs.

[0039] (1) To understand the sensitivity to paclitaxel drugs, a biopsy of the suspicious metastatic lesions in the lungs was performed, and a tissue strip of 2×2×3 mm was obtained.

[0040] (2) The tissue to be tested was placed in 1 ml of ddH2O and ultrasonically broken on ice to obtain the tissue lysate to be tested.

[0041] (3) Take a spherical silver nanoparticle solution with a concentration of 6×10 8 particles / ml, centrifuge at 8500 r / min for 10 min at room temperature, discard the liquid in the tube, add 80 times its own volume of ddH2O to the precipitate and mix well to form a silver nanoparticle suspension;

[0042] (4) Take 5 μl of the above suspension and gently drop it onto the silica wafer, then transfer it to an oven preheated to 60 °C and bake it dry; after the nanoparticles are baked dry, a coffee ring slightly protruding from the surface of the silicon wafer is formed;

[0043] (5) Take 5 μl of the tissue lysate to be tested and gently drop it in the center of the coffee ring, then place the silicon wafer with the tissue lysate to be tested dropped on it in the oven at 60 °C again and bake it dry.

[0044] (6) After taking out the silicon wafer, use a Raman spectrometer of model RPB-785-1.5-FS for SERS detection. The excitation wavelength of the Raman spectrometer is about 785 nm, the detection power is selected as 200 mW, and the integration time is 5 s to obtain the spectrum of the tissue to be tested.

[0045] (7) Collect the SERS spectra of the patient's tissue and compare them with the spectral data of the patient's tissue that is clearly resistant / sensitive to paclitaxel. If detection peaks appear at Raman shifts such as 820 cm -1 , 1180 cm -1 , 1270 cm -1 , 1450 cm -1 etc., the diagnosis is paclitaxel resistance.

[0046] Example 3:

[0047] Patient Zhang xx, female, 73 years old, was diagnosed with breast cancer in March 2024 and received 6 cycles of paclitaxel chemotherapy. During the review, suspicious metastatic lesions were found in the axillary lymph nodes.

[0048] (1) To understand the sensitivity to paclitaxel drugs, a biopsy of the suspicious metastatic lesions in the lymph nodes was performed, and a tissue strip of 2×2×4 mm was obtained.

[0049] (2) Place the tissue to be tested in 1 ml of ddH2O and ultrasonically disrupt it on ice to obtain a disrupted solution of the tissue to be tested.

[0050] (3) Take a spherical silver nanoparticle solution with a concentration of 6×10 8 particles / ml, centrifuge it at 8500 r / min for 10 min at room temperature, discard the liquid in the tube, add 80 times its own volume of ddH2O to the precipitate and mix well to form a silver nanoparticle suspension;

[0051] (4) Take 5 μl of the above suspension and gently drop it onto a silica wafer, then transfer it to an oven preheated to 60 °C and bake it dry; after the nanoparticles are baked dry, a coffee ring slightly protruding from the surface of the silicon wafer is formed;

[0052] (5) Take 5 μl of the disrupted solution of the tissue to be tested and gently drop it in the center of the coffee ring, then place the silicon wafer with the dropped disrupted solution of the tissue to be tested in the oven at 60 °C again and bake it dry.

[0053] (6) After taking out the silicon wafer, perform SERS detection using a Raman spectrometer of model RPB-785-1.5-FS. The excitation wavelength of the Raman spectrometer is about 785 nm, the detection power is selected as 200 mW, and the integration time is 5 s to obtain the spectrum of the tissue to be tested.

[0054] (7) Collect the SERS spectra of the patient's tissue and compare them with the spectral data of the tissue of patients clearly resistant / sensitive to paclitaxel. If detection peaks appear at Raman shifts such as 820 cm -1 、1180 cm -1 、1270 cm -1 、1450 cm -1 etc., it is diagnosed as paclitaxel resistance.

[0055] Result analysis

[0056] Example 1, SERS spectra and comparison of breast cancer patient tissues to be detected and breast cancer paclitaxel-resistant cells.

[0057] 1. Specificity of characteristic peaks:

[0058] Obvious characteristic peaks appear at 820 cm -1 、1180 cm -1 、1450 cm -1 in the resistant tissue, while the signal in this region is weak or there is no peak in the sensitive tissue.

[0059] There is a specific peak at 1270 cm -1 in the sensitive tissue, which may be related to the molecular vibration mode during the effective action of paclitaxel.

[0060] 2. Role of the coffee ring substrate:

[0061] Figure 1 The signal intensity of the drug-resistant tissue was significantly higher than that of the sensitive tissue, indicating that the coffee ring substrate enhanced the Raman signal through the local surface plasmon resonance effect (LSPR) of silver nanoparticles, especially with higher detection sensitivity for drug-resistant related small molecules.

[0062] 3. Verification of technical effects:

[0063] Rapid discrimination ability: Through the characteristic peak differences (such as 820 cm -1 and 1270 cm -1 ), drug resistance can be directly determined in a single detection without complex data processing (detection time ≤ 10 minutes in Examples 1-3).

[0064] Sensitivity verification: The signal intensity of the drug-resistant tissue at 1450 cm -1 was 3-5 times higher than that of the sensitive tissue, proving the enrichment ability of the substrate for drug-resistant markers.

[0065] The above are only specific embodiments of the present application, enabling those skilled in the art to understand or implement the present application. Various modifications to these embodiments will be obvious to those skilled in the art, and the general principles defined herein can be implemented in other embodiments without departing from the spirit or scope of the present application. Therefore, the present application will not be limited to these embodiments shown herein, but rather to the broadest scope consistent with the principles and novel features claimed herein.

Claims

1. A preparation method of a taxol-resistant SERS substrate, characterized in that: It includes the following steps: (1) After centrifuging the silver nanoparticle-sodium citrate suspension, remove the supernatant, and add ultrapure water to mix to obtain a suspension; (2) Drop the suspension onto the silicon wafer, and after drying, form a coffee-ring structure protruding from the surface of the silicon wafer, thus obtaining the SERS substrate.

2. The preparation method according to claim 1, characterized in that: The silver nanoparticles are spherical in structure, and the concentration of the silver nanoparticle solution is 6×10 8 particles / ml, the centrifugation speed is 8500 - 9500 r / min, and the centrifugation time is 10 - 15 min.

3. The preparation method according to claim 1, characterized in that: The volume ratio of the silver nanoparticles to the ultrapure water is 1:80 - 90.

4. The preparation method according to claim 1, wherein: The dropping amount of the suspension is 5 - 6 μl.

5. An SERS substrate prepared by the preparation method according to any one of claims 1 - 4.

6. An application of the SERS substrate as claimed in claim 5 in the detection of paclitaxel resistance.

7. The application according to claim 6, wherein: The detection method includes the following steps: (1) Establish a SERS spectral database for paclitaxel-resistant / sensitive; (2) Obtain the cancer tissue lysate; (3) Take the cancer tissue lysate and drop it onto the center of the coffee ring on the SERS substrate; (4) After drying, perform SERS detection with a Raman spectrometer; (5) Compare the detected spectrum with the database. If characteristic peaks at 820 cm -1 , 1180 cm -1 , 1270 cm -1 , 1450 cm -1 are present, it is determined to be paclitaxel-resistant.

8. The application according to claim 7, wherein: The preparation method of the cancer tissue lysate is: Place cancer tissue with a volume ≥2×2×3 mm in 1 ml of ultrapure water and ultrasonically disrupt it on ice.

9. The application according to claim 7, wherein: The Raman spectroscopy detection parameters include an excitation wavelength of 785 nm, a detection power of 200 mW, and an integration time of 5 seconds.

10. The application according to claim 7, wherein: The SERS spectral database outputs the resistance / sensitivity results through PCA analysis and comparison.

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