A paclitaxel-resistant SERS substrate and detection method

Abstract

The present invention discloses a paclitaxel resistance SERS substrate and detection method. It belongs to the field of drug resistance detection technology. The SERS substrate is prepared by centrifuging a silver nanoparticle-sodium citrate suspension, mixing it with ultrapure water, dropping it onto a silicon wafer, and drying it to form a coffee ring structure. During detection, a cancer tissue fragment is dropped onto the center of the substrate, and a Raman spectrometer is used to collect SERS signals, which are then compared with a pre-established drug resistance / sensitivity spectrum database. According to the 820cm ‑1 、1180cm ‑1 、1270cm ‑1 、1450cm ‑1 The method is simple to operate, has a short detection time (within 10 minutes), and is highly sensitive, making it suitable for rapid screening of paclitaxel resistance in breast cancer patients.

Description

Technical Field

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

[0002] Breast cancer is one of the most common malignancies worldwide, with the highest incidence among female cancers. Paclitaxel is one of the most commonly used chemotherapy drugs, widely used to treat various types of breast cancer. However, due to the development of drug resistance in some breast cancers, this drug cannot effectively control disease progression, seriously affecting patients' survival and quality of life.

[0003] Currently, the identification and assessment of drug resistance in breast cancer relies primarily on evaluating the efficacy of treatment after every two cycles. Patients who develop drug resistance often experience disease progression during this period of ineffective treatment, potentially losing optimal treatment opportunities and potentially even their lives. Additionally, some research is attempting to identify drug resistance in cancer cells through laboratory tests, such as genetic testing of cancer tissue, in vitro culturing of cancer cells, and drug sensitivity testing. This entire process, involving tumor sample pretreatment and extensive instrumentation, is cumbersome, with limited success rates, long testing times, and high costs. Other research is exploring the identification of drug resistance-associated proteins in patients' peripheral blood and the development of corresponding antibody-conjugated nanoparticles to detect drug resistance. However, this approach has several drawbacks: 1. It requires a large blood sample, which is harmful to the patient; 2. The cost of antibody production is high; 3. It does not directly detect the cancer lesion, resulting in a certain false-negative rate; and 4. The peripheral blood sample requires complex pretreatment steps, which hinders rapid analysis.

[0004] Therefore, the development of a method to detect rapid chemotherapy drug resistance in breast cancer patients is of great clinical and social significance. It 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 clinical application of SERS (surface-enhanced Raman spectroscopy) technology has become increasingly widespread. Compared with clinical efficacy evaluation, genetic testing and other technologies, it has obvious advantages such as on-site, rapid, high sensitivity and low detection cost. Summary of the Invention

[0006] The present invention provides a paclitaxel-resistant SERS substrate and detection method. Currently, there are no reports on the use of SERS technology to detect paclitaxel resistance in breast cancer. The few research methods that exist suffer from complex pre-processing and low success rates, hindering their widespread clinical application. Breast cancer, the most common malignant tumor in women, poses a serious threat to the lives of many patients. Clinical treatment requires seizing critical moments to deliver effective treatment as quickly as possible while maintaining the patient's overall health. Therefore, a method is urgently needed to rapidly and accurately obtain drug sensitivity information from patients.

[0007] This paper addresses these issues and proposes a SERS substrate and a detection method based on this SERS substrate. SERS technology combines the advantages of conventional Raman spectroscopy, such as high specificity, low sample usage, and sample-safety, with a lower detection limit than conventional Raman spectroscopy. Using SERS technology to detect cancerous tissue from breast cancer patients, the tumor tissue sample undergoes pre-treatment, such as ultrasonic disruption, and is then dropped onto a coffee-ring SERS substrate constructed from silver nanoparticles for detection. This method offers accurate, rapid, and low-cost detection.

[0008] The method first pre-treats breast cancer tissue from patients, then constructs a highly efficient and sensitive silver nanoparticle SERS substrate. This substrate is then used to perform SERS detection on the cancer tissue. This method can complete SERS detection of patient tissue within minutes and output 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 comprises the following steps:

[0011] 1. Pre-treatment of breast cancer tissue

[0012] Cancer tissue was punctured in patients with breast cancer who were clinically assessed to have paclitaxel resistance. Cancer tissue strips larger than 2×2×3 mm in volume were obtained and placed in 1 ml of ddH2O. The strips were ultrasonically disrupted on ice to obtain the tissue fragments to be tested.

[0013] 2. Silver nanoparticles construct an efficient coffee ring SERS substrate

[0014] (1) Take the concentration as 6×10 8 The spherical silver nanoparticle-sodium citrate solution was centrifuged at 8500 rpm for 10 min at room temperature, the liquid in the tube was discarded, and 80 times the volume of ddH2O was added to the precipitate and mixed to form a silver nanoparticle suspension;

[0015] (2) Take 5 μl of the above suspension and gently add it onto the silicon dioxide wafer, then transfer it to a preheated oven at 60°C and bake it dry; after drying, the nanoparticles form a coffee ring that slightly protrudes from the surface of the silicon wafer;

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

[0017] (4) Place the silicon wafer with the tissue fragmentation solution to be tested in an oven at 60°C and bake it dry again.

[0018] 3. SERS detection and data output

[0019] (1) After removing the silicon wafer, SERS detection was performed using a Raman spectrometer model RPB-785-1.5-FS. The excitation wavelength of the Raman spectrometer was approximately 785 nm, the detection power was selected to be 200 mW, and the integration time was 5 s to obtain the spectrum of the tissue to be tested.

[0020] (2) SERS spectra of patient tissues are collected and imported into a database of patient tissue spectra that has been clearly identified as paclitaxel-resistant / sensitive for comparison. PCA is used for inter-group analysis, and the analysis data will output the test results of whether the tissue to be tested is "paclitaxel-resistant" or "paclitaxel-sensitive."

[0021] This method uses ultrasonic disruption to disrupt breast cancer tissue, allowing the release of specific small molecules from the cancer cells. By leveraging the principle that evaporation of a solution from the inside out drives small particles around, a porous coffee ring substrate is constructed to improve the SERS detection efficiency of drug-resistant cancer tissue.

[0022] Beneficial effects:

[0023] (1) The method for treating tumor tissue of the present invention is simple to operate, has a short treatment time, and has low requirements on the professional level of the operator.

[0024] (2) The present invention has constructed a new type of coffee ring silver nanoparticle substrate with high detection sensitivity and short detection time. It only takes 10 minutes from tissue sampling to full detection. When testing multiple samples, it takes an average of 3 minutes for each sample. The test results can be exported in real time, which is suitable for accurate and rapid detection of drug resistance in breast cancer patients. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 These are the SERS spectra and comparison of the breast cancer patient tissue and paclitaxel-resistant breast cancer cells to be tested in Example 1. DETAILED DESCRIPTION

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

[0027] The present invention is further described below with reference to the following examples, but the present invention is not limited to the following examples.

[0028] Example 1:

[0029] Patient Li xx, female, 55 years old, was diagnosed with breast cancer in October 2023. She underwent 4 cycles of paclitaxel chemotherapy. During the follow-up examination, suspicious metastases were found in the liver.

[0030] (1) To understand the patient's sensitivity to paclitaxel, a puncture of the suspected liver metastasis was performed to obtain a 2×2×4 mm tissue strip.

[0031] (2) Place the tissue to be tested in 1 ml of ddH2O and perform ultrasonic disruption on ice to obtain the tissue disruption solution to be tested.

[0032] (3) Take the concentration as 6×10 8 The spherical silver nanoparticle solution of 1000 nanometers / ml was centrifuged at 8500 rpm for 10 min at room temperature, the liquid in the tube was discarded, and 80 times the volume of ddH2O was added to the precipitate and mixed to form a silver nanoparticle suspension;

[0033] (4) Take 5 μl of the above suspension and gently drop it onto the silicon dioxide wafer. Then transfer it to a preheated oven at 60°C and bake it dry. After drying, the nanoparticles form a coffee ring that slightly protrudes from the surface of the silicon wafer.

[0034] (5) Take 5ul of the tissue fragmentation solution to be tested and gently drop it in the center of the coffee ring. Then place the silicon wafer with the tissue fragmentation solution to be tested in a 60℃ oven to dry it.

[0035] (6) After removing 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 approximately 785 nm, the detection power was selected to be 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 tissue and compare them with the spectral data of the patient tissue that is clearly resistant / sensitive to paclitaxel. If there is a 820cm -1 、1180cm -1 、1270cm -1 、1450cm -1 If the Raman shift shows a detection peak, it is diagnosed as paclitaxel resistance.

[0037] Example 2:

[0038] Patient Wang xx, female, 68 years old, was diagnosed with breast cancer in January 2024. She underwent 6 cycles of paclitaxel chemotherapy. During the follow-up examination, suspicious metastases were found in the lungs.

[0039] (1) To understand the patient's sensitivity to paclitaxel, a puncture of the suspected lung metastasis was performed to obtain a 2×2×3 mm tissue strip.

[0040] (2) Place the tissue to be tested in 1 ml of ddH2O and perform ultrasonic disruption on ice to obtain the tissue disruption solution to be tested.

[0041] (3) Take the concentration as 6×10 8 The spherical silver nanoparticle solution of 1000 nanometers / ml was centrifuged at 8500 rpm for 10 min at room temperature, the liquid in the tube was discarded, and 80 times the volume of ddH2O was added to the precipitate and mixed to form a silver nanoparticle suspension;

[0042] (4) Take 5 μl of the above suspension and gently drop it onto the silicon dioxide wafer. Then transfer it to a preheated oven at 60°C and bake it dry. After drying, the nanoparticles form a coffee ring that slightly protrudes from the surface of the silicon wafer.

[0043] (5) Take 5ul of the tissue fragmentation solution to be tested and gently drop it in the center of the coffee ring. Then place the silicon wafer with the tissue fragmentation solution to be tested in a 60℃ oven to dry it.

[0044] (6) After removing 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 approximately 785 nm, the detection power was selected to be 200 mW, and the integration time was 5 s to obtain the spectrum of the tissue to be tested.

[0045] (7) Collect the SERS spectra of the patient tissue and compare them with the spectral data of the patient tissue that is clearly resistant / sensitive to paclitaxel. If there is a 820cm -1 、1180cm -1 、1270cm -1 、1450cm -1 If the Raman shift shows a detection peak, it is diagnosed as paclitaxel resistance.

[0046] Example 3:

[0047] Patient Zhang xx, female, 73 years old, was diagnosed with breast cancer in March 2024. She underwent 6 cycles of paclitaxel chemotherapy. During the follow-up examination, suspicious metastatic lesions were found in the axillary lymph nodes.

[0048] (1) To understand the patient's sensitivity to paclitaxel, a puncture of the suspected metastatic lesion in the lymph node was performed, and a 2×2×4 mm tissue strip was obtained.

[0049] (2) Place the tissue to be tested in 1 ml of ddH2O and perform ultrasonic disruption on ice to obtain the tissue disruption solution to be tested.

[0050] (3) Take the concentration as 6×10 8 The spherical silver nanoparticle solution of 1000 nanometers / ml was centrifuged at 8500 rpm for 10 min at room temperature, the liquid in the tube was discarded, and 80 times the volume of ddH2O was added to the precipitate and mixed to form a silver nanoparticle suspension;

[0051] (4) Take 5 μl of the above suspension and gently drop it onto the silicon dioxide wafer. Then transfer it to a preheated oven at 60°C and bake it dry. After drying, the nanoparticles form a coffee ring that slightly protrudes from the surface of the silicon wafer.

[0052] (5) Take 5ul of the tissue fragmentation solution to be tested and gently drop it in the center of the coffee ring. Then place the silicon wafer with the tissue fragmentation solution to be tested in a 60℃ oven to dry it.

[0053] (6) After removing 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 approximately 785 nm, the detection power was selected to be 200 mW, and the integration time was 5 s to obtain the spectrum of the tissue to be tested.

[0054] (7) Collect the SERS spectra of the patient tissue and compare them with the spectral data of the patient tissue that is clearly resistant / sensitive to paclitaxel. If there is a 820cm -1 、1180cm -1 、1270cm -1 、1450cm -1 If the Raman shift shows a detection peak, it is diagnosed as paclitaxel resistance.

[0055] Result Analysis

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

[0057] 1. Characteristic peak specificity:

[0058] Resistant tissue at 820 cm -1 、1180 cm -1 、1450 cm -1 There is an obvious characteristic peak in the sensitive tissue, while the signal of sensitive tissue in this area is weak or has no peak.

[0059] Sensitive tissue at 1270cm -1 There is a specific peak at , which may be related to the molecular vibration mode when paclitaxel acts effectively.

[0060] 2. The function of coffee ring base:

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

[0062] 3. Verify the technical effect:

[0063] Rapid differentiation capability: by the difference of characteristic peaks (such as 820cm -1 and 1270 cm -1 ), drug resistance can be directly determined in a single test without complex data processing (the detection time in Examples 1-3 is ≤ 10 minutes).

[0064] Sensitivity verification: drug-resistant tissue at 1450cm -1 The signal intensity is 3-5 times higher than that of sensitive tissue, which proves the ability of the substrate to enrich drug resistance markers.

[0065] The foregoing is merely a list of specific embodiments of the present application, intended to enable those skilled in the art to understand or implement the present application. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present application. Therefore, the present application is not limited to the embodiments shown herein, but is intended to conform to the broadest scope consistent with the principles and novel features of the present application.

Claims

1. A method for detecting paclitaxel resistance using a SERS substrate, characterized in that: The following steps are involved: (1) Establish a SERS spectral database of paclitaxel resistance / sensitivity; (2) Obtaining cancer tissue fragmentation fluid; (3) Take a drop of the cancer tissue fragmentation solution and add it to the center of the coffee ring on the SERS substrate; (4) After drying, SERS detection is performed using a Raman spectrometer; (5) Compare the detected spectrum with the database. If there is 820cm -1 、1180cm -1 、1270cm -1 、1450cm -1 If the characteristic peak at , it is determined to be paclitaxel-resistant; The preparation method of the SERS substrate is as follows: (1) Centrifuging the silver nanoparticle-sodium citrate suspension, removing the supernatant, and adding ultrapure water to obtain a suspension; (2) The suspension is dropped onto a silicon wafer and dried to form a coffee ring structure protruding from the surface of the silicon wafer, thus obtaining a SERS substrate.

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

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

4. The method according to claim 1, wherein: The amount of the suspension added is 5-6 ul.

5. The method according to claim 1, wherein: The cancer tissue disruption solution is prepared by placing cancer tissue with a volume of ≥2×2×3 mm in 1 ml of ultrapure water and disrupting the tissue with ultrasound on ice.

6. The method according to claim 1, wherein: The Raman spectroscopy detection parameters included an excitation wavelength of 785 nm, a detection power of 200 mW, and an integration time of 5 s.

7. The method according to claim 1, wherein: The SERS spectrum database is compared through PCA analysis to output drug resistance / sensitivity results.

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