A SERS substrate and detection method for epirubicin blood drug concentration

Abstract

The application provides a SERS substrate and a detection method for epirubicin blood drug concentration, and belongs to the technical field of detection. ‑1 The characteristic peak intensity is determined. By establishing a quantitative curve of the epirubicin concentration in serum and the peak intensity, rapid quantitative detection of clinical samples is realized. The method has simple sample processing, high detection sensitivity, good repeatability, is consistent with the LC-MS / MS result, and is suitable for bedside treatment drug monitoring.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of detection, and particularly relates to a SERS (Surface Enhanced Raman Scattering) substrate for detecting the blood concentration of epirubicin and a detection method. BACKGROUND

[0002] Epirubicin (EPI) is an anthracycline antibiotic antitumor drug, which is an isomer of doxorubicin. It can inhibit the synthesis of DNA and RNA by directly inserting between DNA base pairs, interfering with the transcription process and preventing the formation of mRNA. It has inhibitory effect on various solid tumors such as leukemia, malignant lymphoma, breast cancer, lung cancer, ovarian cancer, gastric cancer, etc.

[0003] The therapeutic window of epirubicin is narrow, and the effective peak blood concentration range is usually 0.1-1 μg / mL (about 1.72×10 -7 -1.72×10 -6 M). When the peak blood concentration exceeds this range, serious toxicity reactions are easily induced. When the blood concentration is >2 μg / mL (3.45×10 -6 M), the risk of cardiac toxicity in patients significantly increases. The incidence and severity of cardiotoxicity of epirubicin are positively correlated with the cumulative dose in the body. Most of the delayed severe heart failure occurs after half a year of medication or when the total dose exceeds 700-800 mg / m 2 .

[0004] The pharmacokinetic characteristics of epirubicin are complex, and it is distributed in a three-compartment model in the body. It is mainly excreted through the hepatobiliary system, and 40%-45% of the administered dose is excreted through feces, and only 7%-23% is excreted through the kidney with urine. This long terminal half-life characteristic means that the drug may accumulate in the body, especially for patients who need multiple cycles of treatment, so long-term monitoring of blood concentration is crucial for individualized dosing and safety management.

[0005] The pharmacokinetic individual differences of epirubicin are significant and are affected by multiple factors: liver function status, moderate liver dysfunction requires a 50% reduction in dose, severe liver dysfunction requires a 75% reduction in dose; kidney function status, moderate renal impairment does not require dose adjustment, but severe renal dysfunction should consider dose reduction; age factor, elderly patients, especially those with reduced cardiac function, should be used with caution, and the total cumulative dose should be appropriately reduced; combination therapy, when combined with other drugs with potential cardiotoxicity or when chest radiotherapy is received, the risk of cardiotoxicity increases, and the dose should be adjusted.

[0006] In view of the narrow therapeutic window of epirubicin, significant individual pharmacokinetic differences and potential severe toxicity (especially dose cumulative cardiomyopathy and bone marrow suppression), it is extremely important to conduct therapeutic drug monitoring (TDM) during treatment. Reliable TDM can guide clinicians to adjust the dosage regimen in a timely manner according to the actual blood drug concentration of the patient, maximize the efficacy while minimizing the risk of toxicity, and achieve individualized precision treatment. However, the existing mainstream methods have obvious limitations: ①High performance liquid chromatography (HPLC) requires complex sample pretreatment (such as solid phase extraction), the analysis time is as long as 30 minutes or more, and the equipment is expensive, which is difficult to meet the clinical instant detection demand. ②Liquid chromatography-mass spectrometry (LC-MS) has strict requirements for instrument environment and maintenance, and the cost of single detection is more than 1000 yuan. ③Enzyme-linked immunoassay (ELISA) has low sensitivity and high risk of false negative. ④In recent years, although there have been explorations of electrochemical sensors (such as based on molecularly imprinted polymers), but in actual application, there are still many problems such as complex technology, dependence on large-scale instrument equipment detection, need for professional operation, high cost, poor stability and so on.

[0007] The present application aims to provide a rapid detection system for epirubicin blood drug concentration, which distinguishes and identifies epirubicin by using the SERS characteristic peak of epirubicin, establishes a drug gradient concentration quantitative model for drug concentration quantification, and has the characteristics of rapidness, low cost, high sensitivity and high specificity. SUMMARY

[0008] Based on the limitations of existing detection methods and the shortcomings of previous related research, the present application proposes a SERS substrate and detection method for epirubicin blood drug concentration. The constructed ABA sandwich SERS substrate is used to detect trace epirubicin in patient blood, that is, after simple treatment of the blood sample, the sample is dropped on the SERS substrate for detection, and the quantitative detection result is obtained. The detection method has the characteristics of convenient operation, rapidness, low cost, high sensitivity and high specificity, and the physician can complete the detection at the bedside without the participation of physicians in related departments such as pathology department and laboratory department.

[0009] The present application pretreats the blood sample to be tested, and uses the SERS substrate to detect epirubicin in the blood sample. The method can obtain the blood drug concentration result of epirubicin within a few minutes.

[0010] In order to solve the above technical problems, the specific technical solutions of the present application are as follows:

[0011] A SERS detection method for epirubicin blood drug concentration, comprising the following steps:

[0012] 1. Treatment of blood sample:

[0013] (1) Take 1 ml of peripheral blood sample, centrifugal treatment, centrifugal condition is 4℃, 3000r / min, 10min in total;

[0014] (2) Take the supernatant obtained in item (1), discard the precipitate, add 0.1ml acetonitrile to 0.2ml serum sample, mix well and stand for 1min, centrifuge after obvious precipitate appears, centrifugal condition is 5000r / min, 5min in total;

[0015] (3) Take the supernatant obtained in item (2), discard the precipitate, add 0.01ml acetonitrile again, mix well and stand for 1min, centrifuge at 8000r / min for 5min;

[0016] (4) Take the supernatant of item (3) as the detection sample, discard the precipitate.

[0017] 2. Constructing high-efficiency SERS substrate with gold nanoparticles of different diameters:

[0018] (1) Use HAuCl4 as seed and sodium citrate as reducing agent to prepare 40nm and 20nm gold nanoparticles (A liquid: diameter 40nm, B liquid: diameter 20nm) in 1:1 and 1:4 adding environment respectively. Take 6×10 8 9 / ml and 2.4×10 9 9 / ml A liquid and B liquid gold nanoparticle-sodium citrate solution, centrifuge at 4500r / min for 10min at room temperature, discard the liquid in the tube, and add 80 times of ddH2O to the precipitate and mix well to form gold nanoparticle suspension;

[0019] (2) Take 5μL of the above A liquid and B liquid suspension, first gently add A liquid to silica sheet, dry at room temperature; then add B liquid, dry again, add A liquid again and dry, and the ABA sandwich structure high-efficiency SERS substrate is formed on the surface of the silica sheet after the nanoparticles are dried.

[0020] 3. Identification of characteristic peaks of epirubicin standard:

[0021] (1) Preparation of epirubicin standard solution: dissolve 5.80mg of epirubicin drug in 10ml of ultrapure water to prepare 1×10 -3 M epirubicin standard solution; then dilute it into 1×10 -5 M to 1×10 -9 M cyclophosphamide standard solution; avoid light and high temperature during the process;

[0022] (2) Take 5μL of the sample prepared in item (1) and add it to the constructed SERS substrate to dry;

[0023] (3) SERS detection was performed using a portable RPB-785-1.5-FS Raman spectrometer, the excitation wavelength of the Raman spectrometer was 785 nm, the detection power was selected as 30 mw, the integration time was 0.5 s, the spectrum of the sample to be detected was obtained, and the characteristic displacement of epirubicin was 1202 cm -1 , 1236 cm -1 .

[0024] 4. Serum epirubicin drug SERS spectrum quantitative curve construction:

[0025] (1) Preparation of serum epirubicin solution: according to the method of the third (1), 1×10 -3 M epirubicin standard solution was prepared; serum was added to prepare 1×10 -4 M serum mixed drug solution; then gradient dilution was performed to prepare 1×10 -5 M to 1×10 -9 M epirubicin serum solution; the process avoids light and high temperature;

[0026] (2) The serum epirubicin solution with different concentrations obtained in step (1) was pretreated according to the above blood sample processing method, and the pretreated 1×10 -5 M to 1×10 -9 M epirubicin serum solution was obtained;

[0027] (3) 5 μL of the sample to be detected was dropped on the constructed SERS substrate and dried;

[0028] (4) SERS detection was performed using a portable RPB-785-1.5-FS Raman spectrometer, the excitation wavelength of the Raman spectrometer was 785 nm, the detection power was selected as 30 mw, the integration time was 0.5 s, the spectrum of the sample to be detected was obtained, and the characteristic displacement of epirubicin was 1207 cm -1 , 1236 cm -1 .

[0029] (5) The SERS spectra of epirubicin serum solutions with different concentrations were obtained, and a quantitative curve was drawn.

[0030] 5. SERS detection and data output of epirubicin blood drug concentration of tumor patients:

[0031] (1) The next day after epirubicin chemotherapy of tumor patients, peripheral blood samples were taken and blood treatment was performed according to the method of the first part;

[0032] (2) 5 μL of the sample to be detected was dropped on the constructed SERS substrate and dried at room temperature and in the dark;

[0033] (3) SERS detection is performed by using a portable Raman spectrometer of RPB-785-1.5-FS type, the excitation wavelength of the Raman spectrometer is 785 nm, the detection power is selected as 30 mw, the integral time is 0.5 s, the spectrum of the sample to be detected is obtained, and the spectral peak at 1207 cm -1 , 1236 cm -1 is observed.

[0034] (4) The spectral peak obtained by measurement is brought into the quantitative curve to obtain the drug concentration of epirubicin.

[0035] Beneficial effects:

[0036] 1. The serum sample processing method is simple, portable, low in cost and good in effect.

[0037] 2. The application provides a SERS detection method for the blood drug concentration of epirubicin. DETAILED DESCRIPTION

[0038] Figure 1 : 1x10 -7 M concentration epirubicin SERS detection spectrum in different solutions and serum control.

[0039] Figure 2 : Serum epirubicin drug SERS spectrum quantitative curve.

[0040] Figure 3 : The 1x10 -7 M concentration epirubicin serum sample is repeatedly measured (n=20 times), and the Raman characteristic peak intensity at 1236 cm -1 is collected.

[0041] Figure 4 : Epirubicin SERS detection and concentration prediction of clinical serum samples, original SERS spectrum of serum samples of three patients (P001-P003). DETAILED DESCRIPTION

[0042] The application will be described in detail below in combination with the drawings and specific embodiments. However, the following embodiments are only used to explain the application, and the protection scope of the application should include all contents of the claims, and through the description of the following embodiments, a person skilled in the art can fully realize all contents of the claims of the application.

[0043] Example 1:

[0044] Patient P001, female, 55 years old, was diagnosed with breast cancer in April 2025 and underwent epirubicin chemotherapy.

[0045] (1) 1 ml of peripheral blood was collected using a coagulation-promoting tube 24 h after the patient received intravenous epirubicin and centrifuged for 10 min at 4°C and 3000 r / min.

[0046] Take 0.2 ml of serum, add 0.1 ml of acetonitrile, mix well and let stand for 1 min. After obvious precipitation, centrifuge at 5000 r / min for 5 min and discard the precipitate. Add 0.01 ml of acetonitrile again, mix well and let stand for 1 min, centrifuge at 8000 r / min for 5 min and discard the precipitate again to obtain the sample to be tested.

[0047] (2) Using HAuCl4 as a seed and sodium citrate as a reducing agent, two types of gold nanoparticles, 40 nm and 20 nm in solution A and solution B, were prepared in 1:1 and 1:4 addition environments, respectively. (Solution A: diameter 40 nm, Solution B: diameter 20 nm) 8 cells / ml, 2.4×10 9 Two gold nanoparticle-sodium citrate solutions, A and B, were prepared at 4500 r / min for 10 min at room temperature. The liquid in the tube was discarded, and ddH2O was added to the precipitate at 80 times its own volume and mixed well to form a gold nanoparticle suspension.

[0048] (3) Take 5 μL of the above two suspensions, A and B. First, gently drop A onto the silicon dioxide wafer and let it air dry at room temperature. Then, drop B onto the wafer and let it air dry again. Then, drop A onto the wafer and let it air dry again. After the nanoparticles are dried, an ABA sandwich structure high-efficiency SERS substrate is formed on the surface of the silicon wafer.

[0049] (4) Take 5 μL of the sample to be tested and drop it onto the substrate to air dry at room temperature in the dark; set the excitation wavelength of the Raman spectrometer to 785 nm, the detection power to 30 mW, and the integration time to 0.5 s to obtain the spectrum of the sample to be tested, and observe it at 1207 cm⁻¹. -1 1236cm -1 Spectral peak at the displacement;

[0050] (5) The measured 1236cm -1 The spectral peak at the displacement was incorporated into the quantitative curve, yielding a serum epirubicin drug concentration of 5.36 × 10⁻⁶. -8 M.

[0051] Example 2:

[0052] Patient P002, female, 49 years old, was diagnosed with breast cancer in April 2025 and underwent epirubicin chemotherapy.

[0053] (1) After 24h of intravenous infusion of epirubicin to the patient, 1ml of peripheral blood was collected with a coagulation tube, and centrifuged for 10min at 4℃ and 3000r / min. 0.2ml of serum was taken, 0.1ml of acetonitrile was added, and after mixing, it was left for 1min. After obvious precipitation appeared, centrifugation was performed at 5000r / min for 5min, and the precipitate was discarded. 0.01ml of acetonitrile was added again, mixed, and left for 1min. Centrifugation was performed at 8000r / min for 5min, and the precipitate was discarded again to obtain the sample to be tested;

[0054] (2) 40nm and 20nm gold nanoparticles (A liquid: 40nm in diameter, B liquid: 20nm in diameter) were prepared in 1:1 and 1:4 addition environments respectively using HAuCl4 as seeds and sodium citrate as a reducing agent. 6×10 8 and 2.4×10 9 / ml of A liquid and B liquid gold nanoparticle-sodium citrate solutions were taken respectively, and centrifuged at 4500r / min for 10min 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 gold nanoparticle suspension;

[0055] (3) 5μL of the above-mentioned A liquid and B liquid suspensions were taken. First, the A liquid was gently added to the silica sheet, and left to dry at room temperature. Then, the B liquid was added, and left to dry again. The A liquid was added again and left to dry. After the nanoparticles were dried, an ABA sandwich structure high-efficiency SERS substrate was formed on the surface of the silica sheet;

[0056] (4) 5μL of the sample to be tested was added to the substrate and left to dry at room temperature in the dark. The excitation wavelength of the Raman spectrometer was 785nm, the detection power was selected as 30mw, the integration time was 0.5s, the spectrum of the sample to be tested was obtained, and the spectral peak at 1207cm -1 and 1236cm -1 was observed;

[0057] (5) The spectral peak at 1236cm -1 obtained by measurement was brought into the quantitative curve, and the serum epirubicin drug concentration was obtained as 1.85×10 -8 M.

[0058] Example 3:

[0059] Patient P003, female, 52 years old, diagnosed with breast cancer in May 2025, and underwent epirubicin chemotherapy.

[0060] (1) 24 hours after the patient received an intravenous infusion of epirubicin, 1 ml of peripheral blood was collected using a coagulation-promoting tube and centrifuged for 10 min at 4°C and 3000 r / min. 0.2 ml of serum was taken, 0.1 ml of acetonitrile was added, mixed, and allowed to stand for 1 min. After a clear precipitate appeared, centrifugation was performed at 5000 r / min for a total of 5 min. The precipitate was then discarded. 0.01 ml of acetonitrile was added again, mixed, and allowed to stand for 1 min. The mixture was then centrifuged at 8000 r / min for a total of 5 min. The precipitate was discarded again to obtain the sample to be tested.

[0061] (2) Using HAuCl4 as a seed and sodium citrate as a reducing agent, two types of gold nanoparticles, 40 nm and 20 nm in solution A and solution B, were prepared in 1:1 and 1:4 addition environments, respectively. (Solution A: diameter 40 nm, Solution B: diameter 20 nm) 8 cells / ml, 2.4×10 9 Two gold nanoparticle-sodium citrate solutions, A and B, were prepared at 4500 r / min for 10 min at room temperature. The liquid in the tube was discarded, and ddH2O was added to the precipitate at 80 times its own volume and mixed well to form a gold nanoparticle suspension.

[0062] (3) Take 5 μL of the above two suspensions, A and B. First, gently drop A onto the silicon dioxide wafer and let it air dry at room temperature. Then, drop B onto the wafer and let it air dry again. Then, drop A onto the wafer and let it air dry again. After the nanoparticles are dried, an ABA sandwich structure high-efficiency SERS substrate is formed on the surface of the silicon wafer.

[0063] (4) Take 5 μL of the sample to be tested and drop it onto the substrate to air dry at room temperature in the dark; set the excitation wavelength of the Raman spectrometer to 785 nm, the detection power to 30 mW, and the integration time to 0.5 s to obtain the spectrum of the sample to be tested, and observe it at 1207 cm⁻¹. -1 1236cm -1 Spectral peak at the displacement;

[0064] (5) The measured 1236cm -1 The spectral peak at the displacement was incorporated into the quantitative curve, yielding a serum epirubicin drug concentration of 6.66 × 10⁻⁶. -8 M.

[0065] Analysis of test and experimental results:

[0066] like Figure 1 As shown, Figure 1 1×10 -7 SERS spectra of epirubicin at concentration M in double-distilled water and serum. Epirubicin was detected by SERS in double-distilled water, with a characteristic shift of 1202 cm⁻¹. -1 1236cm -1, 1207 cm -1 , 1236 cm -1 .

[0067] As Figure 2 shown, Figure 2 the serum epirubicin drug SERS spectrum quantitative curve. SERS spectrum detection of different concentrations of epirubicin serum solution, selected the characteristic Raman vibration peak of epirubicin molecule at 1236 cm -1 , as the target peak of quantitative analysis, which is attributed to the C-H, C-O-H and C-O vibration characteristics of anthracene ring skeleton, with significant and stable Raman signal. As Figure 2 shown, the intensity of the characteristic peak (y axis, I) and the concentration of epirubicin in serum (x axis, lg(c)) showed a good linear relationship in the range of 1×10 -9 to 1×10 -4 M. Linear fitting, the linear regression equation is I = 19302 + 1782 × lg(c), the correlation coefficient (R 2 ) is 0.9933, the quantitative model has high linearity and reliability.

[0068] As Figure 3 shown, Figure 3 shown, the Raman characteristic peak intensity at 1236 cm -7 was collected for the epirubicin serum sample with a concentration of 1×10 -1 M was repeated many times (n = 20 times). The signal intensity obtained by multiple measurements is closely distributed around the average value, and the relative standard deviation (RSD) is 7.65%. This method has good repeatability and high precision in detecting epirubicin serum concentration.

[0069] As Figure 4 shown, to verify the clinical applicability of the SERS detection method, the blood samples of patients receiving epirubicin chemotherapy for 24 hours were collected for blind test analysis. As Figure 4 shown, the original SERS spectra of three patients (P001-P003) successfully collected the characteristic signal of epirubicin in the range of 600-1600 cm -1 . -1The intensity of the Raman characteristic peak of epirubicin in the serum sample was measured, and the standard curve equation (I = 19302 + 1782xlg(c)) was substituted into the equation to calculate the trough concentration (Ctrough) of epirubicin in the serum of the three patients, as shown in Table 1. To verify the accuracy of the SERS method established in this study, we used liquid chromatography-tandem mass spectrometry (LC-MS / MS), which is a recognized gold standard, to perform blind verification analysis on the serum samples of the three patients (P001, P002, P003). As shown in Table 1, the prediction results obtained by the SERS method were compared with the mass spectrometry results, and both showed a high degree of consistency. The serum epirubicin SERS detection method can quickly detect results, providing real-time basis for individualized precise drug delivery in clinical practice.

[0070] Table 1 shows the predicted concentration of epirubicin calculated from the intensity of the characteristic peak and the standard curve, the LC-MS / MS verification concentration, and the relative error.

[0071] Table 1

[0072]

[0073] The above description is merely a specific implementation of the present application, enabling one 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 the embodiments shown herein, but will conform to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A SERS substrate for epirubicin blood concentration, characterized in that, Its preparation process includes the following steps: Preparation of 40nm gold nanoparticles in solution A: Synthesized in a 1:1 addition environment using HauCl4 as seed and sodium citrate as reducing agent; Preparation of 20nm gold nanoparticles in solution B: Synthesized in a 1:4 addition environment using HAuCl4 as seed and sodium citrate as reducing agent. The concentration is 6×10 8 A suspension of particles A at a concentration of 2.4 × 10⁻⁶ / mL and a concentration of 2.4 × 10⁻⁶ / mL. 9 The B particle suspension was centrifuged at 4500 r / min for 10 min, the supernatant was discarded, and the precipitate was resuspended in 80 volumes of ddH2O. 5 μL of solution A was added dropwise to a silica substrate and allowed to dry. Then 5 μL of solution B was added dropwise and allowed to dry. Then 5 μL of solution A was added dropwise and allowed to dry, forming an ABA sandwich structure SERS substrate.

2. The SERS substrate according to claim 1, characterized in that, The diameter of gold nanoparticle A is 40±5nm, and the diameter of gold nanoparticle B is 20±3nm.

3. A SERS method for detecting epirubicin blood concentration, characterized in that, Includes the following steps: Blood sample pretreatment: Take 0.2 mL of serum, add 0.1 mL of acetonitrile, mix well, let stand for 1 min, then centrifuge at 5000 r / min for 5 min; take the supernatant, add 0.01 mL of acetonitrile, mix well, then centrifuge at 8000 r / min for 5 min to obtain the sample to be tested; Using the SERS substrate described in claim 1 or 2, add 5 μL of the sample to be tested and air dry in the dark; Detection was performed using a Raman spectrometer with an excitation wavelength of 785 nm and a power of 30 mW. The integration time was 0.5 s, and a sample size of 1236 cm⁻¹ was collected. -1 Characteristic peak intensity; The concentration of tabularubicin was calculated based on a pre-established quantitative curve.

4. The method according to claim 3, characterized in that, Before pretreatment, the blood samples need to be centrifuged at 4°C and 3000 r / min for 10 min to separate the serum.

5. The method according to claim 3, characterized in that, The quantitative curve is established through the following steps: Prepare 1×10 -4 M to 1×10 -9 Epirubicin serum solutions with M gradient concentrations; The serum solution underwent the same pretreatment. Detection of 1236 cm on SERS substrate -1 Peak intensity was used to establish a linear equation: I = 19302 + 1782 × lg(c).

6. The method according to claim 3, characterized in that, A 1207cm sample was collected simultaneously during the test. -1 Characteristic peaks serve as auxiliary quantitative references.

7. A method for identifying characteristic peaks of a pirouette star, characterized in that, include: Prepare 1×10 -4 M to 1×10 -9 M-type epirubicin aqueous solution; Using the SERS substrate described in claim 1 or 2, a characteristic peak at 1202 cm⁻¹ was obtained at an excitation wavelength of 785 nm. -1 and 1236cm -1 .

8. A reagent kit for detecting epirubicin blood drug concentration, characterized in that, Include: The ABA sandwich structure SERS substrate as described in claim 1 or 2; Acetonitrile sample processing solution; Epirubicin quantitative curve standard card, concentration range 1×10⁻⁶ -9 M to 1×10 -4 M.

9. The reagent kit according to claim 8, characterized in that, The quantitative curve standard card is marked 1236cm. -1 The linear equation relating peak intensity to concentration is I = 19302 + 1782 × lg(c).

10. A portable epirubicin blood drug concentration analyzer, characterized in that, integrated: The SERS substrate loading module as described in claim 1 or 2; 785nm laser source and spectral detector; The quantitative curve described in claim 5 is incorporated.

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