SERS (Surface Enhanced Raman Scattering) substrate for detecting benign and malignant incisional edges in breast conserving surgery and detection method

Abstract

The invention discloses an SERS (Surface Enhanced Raman Scattering) substrate for detecting benign and malignant incisional margins in breast conserving surgery and a detection method, and belongs to the technical field of detection, the SERS substrate is prepared by mixing centrifugal gold nanoparticle suspension liquid (1 * 10 < 8 >-6 * 10 < 8 > / mL) with ultrapure water according to the volume ratio of 1: (20-30), taking 5 mu L of the mixture, dropwise adding the mixture to a silicon wafer, and drying the silicon wafer to form a coffee ring structure. During detection, the incisal edge tissue is subjected to ice-bath ultrasonication and then dropwise added to a substrate, and after drying, a spectrum is collected through a Raman spectrometer (785nm, 250mW). And when characteristic peaks appear at 800cm <-1 > and 840cm <-1 >, determining the malignant cutting edge. The method is easy and convenient to operate, capable of completing detection within 5 minutes, high in sensitivity and suitable for evaluating the incisal edge state in real time in an operation, and the secondary operation rate is reduced.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of detection, and particularly relates to a SERS substrate for detecting the benignity and malignancy of a surgical margin in a breast-conserving surgery and a detection method. BACKGROUND

[0002] Breast cancer is one of the most common malignant tumors in women worldwide. Breast-conserving radical surgery for breast cancer (i.e. breast-conserving surgery) has become one of the standard surgical methods for early breast cancer because it can maximize the preservation of breast appearance and function while ensuring efficacy. The basic principle of breast-conserving surgery is to completely remove tumor tissue and maximize the preservation of normal breast tissue. The key to success lies in negative surgical margins. Positive surgical margins will lead to secondary surgery.

[0003] Current intraoperative surgical margin evaluation methods have limitations. For example, intraoperative frozen section analysis (FSA) is widely used, but it is time-consuming and requires experienced pathologists. Intraoperative imaging examinations (ultrasound, molybdenum target, etc.) can assist in determining whether the surgical margin is negative, but the removed specimen needs to be sent to the relevant imaging department during surgery. The detection process is complex, cannot achieve rapid and efficient detection, and has limited sensitivity. In order to overcome the limitations of existing methods, researchers have explored various emerging technologies in recent years. Among them, surface-enhanced Raman scattering spectroscopy (SERS) has shown certain clinical application potential in intraoperative surgical margin determination due to its rapidity and high sensitivity. For example, studies have shown that SERS detection combined with near-infrared fluorescence imaging can assist in surgical margin division and shorten intraoperative decision-making time. Specific SERS probes can also target specific tumor cells to identify whether there are residual cancer cells in the surgical margin. However, these studies have problems such as complex substrates and non-universal detection. Therefore, it is necessary to develop a simple, rapid, efficient, and accurate determination method to meet clinical needs. This method can quickly determine whether the tumor has been completely removed during surgery or at the bedside to guide surgical decision-making (such as whether to expand the resection range). SUMMARY

[0004] The application aims to provide a SERS substrate and detection method for determining the benignity and malignancy of a surgical margin in a breast-conserving surgery.

[0005] The present application is based on the limitations of the prior art and the deficiencies in the prior related research, and proposes a SERS substrate and a method for detecting the margin based on the substrate. The SERS substrate is used to detect the margin of the tissue removed during the breast-conserving surgery, that is, the margin sample is subjected to ultrasonic crushing treatment, and the sample is dropped on the SERS substrate for detection. The detection method has the characteristics of convenient operation and rapidness, and the surgeon can complete the detection during the operation without the participation of the doctors of related departments such as pathology and imaging. The present application firstly constructs an efficient and sensitive SERS substrate, and then pretreats the margin sample of the tissue removed during the breast-conserving surgery, and detects the margin sample by using the SERS substrate. The method can complete the detection of the margin in a few minutes and output the benign / malignant result of the margin in real time.

[0006] In order to solve the above technical problems, the specific technical solutions of the present application are as follows:

[0007] A SERS detection method for detecting the benign / malignant margin during the breast-conserving surgery, comprising the following steps:

[0008] 1. Treatment of the margin tissue

[0009] Firstly, the edge (i.e. the margin) of the tissue removed during the breast-conserving surgery is sampled; and then it is subjected to ultrasonic crushing in 500 μL of ultrapure water on ice.

[0010] 2. Construction of an efficient SERS substrate by using gold nanoparticles

[0011] (1) A spherical gold nanoparticle-sodium citrate solution with a concentration of 6×10 8 / ml is centrifuged at 2500 r / min for 10 min at room temperature, the supernatant is discarded, the precipitate is diluted with ultrapure water at a dilution ratio of 1:20, and then it is mixed;

[0012] (2) 5 μL of the above suspension is dropped on the silica sheet, and then it is dried in a 60°C oven; the ring structure formed on the silica sheet after drying is a coffee ring;

[0013] (3) 5 μL of the tissue crushing solution is dropped into the coffee ring;

[0014] (4) The silica sheet in (3) is dried again in a 60°C oven.

[0015] 3. SERS detection and data output

[0016] (1) After the silica sheet is taken out, SERS detection is performed by using a RPB-785-1.5-FS type Raman spectrometer, the excitation wavelength of the Raman spectrometer is 785 nm, the detection power is selected to be 250 mw, the integration time is 3 s, and the spectrum of the margin to be detected is obtained.

[0017] (2) Collect the SERS spectrum of the detected incisal margin, and import the spectrum into the tissue spectrum database which has been identified as cancer / cancer-adjacent, compare, analyze between groups by PCA, and then output the detection result of the tested tissue as "benign incisal margin" or "malignant incisal margin".

[0018] The present application first performs ultrasonic crushing on the incisal margin tissue of breast-conserving surgery, and fully releases the cell contents. Then, the "coffee ring effect" of inducing solute migration to the edge when droplets evaporate on a solid surface is used to construct a SERS substrate. Based on the substrate, rapid detection of whether the incisal margin tissue has cancer cell residues can be realized.

[0019] The present application also discloses the use of the above-mentioned SERS substrate in the preparation of a breast-conserving surgery incisal margin benignity / malignancy detection kit.

[0020] The present application also discloses a breast-conserving surgery incisal margin benignity / malignancy detection kit, which comprises the above-mentioned SERS substrate and instructions for use, and a standard cancer / cancer-adjacent tissue SERS spectrum database.

[0021] The present application also discloses the application of the above-mentioned detection method in intraoperative real-time incisal margin evaluation.

[0022] Technical effects:

[0023] (1) The processing method of the present application for incisal margin tissue is simple, short in time and easy to master.

[0024] (2) The present application provides a novel SERS substrate and a detection method thereof. The method is simple in operation, high in sensitivity and fast in detection speed, and only needs 5 minutes from tissue sampling to result output; in batch detection of multiple incisal margins, the average time consumption of each sample is only about 2 minutes, and it is suitable for rapid and accurate detection of incisal margin tissue in breast-conserving surgery. BRIEF DESCRIPTION OF DRAWINGS

[0025] Figure 1 is the SERS spectrum comparison of the benign incisal margin, the malignant incisal margin and the cancer / cancer-adjacent in Example 1. DETAILED DESCRIPTION

[0026] The present application will be described in detail below in combination with the drawings and specific examples. However, the following examples are only used to explain the present application, and the protection scope of the present application should include the entire content of the claims, and through the description of the following examples, those skilled in the art can fully realize the entire content of the claims of the present application.

[0027] The present application will be described in detail below in combination with the drawings and specific examples. However, the following examples are only used to explain the present application, and the protection scope of the present application should include the entire content of the claims, and through the description of the following examples, those skilled in the art can fully realize the entire content of the claims of the present application.

[0028] Example 1:

[0029] Patient Wang XX, female, 31 years old, diagnosed with breast cancer in October 2024, underwent breast-conserving radical mastectomy for breast cancer.

[0030] (1) Take each cut edge of the intraoperative resection tissue;

[0031] (2) Place the test tissue in 500 μL of ultrapure water and perform ultrasonic fragmentation on ice to obtain the test tissue fragmentation liquid;

[0032] (3) Take 6x10 8 / ml of spherical gold nanoparticle-sodium citrate solution, centrifuge at 2500 r / min at room temperature for 10 min, discard the supernatant, dilute the precipitate with ultrapure water at a dilution ratio of 1:20, and then mix well;

[0033] (4) Take 5 μL of the above suspension and drop it onto the silica wafer, then place it in a 60°C oven to dry; the ring structure formed on the silica wafer after drying is the coffee ring;

[0034] (5) Take 5 μL of the tissue fragmentation liquid and drop it into the coffee ring;

[0035] (6) Place the silica wafer in the 60°C oven again to dry;

[0036] (7) After taking out the silica wafer, use the RPB-785-1.5-FS model Raman spectrometer for SERS detection, with an excitation wavelength of 785 nm, a detection power of 250 mw, and an integration time of 3 s, to obtain the spectrum of the test cut edge;

[0037] (8) Collect the SERS spectra of each cut edge tissue and compare them with the cancer / cancer-adjacent spectrum data. If characteristic peaks appear at 800 cm -1 , 840 cm -1 , etc., it is diagnosed as a malignant cut edge. The detection results are shown in Figure 1 , which is the SERS spectrum comparison of the benign cut edge, malignant cut edge, and cancer / cancer-adjacent.

[0038] Example 2:

[0039] Patient Wu XX, female, 32 years old, diagnosed with breast cancer in November 2024, underwent breast-conserving radical mastectomy for breast cancer.

[0040] (1) Take each cut edge of the intraoperative resection tissue;

[0041] (2) Place the test tissue in 500 μL of ultrapure water and perform ultrasonic fragmentation on ice to obtain the test tissue fragmentation liquid;

[0042] (3) Take 6x10 8The 5 μL suspension solution was added dropwise to a silica slice, and then dried in a 60°C oven. The ring structure formed on the silica slice after drying was a coffee ring.

[0043] (4) 5 μL of the above suspension solution was added dropwise to a silica slice, and then dried in a 60°C oven. The ring structure formed on the silica slice after drying was a coffee ring.

[0044] (5) 5 μL of the tissue broken solution was added dropwise into the coffee ring.

[0045] (6) The silica slice in (3) was again placed in a 60°C oven for drying.

[0046] (7) After the silica slice was taken out, SERS detection was performed by using a RPB-785-1.5-FS type Raman spectrometer. The excitation wavelength of the Raman spectrometer was 785 nm, the detection power was 250 mw, the integration time was 3 s, and the spectrum of the detected incisal edge was obtained.

[0047] (8) The SERS spectra of each incisal edge tissue were collected and compared with the cancer / cancer-adjacent spectrum data. If characteristic peaks appeared at 800 cm -1 , 840 cm -1 , etc., the incisal edge was diagnosed as malignant.

[0048] Example 3:

[0049] Patient Liu XX, female, 45 years old, diagnosed with breast cancer in February 2025, underwent breast-conserving radical mastectomy for breast cancer.

[0050] (1) Each incisal edge of the intraoperative resected tissue was sampled;

[0051] (2) The test tissue was placed in 500 μL of ultrapure water, and ultrasonic broken was performed on ice to obtain a test tissue broken solution;

[0052] (3) A spherical gold nanoparticle-sodium citrate solution with a concentration of 6×10 8 / ml was centrifuged at 2500 r / min for 10 min at room temperature, the supernatant was discarded, the precipitate was diluted with ultrapure water at a dilution ratio of 1:20, and then mixed;

[0053] (4) 5 μL of the above suspension solution was added dropwise to a silica slice, and then dried in a 60°C oven. The ring structure formed on the silica slice after drying was a coffee ring.

[0054] (5) 5 μL of the tissue broken solution was added dropwise into the coffee ring.

[0055] (6) The silica slice in (3) was again placed in a 60°C oven for drying.

[0056] (7) After the silicon wafer is taken out, SERS detection is performed by using a RPB-785-1.5-FS type Raman spectrometer, the excitation wavelength of the Raman spectrometer is 785 nm, the detection power is selected as 250 mw, the integration time is 3 s, and the spectrum of the to-be-detected incisal edge is obtained;

[0057] (8) The SERS spectra of the incisal edge tissues are collected, and are compared with the cancer / cancer-adjacent spectrum data. If characteristic peaks appear at 800 cm -1 , 840 cm -1 , and other Raman shifts, the incisal edge is diagnosed as malignant.

[0058] The above description is merely 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 apparent 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 will conform to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for preparing a SERS substrate for detecting benign or malignant resection margins in breast-conserving surgery, characterized in that: The following steps are involved: (1) The concentration is 1×10 8 -6×10 8 The spherical gold nanoparticle-sodium citrate suspension of 1000 / mL was centrifuged at 2000-2500 r / min for 10 min, and the supernatant was discarded; (2) Adding ultrapure water to the precipitate, with the volume ratio of gold nanoparticles to ultrapure water being 1:20-30, and mixing to form a suspension; (3) Take 5 μL of the suspension and drop it onto a silicon wafer. Dry it at 60 °C to form a coffee ring structure and obtain a SERS substrate.

2. The preparation method according to claim 1, characterized in that The particle size of the gold nanoparticles is 10-50 nm.

3. A SERS substrate prepared by the method according to any one of claims 1 to 2, characterized in that: The substrate is a gold nanoparticle array with a coffee ring structure.

4. A method for detecting benign or malignant margins in breast-conserving surgery, characterized in that: The following steps are involved: (1) Place the cut edge tissue in ultrapure water and ultrasonically disrupt it in an ice bath to obtain tissue disruption solution; (2) adding 5 μL of tissue disruption solution to the center area of ​​the coffee ring of the SERS substrate according to claim 3; (3) After drying at 60°C, the samples were detected using a Raman spectrometer with an excitation wavelength of 785 nm and a power of 250 mW; (4) When the Raman shift is 800 cm -1 and 840cm -1 When a characteristic peak appears at the margin, it is judged as a malignant resection margin.

5. The detection method according to claim 4, characterized in that The ultrasonic crushing conditions are as follows: ice bath, power 100-200W, and time 1-2min.

6. The detection method according to claim 4, characterized in that The integration time of the Raman spectrometer is 3 s.

7. Use of the SERS substrate according to claim 3 in preparing a kit for detecting benign and malignant resection margins in breast-conserving surgery.

8. A kit for detecting benign or malignant margins in breast-conserving surgery, characterized in that: Comprising the SERS substrate according to claim 3.

Images