SERS (Surface Enhanced Raman Scattering) detection method for paclitaxel drug concentration in blood of breast cancer patient

Abstract

The invention discloses an SERS (Surface Enhanced Raman Scattering) detection method for paclitaxel drug concentration in blood of a breast cancer patient, and belongs to the technical field of detection. The method comprises the following steps: performing acetonitrile two-step precipitation centrifugal treatment on a blood sample to obtain a detection sample; the method comprises the following steps: constructing a high-sensitivity SERS substrate (the volume ratio of gold nanoparticles to ethanol is 1: (90-100), and a film is formed on a water-cyclohexane interface) through self-assembly of gold nanoparticles on a liquid-liquid interface; the method comprises the following steps: dropwise adding a sample into a substrate, and detecting characteristic peaks of 1024cm <-1 > and 1053cm <-1 > by using a portable Raman spectrometer (785nm, 30mW, 0.5 s); and calculating the paclitaxel concentration in combination with a pre-established quantitative curve. The method is easy and convenient to operate and suitable for clinical bedside rapid monitoring.

Description

Technical Field

[0001] The present invention belongs to detection technology, and in particular relates to a SERS substrate and a detection method for the paclitaxel drug concentration in the blood of breast cancer patients. Background Art

[0002] Paclitaxel, a diterpenoid compound extracted from the bark of the yew tree, inhibits tumor cell division by stabilizing microtubules and is widely used in the treatment of solid tumors such as breast cancer, ovarian cancer, and lung cancer. However, paclitaxel has a narrow therapeutic window, with its effective concentration (50-100 nM) approaching its toxic concentration (>1 μM). Metabolism varies significantly between individuals, and insufficient blood concentrations can lead to treatment failure, while excessive concentrations can trigger adverse reactions such as myelosuppression and neurotoxicity. Therefore, real-time monitoring of paclitaxel concentrations in the blood is crucial for optimizing dosing regimens.

[0003] Currently, paclitaxel blood concentration detection relies primarily on the following technologies, all of which have significant drawbacks: 1. High-performance liquid chromatography (HPLC): Traditional HPLC requires complex sample pretreatment (such as solid-phase extraction), analysis times of more than 30 minutes, and expensive equipment, making it difficult to meet the needs of clinical point-of-care testing. 2. Immunoassay: Susceptible to cross-reaction interference from paclitaxel metabolites (such as 6α-hydroxypaclitaxel), lack of specificity, and the risk of false positives. In addition, existing technologies require multiple venous blood draws, and frequent testing increases the burden on patients and cannot achieve real-time dynamic monitoring. Although electrochemical sensor technology (such as that based on molecular imprinting polymers) has been explored in recent years, practical applications still face the following challenges: 1. Technical complexity: Relying on large-scale instrumentation and equipment for detection, requiring professional operation, it is not suitable for all clinical testing scenarios; 2. High cost: The sensor preparation process is complex, stability is poor, and each synthesis increases patient costs.

[0004] The present invention aims to propose a rapid PTX blood concentration detection system developed based on a SERS detection substrate self-assembled at a liquid-liquid interface. By utilizing the gaps between incompatible liquids to construct an automatically assembled SERS detection substrate, the system can significantly improve substrate synthesis efficiency, reduce costs, and enhance specificity and sensitivity, thus filling the current technological gap. Summary of the Invention

[0005] The present invention belongs to detection technology, and in particular relates to a SERS substrate and a detection method for the paclitaxel drug concentration in the blood of breast cancer patients.

[0006] This paper addresses the limitations of existing methods and deficiencies in previous research by proposing a SERS substrate and a method for detecting paclitaxel concentrations in blood based on this substrate. This SERS substrate can be used to detect trace amounts of paclitaxel in a patient's blood. Specifically, a blood sample is processed and then dropped onto the SERS substrate for detection. This method is convenient and rapid, allowing physicians to perform the test at the bedside without requiring the involvement of physicians from other departments, such as pathology and laboratory medicine.

[0007] This application first constructs an efficient and sensitive SERS substrate, then pre-treats the blood sample to be tested, and uses the SERS substrate to detect the blood sample. This method can complete drug concentration detection within minutes.

[0008] A SERS method for detecting the concentration of paclitaxel in the blood of a breast cancer patient comprises the following steps:

[0009] 1. Blood Sample Processing

[0010] (1) Add 0.1 ml of acetonitrile to 0.1 ml of blood sample, mix well, let it stand for 1 min, and centrifuge after obvious precipitation occurs. The centrifugation condition is 6000 r / min for a total of 5 min.

[0011] (2) Take the supernatant obtained in (1), discard the precipitate, add 0.01 ml of acetonitrile again, mix well, let it stand for 1 min, and centrifuge at 8000 r / min for a total of 5 min.

[0012] (3) Obtain the supernatant from (2) as the test sample and discard the precipitate.

[0013] 2. Using gold nanoparticles to construct an efficient SERS substrate

[0014] (1) Gold nanoparticles-sodium citrate suspension (1×10 8 -6×10 8 The mixture was centrifuged at a speed of 6000 r / min for 10 min, the supernatant was discarded, and anhydrous ethanol (the volume ratio of gold nanoparticles to anhydrous ethanol was 1:90-100) was added and mixed to form a suspension.

[0015] (2) Add 3 ml of ddH2O to the quartz dish, let it stand for 30 seconds, add 1 ml of cyclohexane to the upper layer of the liquid, let it stand for another 1 minute, and then separate the layers.

[0016] (3) Gently add the suspension obtained in (1) to the liquid layer in (2) and let it stand for 1 minute to form a gold nanoparticle film.

[0017] (4) Use a 0.5×0.5 cm clean silicon wafer to pick up the gold nanoparticle film and dry it to complete the substrate construction.

[0018] 3. Construction of quantitative curve

[0019] (1) Anhydrous ethanol and castor oil polyoxyethylene ether were mixed in a ratio of 2:1 at room temperature to construct a paclitaxel drug solvent.

[0020] (2) Dissolve 1 mg of paclitaxel in 1 ml of the solvent obtained in step (1) to prepare 1×10 -3 mol / L PTX standard solution; then gradient dilution to 1×10 -5 to 1×10 -11 mol / L PTX standard solution.

[0021] (3) Drop 1 μl of sample onto the constructed substrate and let it dry at room temperature.

[0022] (4) SERS detection was performed using a portable Raman spectrometer model RPB-785-1.5-FS. The excitation wavelength of the Raman spectrometer was 785 nm, the detection power was 30 mW, and the integration time was 0.5 s. The spectrum of the sample to be tested was obtained. The shift of paclitaxel was 1024 cm -1 , 1053cm -1 .

[0023] (5) Quantitative curves were drawn using the spectral data obtained from SERS detection of PTX standard solutions with different concentrations.

[0024] 4. SERS detection and data output

[0025] (1) Drop 1 μl of the sample to be tested onto the constructed substrate and let it dry at room temperature.

[0026] (2) SERS detection was performed using a portable Raman spectrometer model RPB-785-1.5-FS. The excitation wavelength of the Raman spectrometer was 785 nm, the detection power was 30 mW, and the integration time was 0.5 s. The spectrum of the sample to be tested was obtained and the observation was performed at 1024 cm -1 , 1053cm -1 The spectral peak at the shift.

[0027] (3) The measured spectral peaks were incorporated into the quantitative curve to obtain the PTX drug concentration.

[0028] A SERS substrate is prepared by the following method:

[0029] The concentration was 1×10 8 ~6×10 8The gold nanoparticle-sodium citrate suspension of 1000 nanoparticles / ml was centrifuged at 6000 rpm for 10 min, and the supernatant was discarded.

[0030] Anhydrous ethanol was added, with the volume ratio of gold nanoparticles to ethanol being 1:90-100, to form a suspension;

[0031] Add 3 ml of deionized water to the quartz dish, let it stand for 30 seconds, then add 1 ml of cyclohexane and let it stand for 1 minute to separate the layers;

[0032] The suspension was gently added to the liquid layer and allowed to stand for 1 min to form a gold nanoparticle film;

[0033] The membrane was picked up with a 0.5×0.5 cm silicon wafer and air-dried.

[0034] A paclitaxel blood concentration detection kit comprises: the above-mentioned SERS substrate, acetonitrile and paclitaxel quantitative curve data.

[0035] An application of the above-mentioned SERS substrate in preparing a paclitaxel detection device.

[0036] A real-time paclitaxel concentration detection system comprising:

[0037] The blood processing module performs the following steps:

[0038] Take 0.1 ml of blood sample, add 0.1 ml of acetonitrile, mix well, let stand for 1 min, centrifuge at 6000 r / min for 5 min, and collect the supernatant;

[0039] Add 0.01 ml of acetonitrile to the supernatant, mix well, let stand for 1 min, centrifuge at 8000 rpm for 5 min, and take the supernatant as the test sample;

[0040] SERS substrate construction module, perform the following steps:

[0041] The concentration was 1×10 8 ~6×10 8 The gold nanoparticle-sodium citrate suspension of 1000 nanoparticles / ml was centrifuged at 6000 rpm for 10 min, and the supernatant was discarded.

[0042] Anhydrous ethanol was added, with the volume ratio of gold nanoparticles to ethanol being 1:90-100, to form a suspension;

[0043] Add 3 ml of deionized water to the quartz dish, let it stand for 30 seconds, then add 1 ml of cyclohexane and let it stand for 1 minute to separate the layers;

[0044] The suspension was gently added to the liquid layer and allowed to stand for 1 min to form a gold nanoparticle film;

[0045] Use a 0.5 × 0.5 cm silicon wafer to pick up the membrane and air dry;

[0046] Portable Raman spectrometer;

[0047] The data processing unit outputs the paclitaxel concentration value based on the paclitaxel quantitative curve.

[0048] The beneficial effects of the present invention are:

[0049] (1) The construction method of the SERS detection substrate of the present invention is simple, time-saving, and easy to master.

[0050] (2) The serum sample processing method of the present invention is simple and portable, low in cost and effective.

[0051] (3) The present invention provides a novel SERS substrate and its detection method. This method is simple to operate, highly sensitive, and fast. It takes only 5 minutes from blood sampling to result output. In batch testing of multiple samples, the average time per sample is only about 2 minutes. It is widely applicable to the rapid and accurate detection of PTX blood concentration in clinical scenarios. BRIEF DESCRIPTION OF THE DRAWINGS

[0052] Figure 1 This is the SERS detection result of different concentrations of paclitaxel standards in Example 1.

[0053] Figure 2 This is the standard curve of paclitaxel blood concentration.

[0054] Figure 3 These are the SERS detection results of PTX concentrations in the serum of three clinical patients in Examples 1-3. DETAILED DESCRIPTION

[0055] 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.

[0056] Example 1:

[0057] Patient Wang xx, female, 55 years old, was diagnosed with breast cancer in May 2025 and underwent paclitaxel chemotherapy.

[0058] 1 ml of blood was collected 24 h after the patient received intravenous paclitaxel;

[0059] Add 1 ml of acetonitrile to the blood to be tested, mix well, let it stand for 1 minute, centrifuge at 6000 r / min for 5 minutes after obvious precipitation, discard the precipitate, add 1 ml of acetonitrile again, mix well, let it stand for 1 minute, centrifuge at 8000 r / min for 5 minutes, discard the precipitate again, and obtain the sample to be tested;

[0060] Gold nanoparticles-sodium citrate suspension (1×10 8 -6×10 8 The mixture was centrifuged at a speed of 6000 r / min for 10 min, the supernatant was discarded, and anhydrous ethanol (the volume ratio of gold nanoparticles to anhydrous ethanol was 1:90-100) was added and mixed to form a suspension;

[0061] Add 3 ml of ddH2O to the quartz dish, let it stand for 30 seconds, add 1 ml of cyclohexane to the upper layer of the liquid, let it stand for another 1 minute, and then separate the layers.

[0062] The obtained suspension was gently added to the liquid layer and allowed to stand for 1 min to form a gold nanoparticle film.

[0063] A 0.5×0.5 cm clean silicon wafer was used to remove the gold nanoparticle film and air dry it to complete the substrate construction.

[0064] Take 1 μl of the sample to be tested and drop it on the substrate.

[0065] The excitation wavelength of the Raman spectrometer was 785 nm, the detection power was 30 mW, the integration time was 0.5 s, and the spectrum of the sample to be tested was obtained. The observation was at 1024 cm -1 , 1053cm -1 The spectral peak at the shift (e.g. Figure 1 and Figure 2 As shown, Figure 1 This is the SERS detection result of different concentrations of paclitaxel standards in Example 1. Figure 2 is the standard curve of paclitaxel blood concentration); the peak value of the spectrum obtained by measurement is substituted into the quantitative curve formula, and the PTX drug concentration is calculated to be 5.5×10 -6 M.

[0066] Example 2:

[0067] Patient Li xx, female, 68 years old, was diagnosed with breast cancer in March 2025 and underwent paclitaxel chemotherapy.

[0068] 1 ml of blood was collected 24 h after the patient received intravenous paclitaxel;

[0069] 1 ml of acetonitrile was added to the blood to be tested, mixed and allowed to stand for 1 min, centrifuged after obvious precipitation appeared, centrifuged at 6000 r / min for 5 min, discarded the precipitate, added 1 ml of acetonitrile again, mixed and allowed to stand for 1 min, centrifuged at 8000 r / min for 5 min, discarded the precipitate again, and obtained the sample to be tested;

[0070] Gold nanoparticles-sodium citrate suspension (1×10 8 -6×108 The mixture was centrifuged at a speed of 6000 r / min for 10 min, the supernatant was discarded, and anhydrous ethanol (the volume ratio of gold nanoparticles to anhydrous ethanol was 1:90-100) was added and mixed to form a suspension;

[0071] Add 3 ml of ddH2O to the quartz dish, let it stand for 30 seconds, add 1 ml of cyclohexane to the upper layer of the liquid, let it stand for another 1 minute, and then separate the layers.

[0072] The obtained suspension was gently added to the liquid layer and allowed to stand for 1 min to form a gold nanoparticle film.

[0073] A 0.5×0.5 cm clean silicon wafer was used to remove the gold nanoparticle film and air dry it to complete the substrate construction.

[0074] Take 1 μl of the sample to be tested and drop it on the substrate;

[0075] The excitation wavelength of the Raman spectrometer was 785 nm, the detection power was 30 mW, the integration time was 0.5 s, and the spectrum of the sample to be tested was obtained. The observation was at 1024 cm -1 , 1053cm -1 spectral peak at the displacement;

[0076] The measured spectral peak was incorporated into the quantitative curve, and the PTX drug concentration was calculated to be 6.1×10 -6 M.

[0077] Example 3:

[0078] Patient Li xx, female, 61 years old, was diagnosed with breast cancer in March 2025 and underwent paclitaxel chemotherapy.

[0079] 1 ml of blood was collected 24 hours after the patient received intravenous paclitaxel;

[0080] 1 ml of acetonitrile was added to the blood to be tested, mixed and allowed to stand for 1 min, centrifuged after obvious precipitation appeared, centrifuged at 6000 r / min for 5 min, discarded the precipitate, added 1 ml of acetonitrile again, mixed and allowed to stand for 1 min, centrifuged at 8000 r / min for 5 min, discarded the precipitate again, and obtained the sample to be tested;

[0081] Gold nanoparticles-sodium citrate suspension (1×10 8 -6×10 8 The mixture was centrifuged at a speed of 6000 r / min for 10 min, the supernatant was discarded, and anhydrous ethanol (the volume ratio of gold nanoparticles to anhydrous ethanol was 1:90-100) was added and mixed to form a suspension;

[0082] Add 3 ml of ddH2O to the quartz dish, let it stand for 30 seconds, add 1 ml of cyclohexane to the upper layer of the liquid, let it stand for another 1 minute, and then separate the layers.

[0083] The obtained suspension was gently added to the liquid layer and allowed to stand for 1 min to form a gold nanoparticle film.

[0084] A 0.5×0.5 cm clean silicon wafer was used to remove the gold nanoparticle film and air dry it to complete the substrate construction.

[0085] Take 1 μl of the sample to be tested and drop it on the substrate;

[0086] The excitation wavelength of the Raman spectrometer was 785 nm, the detection power was 30 mW, the integration time was 0.5 s, and the spectrum of the sample to be tested was obtained. The observation was at 1024 cm -1 , 1053cm -1 spectral peak at the displacement;

[0087] The measured spectral peak was incorporated into the quantitative curve, and the PTX drug concentration was calculated to be 6.7×10 -6 M (such as Figure 3 As shown, Figure 3 (Examples 1-3, SERS detection results of PTX concentrations in serum of three clinical patients).

[0088] 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 SERS detection method for paclitaxel drug concentration in the blood of breast cancer patients, characterized in that: The following steps are involved: (1) Blood sample processing: Take 0.1 ml of blood sample, add 0.1 ml of acetonitrile, mix well, let stand for 1 min, centrifuge at 6000 r / min for 5 min, and collect the supernatant; Add 0.01 ml of acetonitrile to the supernatant, mix well, let stand for 1 min, centrifuge at 8000 rpm for 5 min, and take the supernatant as the test sample; (2) SERS substrate construction: The concentration was 1×10 8 ~6×10 8 The gold nanoparticle-sodium citrate suspension of 1000 nanoparticles / ml was centrifuged at 6000 rpm for 10 min, and the supernatant was discarded. Anhydrous ethanol was added, with the volume ratio of gold nanoparticles to ethanol being 1:90-100, to form a suspension; Add 3 ml of deionized water to the quartz dish, let it stand for 30 seconds, then add 1 ml of cyclohexane and let it stand for 1 minute to separate the layers; The suspension was gently added to the liquid layer and allowed to stand for 1 min to form a gold nanoparticle film; Use a 0.5 × 0.5 cm silicon wafer to pick up the membrane and air dry; (3) Detection and quantification: Take 1 μl of the test sample and drop it onto the SERS substrate and let it dry; Raman spectrometer was used to detect 1024 cm -1 and 1053cm -1 Characteristic peak intensity, where the Raman spectrometer uses an excitation wavelength of 785 nm, a power of 30 mW, and an integration time of 0.5 s; The concentration was calculated according to the pre-established paclitaxel quantitative curve.

2. The method according to claim 1, characterized in that The blood sample in step (1) is blood collected from a breast cancer patient 24 hours after intravenous infusion of paclitaxel.

3. The method according to claim 1, characterized in that The gold nanoparticles in step (2) have a particle size of 20-60 nm.

4. The method according to claim 1, wherein The Raman spectrometer in step (3) is a portable RPB-785-1.5-FS model.

5. The method according to claim 1, wherein The paclitaxel quantitative curve is constructed by the following steps: Prepare 1×10 -11 ~1×10 -5 mol / L paclitaxel standard solution; Take 1 μl of the standard and drop it on the SERS substrate. Detect 1024 cm -1 and 1053cm -1 Peak intensity; Draw a peak intensity-concentration standard curve.

6. The method according to claim 5, characterized in that The paclitaxel standard solution was a mixture of anhydrous ethanol and castor oil polyoxyethylene ether in a volume ratio of 2:

1.

7. A SERS substrate, characterized in that: Prepared by the following steps: The concentration was 1×10 8 ~6×10 8 The gold nanoparticle-sodium citrate suspension of 1000 nanoparticles / ml was centrifuged at 6000 rpm for 10 min, and the supernatant was discarded. Anhydrous ethanol was added, with the volume ratio of gold nanoparticles to ethanol being 1:90-100, to form a suspension; Add 3 ml of deionized water to the quartz dish, let it stand for 30 seconds, then add 1 ml of cyclohexane and let it stand for 1 minute to separate the layers; The suspension was gently added to the liquid layer and allowed to stand for 1 min to form a gold nanoparticle film; The membrane was picked up with a 0.5×0.5 cm silicon wafer and air-dried.

8. A paclitaxel blood concentration detection kit, characterized in that: Include: The SERS substrate according to claim 7; acetonitrile; Paclitaxel quantitative curve data.

9. Use of the SERS substrate according to claim 7 in preparing a paclitaxel detection device.

10. A real-time detection system for paclitaxel concentration, characterized in that: include: The blood processing module performs the following steps: Take 0.1 ml of blood sample, add 0.1 ml of acetonitrile, mix well, let stand for 1 min, centrifuge at 6000 r / min for 5 min, and collect the supernatant; Add 0.01 ml of acetonitrile to the supernatant, mix well, let stand for 1 min, centrifuge at 8000 rpm for 5 min, and take the supernatant as the test sample; SERS substrate construction module, perform the following steps: The concentration was 1×10 8 ~6×10 8 The gold nanoparticle-sodium citrate suspension of 1000 nanoparticles / ml was centrifuged at 6000 rpm for 10 min, and the supernatant was discarded. Anhydrous ethanol was added, with the volume ratio of gold nanoparticles to ethanol being 1:90-100, to form a suspension; Add 3 ml of deionized water to the quartz dish, let it stand for 30 seconds, then add 1 ml of cyclohexane and let it stand for 1 minute to separate the layers; The suspension was gently added to the liquid layer and allowed to stand for 1 min to form a gold nanoparticle film; Use a 0.5 × 0.5 cm silicon wafer to pick up the membrane and air dry; Portable Raman spectrometer; The data processing unit outputs the paclitaxel concentration value based on the paclitaxel quantitative curve.

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