Dynamic liquid integrated single cell surface enhanced Raman scattering detection platform

Abstract

A dynamic liquid integrated single cell surface enhanced Raman scattering (SERS) detection platform comprises a distortion mixing part, a 3D flow focusing part and a single cell SERS detection part which are connected in sequence, the twisting and mixing part is used for mixing inflowing cells and a multifunctional modified nano probe through a spirally twisted mixing micro-fluidic chip, and a mixed cell solution combined with the multifunctional modified nano probe is delivered to the 3D flow focusing part and is subjected to 3D focusing in the center of the 3D flow focusing part to form a single row; finally, flowing through a laser area of the single-cell SERS detection part, and collecting an SERS spectrum for detection; through the arrangement of the micro-fluidic chip and the coaxial needle, a cell solution and a multifunctional modified nano-probe solution are guided into a twisted mixing channel to be efficiently mixed and then focused, the sample treatment process is simplified, and the Raman detection reaches the single cell level in cooperation with the use of the multifunctional modified nano-probe; the method belongs to label-free detection, the requirement for pretreatment of the cells is reduced, the original state of the cells is kept, and downstream detection can be carried out; the kit has the advantages of high sensitivity, low cost, simplicity and convenience in operation, convenience in data analysis, cell viability maintenance and the like.

Description

Technical Field

[0001] The present invention belongs to the technical field of single molecule detection, and in particular relates to a dynamic liquid integrated single cell surface enhanced Raman scattering detection platform. Background Art

[0002] Single-cell analysis can visualize the phenotypic heterogeneity of disease-related tumors and may provide patients with highly personalized and accurate diagnostic information. At present, flow cytometry is often used for cell detection. This method has the characteristics of wide application range, high sensitivity and high throughput, and can meet the needs of quantitative cell analysis in many fields. However, flow cytometry mainly relies on fluorescent dyes to label cells and detect various parameters, which are labeling or invasive to cells, and fluorescent dyes may be affected by factors such as photobleaching and pH changes, resulting in signal attenuation or changes. As a high-precision instrument, flow cytometers are complex in structure, expensive, and require regular calibration and maintenance to ensure their performance and accuracy. When used in the laboratory, high requirements are placed on operators, who need to undergo special training.

[0003] Surface enhanced Raman scattering (SERS) is a vibrational spectroscopy-based technique that has the advantages of high sensitivity, non-invasiveness to biological samples, and resistance to photobleaching. It is considered a powerful technique for real-time detection of cancer cells at the single-cell level. Nanoprobes play a vital role in the SERS detection of cells. However, the main problem is that once nanoparticles enter biological buffers such as phosphate-buffered saline with high ionic strength and chloride ion content, ion exchange on their surface will cause particle aggregation, thereby reducing the sensitivity of SERS and making single-cell detection difficult.

[0004] Nanoprobes play a vital role in cell SERS detection. In the past few decades, based on the research of precious metal nanoprobes, such as: Jackson, HW; Fischer, JR; Zanotelli, VRT; Ali, HR; Mechera, R.; Soysal, SD; Moch, H.; Muenst, S.; Varga, Z.; Weber, WP;

[0005] Bodenmiller, B., The single-cell pathology landscape of breast cancer. Nature 2020, 578 (7796), 615-620. and literature Mardis, ER, DNA sequencing technologies: 2006–2016. Nature Protocols 2017, 12 (2), 213-218. etc., greatly improved the performance of SERS for single cells, so that cells can be manually detected one by one in a static manner on a micro-Raman spectroscopy device. However, due to the aggregation of nanoprobes in biological fluids and the low binding degree of nanoprobes to cells, the sensitivity of SERS detection is reduced, so it is difficult to achieve label-free detection at the single-cell level in dynamic liquids. Summary of the invention

[0006] In order to overcome the problems existing in the above-mentioned prior art, the purpose of the present invention is to provide a dynamic liquid-integrated single-cell surface-enhanced Raman scattering detection platform for label-free detection of cancer cells; through the setting of a microfluidic chip and a coaxial needle, the cell solution and the multifunctional modified nanoprobe solution are guided into a twisted mixing channel for efficient mixing and then focusing, thereby simplifying the sample processing process, and with the use of multifunctional modified nanoprobes, Raman detection reaches the single-cell level; the present invention belongs to label-free detection, reduces the need for cell pretreatment, maintains the original state of the cells, can perform downstream detection, and has the advantages of high sensitivity, low cost, simple operation, convenient data analysis and maintaining cell activity.

[0007] In order to achieve the above object, the technical solution adopted by the present invention is as follows:

[0008] A dynamic liquid integrated single-cell surface enhanced Raman scattering detection platform comprises a twisted mixing part 1, a 3D flow focusing part 2 and a single-cell SERS detection part 3 which are connected in sequence. The twisted mixing part 1 mixes the inflowing cells with a 1-1 multifunctionally modified nanoprobe through a spirally twisted mixing microfluidic chip 1-1. The cell solution combined with the multifunctionally modified nanoprobe after mixing flows to the 3D flow focusing part 2, is 3D focused into a single column at the center of the 3D flow focusing part 2, and finally flows through the laser area of ​​the single-cell SERS detection part 3 to collect its SERS spectrum for detection.

[0009] The twisted mixing part 1, the 3D flow focusing part 2 and the single cell SERS detection part 3 are connected in sequence through a soft silicone tube 4; the twisted mixing part 1 includes a spirally twisted mixing microfluidic chip 1-1, a synchronously spirally twisted hollow channel 1-2 is arranged in the middle of the spirally twisted mixing microfluidic chip 1-1, and a first inlet end 1-3 for injecting a cell solution and a second inlet end 1-4 for injecting a multifunctional modified nanoprobe solution are respectively arranged on both sides of the spirally twisted mixing microfluidic chip 1-1, and the first inlet end 1-3 and the second inlet end 1-4 merge into the hollow channel 1-2; a rectangular observation area 1-5 is arranged at the outlet end of the hollow channel 1-2 for taking fluorescent images and observing the state of the mixed solution.

[0010] The cross section of the hollow channel 1-2 of the spirally twisted hybrid microfluidic chip 1-1 is rectangular, trapezoidal, right-angled triangle or isosceles triangle, and the spiral twisting angle is 30° to 90°.

[0011] The optimal cross-section of the hollow channel 1-2 of the spirally twisted hybrid microfluidic chip 1-1 is an isosceles triangle, and the optimal twisting angle is 30°.

[0012] The spirally twisted hybrid microfluidic chip 1 - 1 is divided into n units, each unit is a complete spiral, and the length of each unit is 0.5-3 mm; the length of the spirally twisted hybrid microfluidic chip 1 - 1 is 3-24 mm.

[0013] The multifunctional modified nanoprobe is a Ag NSs surface modified with PEG and 3COOH (Ag NSs@PEG@3COOH), that is, the PEG and 3COOH molecules replace the CIT molecules on the Ag NSs surface.

[0014] The specific process of modifying PEG and 3COOH on the surface of Ag NSs is as follows:

[0015] In the first step, the surface of Ag NSs was modified with long PEG chain 5KDa PEG;

[0016] 1.1) Concentrating Ag NSs to 1.8-2 a.u.: Centrifuge the Ag NSs solution at 2500-3500 rpm for 13-15 minutes, remove the supernatant, and then measure the concentration of the Ag NSs solution using an ultraviolet-visible spectrometer (UV-Vis) until the absorbance of the solution reaches 1.8-2.2 au.

[0017] 1.2) preparing a 5-10 mM 5KDa PEG aqueous solution: taking 8-8.89 mL of the Ag NSs solution after the concentration in step 1.1), adding 10-20 μL of a 5-10 mM 5KDa PEG aqueous solution, stirring at 600-800 rpm for 1.3-1.5 hours to react, after the reaction is completed, centrifuging at 3000-3500 rpm for 13-15 minutes, removing the supernatant, and adding an equal volume of ultrapure water to the precipitated Ag NSs as the removed supernatant to redisperse the nanoparticles, and storing the modified Ag NSs solution in a refrigerator at 3-5°C in the dark for subsequent use;

[0018] In the second step, based on the modification of the Ag NSs surface with the long PEG chain of 5KDa, the AgNSs surface was modified with the short PEG chain of 550Da;

[0019] 2.1) Concentrating the modified AgNSs solution in step 1.2) to 1.8-2 a.u.: centrifuging the AgNSs solution at 3000-3500 rpm for 13-15 minutes, removing the supernatant, and then measuring the concentration of the AgNSs solution using an ultraviolet-visible spectrometer (UV-Vis) until the absorbance of the solution reaches 1.8-2 a.u.;

[0020] 2.2) preparing a 0.076-0.08 mM 5KDa PEG aqueous solution and a 9.924-10 mM 550Da PEG aqueous solution;

[0021] 2.2.1) Take 8-10 mL of the Ag NSs solution after the concentration in step 2.1), add 16-20 μL of 5 KDa PEG solution, and stir at 600-800 rpm for 1.3-1.5 hours to react; after the reaction is completed, centrifuge at 3000-3500 rpm for 13-15 minutes, remove the supernatant, and set the concentration of the obtained reactant to 1.8-2 a.u.;

[0022] 2.2.2) Short-chain PEG modification: 8-10 mL of the reaction product of step 2.2.1) was added with 16-20 μL of 550 Da PEG solution, and the mixture was stirred at 600-800 rpm for 1.3-1.5 hours for reaction; after the reaction, the mixture was centrifuged at 3000-3500 rpm for 13-15 minutes, the supernatant was removed to remove unreacted PEG molecules, and an equal volume of ultrapure water was added to the removed supernatant to disperse the Ag NSs@PEG solution, which was then stored in a refrigerator at 3-5°C away from light for subsequent use;

[0023] The third step is to modify the Ag NSs@PEG obtained in the second step with 3COOH to obtain Ag NSs@PEG@3COOH;

[0024] The concentration of the Ag NSs@PEG solution obtained in step 2.2.2) was measured using an ultraviolet-visible spectrometer (UV-Vis) until the absorbance of the solution reached 1.8-2 a.u.; 7.2-8 mL of the concentrated Ag NSs@PEG solution was taken into a glass bottle, 20-22 μL of 3COOH aqueous solution was added, and the mixture was stirred at 600-800 rpm for 25-30 minutes for reaction; after the reaction, the mixture was centrifuged at 3000-3500 rpm for 13-15 minutes, the supernatant was removed, and an equal volume of ultrapure water was added to the removed supernatant to obtain an AgNSs@PEG@3COOH solution, which was stored in a refrigerator at 3-5°C away from light.

[0025] The 3D flow focusing part 2 comprises a coaxial needle 2-1, which comprises a coaxially stacked inner needle 2-1-1 and an outer needle 2-1-2, wherein the size of the inner needle 2-1-1 is 22-30G (0.40×0.70mm-0.16×0.31mm), and the size of the outer needle 2-1-2 is 17-21G (0.10×1.50mm-0.50×0.80mm); the inlet end of the inner needle 2-1-1 is connected to the spiral twisted The end of the hollow channel 1-2 of the curved hybrid microfluidic chip 1-1 is connected to the nuclear fluid mixed with the circulating cells and the nanoprobes, so that the cells are 3D focused in a single column at the center of the channel of the inner needle; two side inlets 2-1-3 are provided on both sides of the inlet end of the outer needle 2-1-2 to introduce the outer sheath fluid; the nuclear fluid is surrounded by the outer sheath fluid to form a narrow column, and the flow rate ratio of the nuclear fluid to the sheath fluid is adjusted by setting the flow rate parameters of the injection pump to achieve 3D flow focusing of each cell in the middle of the channel.

[0026] When the flow rate ratio of the nuclear liquid to the sheath liquid is 1:1, it is a 3D flow focusing state. When focusing, the cells are restricted to a single cell narrow band, and when not focusing, the cells are dispersed in the entire channel.

[0027] The single-cell SERS detection part 3 includes a quartz or glass capillary and a Raman spectrometer; the quartz or glass capillary is connected to the end of the coaxial probe 2-1 through a silicone hose, and the cells combined with the nanoprobe flow through the quartz or glass capillary in a single column in the quartz or glass capillary. Whenever a cell passes through the laser area of ​​the Raman spectrometer, the Raman spectrometer will collect a cell spectrum to achieve single-cell detection.

[0028] Compared with the prior art, the present invention has the following beneficial effects.

[0029] 1. The present invention has high sensitivity: the dynamic liquid single-cell Raman detection platform uses multifunctional modified nanoprobes to improve the stability and sensitivity of the nanoprobes, and efficiently mixes cells and nanoprobes through twisted mixing microfluidic chips, so that Raman detection reaches the single-cell level.

[0030] 2. The dynamic liquid single-cell Raman detection platform of the present invention is constructed using a 3D printed bracket and a silicone hose, which is low in cost and easier to popularize.

[0031] 3. The dynamic liquid single-cell Raman detection platform of the present invention uses the design of microfluidic chips and coaxial needles to focus cells in a single row in the channel center of the inner needle in 3D; two side inlets are provided on both sides of the inlet end of the outer needle to introduce the outer sheath fluid; the nuclear fluid is surrounded by the outer sheath fluid to form a narrow column, and the flow rate ratio of the nuclear fluid to the sheath fluid is adjusted by setting the flow rate parameters of the injection pump to achieve 3D flow focusing of each cell in the middle of the channel, which simplifies the sample processing process and makes the operation easier.

[0032] 4. The dynamic liquid single-cell Raman detection platform uses a segmented weighted similarity KNN (SWS-KNN) model for the spectra collected by the Raman analyzer, which can more effectively classify the composite SERS spectra, facilitate data analysis, and achieve label-free single-cell detection.

[0033] 5. The dynamic liquid single-cell Raman detection platform reduces the need for cell pretreatment through label-free detection, maintains the original state and cell activity of the cells, and can be used for downstream detection.

[0034] 6. The multifunctional modified nanoprobe Ag NSs@PEG@3COOH prepared by the method of the present invention can be stably dispersed in biological fluids for at least 30 minutes, and has long-term stability and high SERS sensitivity.

[0035] In summary, the dynamic liquid single-cell Raman detection platform has the advantages of high sensitivity, low cost, easy operation, convenient data analysis and maintaining cell activity. It can effectively reduce cost and time and provide a new solution for cell detection in actual sample applications. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for use in the embodiments of the present invention. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.

[0037] Figure 1 Schematic diagram of the structure of the DLISC-SERS platform for single cell detection of the present invention.

[0038] Figure 2 The design and assembly diagram of the DLISC-SERS platform for single-cell detection of the present invention; wherein: (a) is a schematic diagram of the principle of the developed platform; (b) is a real photo of the assembled platform, with a scale of 10 mm; (c) is the twisting process of a single channel unit of the twisted mixing microfluidic chip; (d) is a schematic diagram of the multifunctional modified nanoprobe.

[0039] Figure 3 The present invention is a schematic diagram of the design of a twisted mixing channel and the mixing performance of the twisted mixing microfluidic chip; wherein: (a) are three important parameters in designing a twisted mixing channel; (b) is the mixing effect of a twisted mixing channel with different i) cross-sectional shapes, ii) twist angles and iii) unit lengths; (c, d, e) are the corresponding normalized mixing indexes; (f) is a fluorescence mixing image and fluorescence intensity statistical graph of the chip inlet and visible area corresponding to lengths 1 to 5 of 3 mm, 7 mm, 12 mm, 18 mm, and 24 mm, respectively; the scale bar is 0.5 mm.

[0040] Figure 4 Schematic diagram of the performance of the multifunctional modified nanoprobe of the present invention; (a) TEM of Ag NSs, (b) Ag NSs@PEG and (c) Ag NSs@PEG@3COOH, all scale bars are 200 nm; (d) normalized UV-visible absorbance spectra of Ag NSs and Ag NSs@PEG with three different PEG concentrations; (e) zeta potential of Ag NSs, Ag NSs@PEG and Ag NSs@PEG@3COOH; (f) modification differences of different ratios; (g) 1174 cm-1 at different pH values. -1 The integrated area intensity at 1391 cm, n = 30; (h) is the SERS spectra of cancer cells with direct mixing and twisted mixing; (i) is the SERS spectra of direct mixing and twisted mixing at 1391 cm -1 The integrated area intensity at 1391 cm, n = 30; (j) is a photograph of a twisted mixing microfluidic chip with five different channel lengths (scale bar is 10 mm); (k) is the SERS spectra of cancer cells in a twisted mixing microfluidic chip with five different channel lengths and (l) is the SERS spectra of cancer cells in a twisted mixing microfluidic chip with five different channel lengths at 1391 cm -1 The integrated area intensity at .

[0041] Figure 5Schematic diagram for verification of 3D hydrodynamic focusing of cells on the platform of the present invention; wherein, (a) is a schematic diagram of 3D hydrodynamic focusing; (b) is a top view and (e) is a superimposed cell microscopic image in a side view; (c) is a top view and (f) is a stacked fluorescence image of particle trajectories in a side view; the scale bar is 100 μm; (d) is the corresponding normalized fluorescence distribution (black) and Gaussian equation line (red) in the top view and (g) in the side view.

[0042] Figure 6 Schematic diagram of experimental verification of the digital discretization method of the DLISC-SERS platform of the present invention; (a) is a schematic diagram of single cell detection; (b) is the classification of blank spectra and cell spectra using the SWS-KNN model; before SWS-KNN classification, the spectra were preprocessed by background correction, normalization, blank subtraction and Min-Max normalization; (c) is the experimental verification of the digital discretization method of the DLISC-SERS platform of the present invention; (a) is a schematic diagram of single cell detection; (b) is the classification of blank spectra and cell spectra using the SWS-KNN model; (c) is the experimental verification of the digital discretization method of the DLISC-SERS platform of the present invention; (d) is the experimental verification of the digital discretization method of the DLISC-SERS platform of the present invention; (e) is the experimental verification of the digital discretization method of the DLISC-SERS platform of the present invention; (f) is the experimental verification of the digital discretization 0 Three groups of classification results. Each stripe represents a blank spectrum (light blue) or a cell spectrum (blue); (d) contains different λ 0 The average count of the cell information spectrum, n = 3; (e) is the experimental λ t With preset λ 0 There is a linear relationship between them, n=3.

[0043] Figure 7 Schematic diagram of label-free dynamic single-cell detection of mixed samples on the DLISC-SERS platform of the present invention; (a) is the workflow of single-cell SERS detection and spectral classification; (b) is the performance evaluation using the confusion matrix; (c) is the classification result between the three groups, λ 0 =0.16, each stripe represents a blank spectrum (light blue), a breast cancer cell spectrum (blue) or a mammary gland cell spectrum (orange); (d) is the count of the breast cancer cell spectrum and the mammary gland cell spectrum in each group. (e) is the ratio of the spectral counts of the two cells in each group. The red dotted line represents the average of the three groups of ratios.

[0044] In the figure: 1. twisted mixing part; 2. 3D flow focusing part; 3. single cell SERS detection part; 4. soft silicone tube; 1-1. spirally twisted mixing microfluidic chip; 1-2. spirally twisted hollow channel; 1-3. first inlet end; 1-4. second inlet end; 1-5. rectangular observation area; 2-1. coaxial needle; 2-1-1. inner needle; 2-1-2. outer needle; 2-1-3. side inlet. DETAILED DESCRIPTION

[0045] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0046] The present invention is described in detail below in conjunction with the accompanying drawings.

[0047] Example 1

[0048] like Figure 1-Figure 4 As shown, a dynamic liquid integrated single-cell surface enhanced Raman scattering detection platform comprises a twisted mixing part 1, a 3D flow focusing part 2 and a single-cell SERS detection part 3 which are connected in sequence. The twisted mixing part 1 mixes the inflowing cells with the multifunctional modified nanoprobes through a spirally twisted mixing microfluidic chip 1-1. The cell solution combined with the multifunctional modified nanoprobes after mixing flows to the 3D flow focusing part 2, is 3D focused into a single column at the center of the 3D flow focusing part 2, and finally flows through the laser area of ​​the single-cell SERS detection part 3 to collect its SERS spectrum for detection.

[0049] The twisted mixing part 1, the 3D flow focusing part 2 and the single cell SERS detection part 3 are connected in sequence through a soft silicone tube 4; the twisted mixing part 1 includes a spirally twisted mixing microfluidic chip 1-1, a synchronously spirally twisted hollow channel 1-2 is arranged in the middle of the spirally twisted mixing microfluidic chip 1-1, and a first inlet end 1-3 for injecting a cell solution and a second inlet end 1-4 for injecting a multifunctional modified nanoprobe solution are respectively arranged on both sides of the spirally twisted mixing microfluidic chip 1-1, and the first inlet end 1-3 and the second inlet end 1-4 merge into the hollow channel 1-2; a rectangular observation area 1-5 is arranged at the outlet end of the hollow channel 1-2 for taking fluorescent images and observing the state of the mixed solution.

[0050] The spirally twisted hybrid microfluidic chip 1-1 is as follows Figure 3 As shown in (a), the cross section of the hollow channel 1-2 is an isosceles triangle, and the spiral twist angle is 30°-90°. Compared with the mixing channels with rectangular, trapezoidal, right-angled triangle and isosceles triangle cross sections, Figure 3 (b)i) shows the channel stretching degree of four cross-sectional shape designs: rectangular, trapezoidal, right-angled triangle, and isosceles triangle. The simulation analyzes the influence of different twist angles such as 30°, 45°, 60°, and 90° on the mixing index, such as Figure 3 (b) as shown in ii). Figure 3 As shown in (b) iii), mixing can be achieved when the unit length is 0.5mm, 1mm, 2mm, and 3mm. The mixing index of the four cross-sectional shapes is as follows Figure 3The mixing index of the triangular cross section shown in (c) is significantly higher than that of the rectangular cross section and the trapezoidal cross section, and the mixing index of the isosceles triangle channel outlet cross section is only 0.00004 higher than that of the right triangle. It can be found that in the twisted mixing channel with a length of 24 mm, the isosceles triangle cross-section channel can achieve full mixing of the two concentration solutions, and the mixing index is as high as 0.9984.

[0051] The optimal cross-section of the hollow channel 1-2 of the spirally twisted hybrid microfluidic chip 1-1 is an isosceles triangle, and the optimal twist angle is 30°. Figure 3 As shown in (d), the twist angles of 30°, 45°, 60°, and 90° can all achieve mixing, and the best mixing effect is achieved at 30°. Figure 3 As shown in (e), when the unit length is greater than 1 mm, the mixing index gradually decreases with the increase of the unit length. When the unit length is less than or equal to 1 mm, the mixing index remains basically unchanged. 3D printing technology adopts a layer-by-layer manufacturing method. In order to avoid the influence of the degree of distortion on the 3D printing preparation effect, a channel structure with a unit length of 1 mm is finally selected. Figure 3 (f) shows mixing experiments using fluorescein solution on the developed microfluidic chip with different channel lengths to confirm the theoretical prediction. The white dashed line represents the inner wall of the channel with a spacing of 1000 μm, and the red dashed line represents the location of the fluorescence distribution statistics. The fluorescence distribution shows that the mixing effect is best when the channel length is 24 mm.

[0052] The spirally twisted hybrid microfluidic chip 1-1 is divided into n units, each unit being a complete spiral; each unit is 1 mm long, and the spirally twisted hybrid microfluidic chip is 24 mm long.

[0053] The multifunctional modified nanoprobe is a Ag NSs surface modified with PEG and 3COOH (Ag NSs@PEG@3COOH), that is, the PEG and 3COOH molecules replace the CIT molecules on the Ag NSs surface, and the modified 3COOH provides carboxyl groups on the nanoprobe surface to improve the binding of the nanoprobe to cells.

[0054] In a preferred embodiment of the present invention, the specific process of modifying the surface of Ag NSs with PEG and 3COOH is as follows:

[0055] In the first step, the surface of Ag NSs was modified with long PEG chain 5KDa PEG;

[0056] 1.1) Concentrating Ag NSs to 2 a.u.: Centrifuge the Ag NSs solution at 3000 rpm for 15 minutes, remove the supernatant, and then measure the concentration of the Ag NSs solution using an ultraviolet-visible spectrometer (UV-Vis) until the absorbance of the solution reaches 2 a.u.;

[0057] 1.2) Prepare 5 mM 5KDa PEG aqueous solution: Take the Ag solution after concentration in step 1.1)

[0058] 8 mL of NSs solution was added with 40 μL of 5 mM 5 KDa PEG aqueous solution and incubated at 800

[0059] The mixture was stirred at 3000 rpm for 3 hours to achieve the binding of PEG molecules to the surface of Ag NSs. After the reaction, the mixture was centrifuged at 3000 rpm for 15 minutes to remove the supernatant to remove the unreacted PEG molecules. Ultrapure water with an equal volume to the removed supernatant was added to the precipitated Ag NSs to redisperse the nanoparticles. The modified Ag NSs solution was stored in a refrigerator at 3-5°C away from light for subsequent use.

[0060] In the second step, based on the modification of the Ag NSs surface with the long PEG chain of 5KDa, the AgNSs surface was modified with the short PEG chain of 550Da;

[0061] 2.1) Concentrating the modified AgNSs solution in step 1.2) to 2 a.u.: centrifuging the AgNSs solution at 3000 rpm for 15 minutes, removing the supernatant, and then measuring the concentration of the AgNSs solution using an ultraviolet-visible spectrometer (UV-Vis) until the absorbance of the solution reaches 2 a.u.;

[0062] 2.2) preparing 0.076 mM 5KDa PEG aqueous solution and 9.924 mM 550Da PEG aqueous solution;

[0063] 2.2.1) Take 8 mL of the Ag NSs solution after the concentration in step 2.1), add 20 μL of 5 KDa PEG solution (0.076 mM), and stir at 800 rpm for 1 hour and 15 minutes to react; after the reaction is completed, centrifuge at 3000 rpm for 15 minutes, remove the supernatant, and set the concentration of the obtained reactant to 1.8 au;

[0064] 2.2.2) Short-chain PEG modification: 8 mL of the reaction product prepared in step 2.2.1) was added with 20 μL of 550 Da PEG solution (9.924 mM), and the mixture was stirred at 800 rpm for 1 hour and 15 minutes for reaction; after the reaction, the mixture was centrifuged at 3000 rpm for 15 minutes, the supernatant was removed to remove unreacted PEG molecules, and an equal volume of ultrapure water was added to the supernatant to disperse the Ag NSs@PEG solution, which was then stored in a refrigerator at 3-5°C away from light for subsequent use;

[0065] The third step is to modify the Ag NSs@PEG obtained in the second step with 3COOH to obtain Ag NSs@PEG@3COOH;

[0066] The concentration of the Ag NSs@PEG solution obtained in step 2.2.2) was measured using an ultraviolet-visible spectrometer (UV-Vis) until the absorbance of the solution reached 1.8 au; 8 mL of the concentrated Ag NSs@PEG solution was taken into a small glass bottle with a volume of 20 mL, 20 μL of 3COOH aqueous solution (20 mM) was added, and the mixture was stirred at 800 rpm for 30 minutes for reaction; after the reaction was completed, centrifuged at 3000 rpm for 15 minutes, the supernatant was removed, and an equal volume of ultrapure water was added to the removed supernatant to obtain an Ag NSs@PEG@3COOH solution, which was stored in a refrigerator at 3-5°C away from light.

[0067] like Figure 4 As shown in (ac), the size and structure of Ag NSs, Ag NSs@PEG, and Ag NSs@PEG@3COOH were characterized by TEM. Figure 4 (d) shows the UV absorption spectra of AgNSs before and after modification when the PEG concentration is 0.1mM, 1mM, and 10mM. The UV absorption peak of AgNSs is located at a wavelength of 384.25nm. As the PEG modification concentration increases, the UV absorption spectrum undergoes an obvious red shift. Figure 4 As shown in (e), PEG and 3COOH molecules replaced the CIT on the surface of Ag NSs, and the electronegativity gradually weakened during the PEG and 3COOH modification. Figure 4 As shown in (f), the stability of the nanoprobes modified with different ratios in PBS was statistically analyzed. When the ratio of 5KDa PEG:550DaPEG was 1:130 and the ratio of 3COOH:PEG was 2:1, the stability of Ag NSs@PEG@3COOH in PBS was the best. Figure 4 (g) shows the effect of pH environment on the SERS detection performance of this experiment. The SERS performance is best when the pH is 8. Figure 4 (h) Comparison of SERS spectra of MDA-MB-231 cells in straight channel mixing and twisted mixing. Figure 4 As shown in (i), after twist mixing, 1391 cm -1 The integrated peak area at is about twice that of the straight mixing. Figure 4 As shown in (j), five twisted mixing microfluidic chips with different lengths were designed and prepared. Figure 4 (k) shows the average SERS spectra of malignant breast cancer cells detected by chips of different lengths. Figure 4 (l) shows 1391cm -1The normalized integrated area intensity at 1391 cm is the best when the length is 5. -1 The normalized intensity of the characteristic peak at is 0.98174 au.

[0068] The 3D flow focusing part 2 comprises a coaxial needle 2-1, and the coaxial needle 2-1 comprises an inner needle 2-1-1 and an outer needle 2-1-2 which are coaxially stacked. Figure 5 As shown in (a), the size of the inner needle 2-1-1 is 30G (0.16×0.31mm), and the size of the outer needle 2-1-2 is 21G (0.50×0.80mm); the inlet end of the inner needle 2-1-1 is connected to the end of the hollow channel 1-2 of the spirally twisted hybrid microfluidic chip to flow the nuclear fluid mixed with the cells and the nanoprobes, so that the cells are 3D focused in the center of the channel of the inner needle in a single column; two side inlets 2-1-3 are provided on both sides of the inlet end of the outer needle 2-1-2 to introduce the outer sheath fluid; the nuclear fluid is surrounded by the outer sheath fluid to form a narrow column, and the flow rate ratio of the nuclear fluid to the sheath fluid is adjusted by setting the flow rate parameters of the injection pump to achieve 3D flow focusing of each cell in the middle of the channel.

[0069] like Figure 5 As shown in (b), a top-down photo of a single cell passing through a glass capillary was taken with a high-speed camera and superimposed. Figure 5 As shown in (c), after the cells were stained with FDA, they were observed to flow in a single column in a glass capillary under an inverted microscope. Figure 5 As shown in (d), the fluorescence distribution of the red dashed line passing through the observation area is abstracted and normalized to obtain a fitted Gaussian curve. The full width at half maximum (FWHM) of the Gaussian curve is 68.87 μm. In the top view, the cell can be focused in the middle of the channel. Figure 5 As shown in (eg), as in the top view case, the cell can also reach a stable focusing state under the side view case, and the full width at half maximum of the Gaussian curve is 84.47 μm.

[0070] When the flow rate ratio of the nuclear liquid to the sheath liquid is 1:1, it is a 3D flow focusing state. When focusing, the cells are restricted to a single cell narrow band, and when not focusing, the cells are dispersed in the entire channel.

[0071] The single-cell SERS detection part includes a quartz capillary and a Raman spectrometer; the quartz capillary is connected to the end of the 2-1 of the coaxial probe through a silicone hose, and the cells combined with the nanoprobe flow through the quartz capillary in a single column in the quartz capillary. Whenever a cell passes through the laser area of ​​the Raman spectrometer, the Raman spectrometer will collect a cell spectrum to achieve single-cell detection.

[0072] Example 2

[0073] In the second embodiment of the present invention, the spirally twisted hybrid microfluidic chip 1-1 is divided into n units, each unit is a complete spiral; each unit is 0.5 mm long, and the spirally twisted hybrid microfluidic chip is 3 mm long.

[0074] The specific process of modifying PEG and 3COOH on the surface of Ag NSs is as follows:

[0075] In the first step, the surface of Ag NSs was modified with long PEG chain 5KDa PEG;

[0076] 1.1) Concentrating Ag NSs to 1.8 au: Centrifuge the Ag NSs solution at 2500 rpm for 13 minutes, remove the supernatant, and then measure the concentration of the Ag NSs solution using an ultraviolet-visible spectrometer (UV-Vis) until the absorbance of the solution reaches 1.8 au;

[0077] 1.2) Prepare 5 mM 5KDa PEG aqueous solution: Take the Ag solution after concentration in step 1.1)

[0078] 8 mL of NSs solution was added with 10 μL of 5 mM 5 KDa PEG aqueous solution and incubated at 600

[0079] The reaction was stirred at 3000 rpm for 1.3 hours. After the reaction was completed, the mixture was centrifuged at 3000 rpm for 13 minutes, the supernatant was removed, and ultrapure water with an equal volume to the removed supernatant was added to the precipitated Ag NSs to redisperse the nanoparticles. The modified Ag NSs solution was stored in a refrigerator at 3-5°C away from light for subsequent use.

[0080] In the second step, based on the modification of the Ag NSs surface with the long PEG chain of 5KDa, the AgNSs surface was modified with the short PEG chain of 550Da;

[0081] 2.1) Concentrating the modified Ag NSs solution in step 1.2) to 1.8 au: centrifuging the Ag NSs solution at 3000 rpm for 13 minutes, removing the supernatant, and then measuring the concentration of the Ag NSs solution using an ultraviolet-visible spectrometer (UV-Vis) until the absorbance of the solution reaches 1.8 au;

[0082] 2.2) preparing 0.076 mM 5KDa PEG aqueous solution and 9.924 mM 550Da PEG aqueous solution;

[0083] 2.2.1) Take 8-10 mL of the Ag NSs solution after the concentration in step 2.1), add 16-20 μL of 5 KDa PEG solution, and stir at 600 rpm for 1.3 hours to react; after the reaction is completed, centrifuge at 3000 rpm for 13 minutes, remove the supernatant, and set the concentration of the obtained reactant to 1.8 au;

[0084] 2.2.2) Short-chain PEG modification: 8 mL of the reaction product prepared in step 2.2.1) was added with 16 μL of 550 Da PEG solution, and the mixture was stirred at 600 rpm for 1.3 hours for reaction; after the reaction, the mixture was centrifuged at 3000 rpm for 13 minutes, the supernatant was removed to remove unreacted PEG molecules, and an equal volume of ultrapure water was added to the supernatant to disperse the Ag NSs@PEG solution, which was then stored in a refrigerator at 3-5°C away from light for subsequent use;

[0085] The third step is to modify the Ag NSs@PEG obtained in the second step with 3COOH to obtain Ag NSs@PEG@3COOH;

[0086] The concentration of the Ag NSs@PEG solution obtained in step 2.2.2) was measured using an ultraviolet-visible spectrometer (UV-Vis) until the absorbance of the solution reached 1.8 au; 7.2 mL of the concentrated Ag NSs@PEG solution was taken into a glass bottle, 20 μL of 3COOH aqueous solution was added, and the mixture was stirred at 600 rpm for 25 minutes for reaction; after the reaction, the mixture was centrifuged at 3000 rpm for 13 minutes, the supernatant was removed, and an equal volume of ultrapure water was added to the removed supernatant to obtain an Ag NSs@PEG@3COOH solution, which was stored in a refrigerator at 3-5°C away from light.

[0087] The 3D flow focusing part 2 includes a coaxial needle 2-1, which includes a coaxially stacked inner needle 2-1-1 and an outer needle 2-1-2, the size of the inner needle 2-1-1 is 22G (0.40×0.70mm), and the size of the outer needle 2-1-2 is 17G (0.10×1.50mm); the inlet end of the inner needle 2-1-1 is connected to the end of the hollow channel 1-2 of the spirally twisted hybrid microfluidic chip to flow the nuclear fluid mixed with the nanoprobe, so that the cells are 3D focused in the channel center of the inner needle in a single column; two side inlets 2-1-3 are provided on both sides of the inlet end of the outer needle 2-1-2 to introduce the outer sheath fluid; the nuclear fluid is surrounded by the outer sheath fluid to form a narrow column, and the flow rate ratio of the nuclear fluid to the sheath fluid is adjusted by setting the flow rate parameters of the injection pump to achieve 3D flow focusing of each cell in the middle of the channel.

[0088] Example 3

[0089] In the third embodiment of the present invention, the spirally twisted hybrid microfluidic chip 1-1 is divided into n units, each unit is a complete spiral; each unit is 3 mm long, and the spirally twisted hybrid microfluidic chip is 24 mm long.

[0090] The specific process of modifying PEG and 3COOH on the surface of Ag NSs is as follows:

[0091] In the first step, the surface of Ag NSs was modified with long PEG chain 5KDa PEG;

[0092] 1.1) Concentrating Ag NSs to 2 a.u.: Centrifuge the Ag NSs solution at 3500 rpm for 15 minutes, remove the supernatant, and then measure the concentration of the Ag NSs solution using an ultraviolet-visible spectrometer (UV-Vis) until the absorbance of the solution reaches 2.2 au.

[0093] 1.2) Prepare 10mM 5KDa PEG aqueous solution: Take the Ag solution after concentration in step 1.1)

[0094] 8-8.89 mL of NSs solution, add 20 μL of 5-10 mM 5KDa PEG aqueous solution, stir at 800 rpm for 1.5 hours to react, after the reaction is completed, centrifuge at 3500 rpm for 15 minutes, remove the supernatant, and add ultrapure water of the same volume as the removed supernatant to the precipitated Ag NSs to redisperse the nanoparticles, and store the modified Ag NSs solution in a 3-5°C refrigerator away from light for subsequent use;

[0095] In the second step, based on the modification of the Ag NSs surface with the long PEG chain of 5KDa, the AgNSs surface was modified with the short PEG chain of 550Da;

[0096] 2.1) Concentrating the modified AgNSs solution in step 1.2) to 2 a.u.: centrifuging the AgNSs solution at 3500 rpm for 15 minutes, removing the supernatant, and then measuring the concentration of the AgNSs solution using an ultraviolet-visible spectrometer (UV-Vis) until the absorbance of the solution reaches 2 a.u.;

[0097] 2.2) preparing 0.08 mM 5KDa PEG aqueous solution and 10 mM 550Da PEG aqueous solution;

[0098] 2.2.1) Take 10 mL of the Ag NSs solution after the concentration in step 2.1), add 20 μL of 5 KDa PEG solution, and stir at 800 rpm for 1.5 hours to react; after the reaction is completed, centrifuge at 3500 rpm for 15 minutes, remove the supernatant, and set the concentration of the obtained reactant to 2 a.u.;

[0099] 2.2.2) Short-chain PEG modification: 10 mL of the reaction product prepared in step 2.2.1) was added with 20 μL of 550 Da PEG solution, and the mixture was stirred at 800 rpm for 1.5 hours for reaction; after the reaction, the mixture was centrifuged at 3500 rpm for 15 minutes, the supernatant was removed to remove unreacted PEG molecules, and an equal volume of ultrapure water was added to the supernatant to disperse the AgNSs@PEG solution, which was then stored in a refrigerator at 3-5°C away from light for subsequent use;

[0100] The third step is to modify the Ag NSs@PEG obtained in the second step with 3COOH to obtain Ag NSs@PEG@3COOH;

[0101] The concentration of the Ag NSs@PEG solution obtained in step 2.2.2) was measured using an ultraviolet-visible spectrometer (UV-Vis) until the absorbance of the solution reached 2 a.u.; 8 mL of the concentrated Ag NSs@PEG solution was taken into a glass bottle, 22 μL of 3COOH aqueous solution was added, and the mixture was stirred at 800 rpm for 30 minutes for reaction; after the reaction, the mixture was centrifuged at 3500 rpm for 15 minutes, the supernatant was removed, and an equal volume of ultrapure water was added to the removed supernatant to obtain an Ag NSs@PEG@3COOH solution, which was stored in a refrigerator at 3-5°C away from light.

[0102] The 3D flow focusing part 2 includes a coaxial needle 2-1, which includes a coaxially stacked inner needle 2-1-1 and an outer needle 2-1-2, the size of the inner needle 2-1-1 is 30G (0.16×0.31mm), and the size of the outer needle 2-1-2 is 21G (0.50×0.80mm); the inlet end of the inner needle 2-1-1 is connected to the end of the hollow channel 1-2 of the spirally twisted hybrid microfluidic chip 1-1 to flow the nuclear fluid mixed with the cells and the nanoprobes, so that the cells are 3D focused in the channel center of the inner needle in a single row; two side inlets 2-1-3 are provided on both sides of the inlet end of the outer needle 2-1-2 to introduce the outer sheath fluid; the nuclear fluid is surrounded by the outer sheath fluid to form a narrow column, and the flow rate ratio of the nuclear fluid to the sheath fluid is adjusted by setting the flow rate parameters of the injection pump to achieve 3D flow focusing of each cell in the middle of the channel.

[0103] SERS measurements

[0104] When the cross-sectional shape is an isosceles triangle, the twist angle is 30°, the unit length is 1 mm, and the channel length is 24 mm, the mixing index of the helically twisted hybrid microfluidic chip 1-1 reaches 0.85. When the ratio of 5KDa PEG to 550Da PEG is 1:130 and the ratio of 3COOH to PEG is 2:1, the multifunctional modified nanoprobe Ag NSs@PEG@3COOH has high stability and sensitivity. When the nanoprobe concentration is 16 a.u. and the flow rate is 20 μL / min, breast cancer cells are detected at 1391 cm -1 The normalized intensity of the characteristic peak at reached 0.98174 au. The dynamic liquid single-cell SERS detection was analyzed using a digital discretization method. A KNN (SWS-KNN) model based on segmented weighted similarity was established for SERS spectrum classification, with a sensitivity, specificity and accuracy of 100%, 99.4% and 99.5%, respectively. The platform can be used for label-free detection of mixed samples containing two cells at the single-cell level. The three-way classification accuracy of the SWS-KNN model is 95.2%. The dynamic liquid integrated single-cell SERS platform of the present invention is a powerful tool for detecting cancer cells at the single-cell level.

[0105] The SERS signal was detected by Finder One Raman spectrometer (Zhuoli Hanguang, Beijing). Subsequent experiments used a 532nm laser, 55mW power, and 10× objective lens. The single-cell SERS detection conditions were an integration time of 1s, a measurement interval of 0.01s, and 50 spectra were collected continuously. Ag NSs@PEG@3COOH was concentrated to 32a.u., and then PBS powder was added. Na 2 HPO 4 The solution adjusts the pH value of the Ag NSs@PEG@3COOH solution to . The Ag NSs@PEG@3COOH solution and the cell solution mixed by the twisted mixing microfluidic chip are the core solution, and PBS is the sheath solution. The volume flow rates of the core solution and the sheath solution are set to 3.636μL / min and 16.364μL / min, respectively, and the total volume flow rate of the SERS detection area is 20μL / min. After entering the twisted mixing microfluidic chip, the Ag NSs@PEG@3COOH and the cell solution will dilute each other by half.

[0106] The optimized inner needle size of the coaxial needle is 22G (0.40×0.70 mm), and the outer needle size is 17G (0.10×1.50 mm). Subsequent experiments were also conducted on this platform of the present invention.

[0107] The concentration was 5.28×10 3 mL -1Breast cancer cell (MDA-MB-231) and mammary gland cell (MCF-10A) solutions were used for label-free detection of single cancer cells in mixed samples. Mixed samples were obtained by mixing equal volumes with a preset nuclear flux of λ=0.16. Three groups of mixed sample spectra were collected, with 100 spectra in each group. In theory, each group of collected spectra contains 15 cell spectra, and the average count ratio of breast cancer cell spectra to mammary gland cell spectra is close to 1:1.

[0108] Data processing and analysis

[0109] The mixing index data derived from the CFD simulation results were evaluated using the statistical software OriginPro 2017 (OriginLab, Massachusetts, USA). All fluorescence images were analyzed using Image-Pro Plus 6.0 (Media Cybernetics, USA). They were stacked into one image, and the fluorescence distribution was extracted and evaluated as in our previous work30.

[0110] SERS spectra were preprocessed and analyzed using PyCharm 2020 (JetBrains, Czech Republic) and MATLAB R2017a (MathWorks, USA). Each spectrum was restricted to 600 cm -1 Up to 1800cm -1 The preprocessing of the spectra included background correction, normalization, blank subtraction, and min-max normalization using the adaptive iterative weighted penalized least squares (airPLS) algorithm.

[0111] The SWS-KNN algorithm is used to classify the preprocessed SERS spectra. Set λ 0 (0.1, 0.16, 0.2) corresponding to different nuclear liquid cell concentrations (1.65×10 3 , 2.64×10 3 , 3.3×10 3 mL -1), and a series of experiments were conducted. Some of the collected spectra were manually labeled as blank, breast cancer cells, and mammary cells, and spectra that were difficult to identify were removed. The two-way classification SWS-KNN model for blank and cell spectra contains a training library of 683 spectra and a test library of 188 spectra. The training library contains 602 blank spectra and 81 cell spectra, and the test library contains 155 blank spectra and 33 cell spectra. The three-way classification SWS-KNN model for mixed samples contains a training library of 726 spectra and a test library of 210 spectra. The training library includes 602 blank spectra, 81 breast cancer cell spectra, and 43 mammary cell spectra, while the test library includes 155 blank spectra, 33 breast cancer cell spectra, and 22 mammary cell spectra. The classification performance of each SWS-KNN model was evaluated using its confusion matrix.

[0112] like Figure 6 As shown in (a), most of the detected spectra are blank spectra. When cells flow through the laser area, cell spectra different from the blank spectra are detected. Figure 6 As shown in (b), the SWS-KNN algorithm is used to classify blank cells and breast cancer cells, and the sensitivity, specificity and accuracy of the confusion matrix reach 100%, 99.4% and 99.5% respectively. Figure 6 As shown in (c), the vertical lines of two colors represent different preset λ 0 Each light blue vertical line represents a blank spectrum, and each blue vertical line represents a cell (MDA-MB-231) spectrum. Figure 6 (d) shows the count of all spectra collected for each group as 100 and shows the 0 The average count of the cell spectrum. Figure 6 As shown in (e), the experimental λ t With the preset λ 0 By comparing the plots, we can get λ t With λ 0 There is an obvious linear relationship between 2 is 0.983).

[0113] like Figure 7 As shown in (a), the hybrid cells and Ag NSs@PEG@3COOH are quickly mixed in the twisted hybrid chip, and then three-dimensional flow focusing is performed by sheath flow to achieve single-file cell passing through the laser spot to detect the cell spectrum. Figure 7 As shown in (b), the SWS-KNN algorithm is also used to classify blank cells, breast cancer cells, and mammary gland cells. The classification results of each group of spectra are shown in Figure 7 (c). Each vertical line represents a spectrum, where light blue represents blank spectrum, blue represents breast cancer cell spectrum, and orange represents breast cell spectrum. The corresponding counts of the classification results are shown in Figure 7 (d) as shown. Figure 7 (e) shows the ratio of the spectral counts of the two types of cells. The average ratio reaches 1.011, which is very close to the 1:1 cell mixing ratio preset in the experiment.

[0114] The present invention achieves label-free detection at the single-cell level and can be further used for label-free detection of single cells in a mixed sample containing two different types of cells. It is expected to be used for single-cell analysis, including monitoring changes in cancer cells, detecting and classifying cells, and non-invasive biosensing of living cells.

[0115] The above description is only a specific implementation mode of the present invention, but the protection scope of the present invention is not limited thereto. Any modifications, equivalent substitutions and improvements made by any technician familiar with the technical field within the technical scope disclosed by the present invention and within the spirit and principle of the present invention should be covered by the protection scope of the present invention.

Claims

1. A dynamic liquid integrated single-cell surface enhanced Raman scattering detection platform, comprising a twisted mixing part (1), a 3D flow focusing part (2) and a single-cell SERS detection part (3) connected in sequence, characterized in that: The twisted mixing part (1) mixes the inflowing cells with the multifunctional modified nanoprobes through a spirally twisted mixing microfluidic chip (1-1), and the cell solution combined with the multifunctional modified nanoprobes after mixing is sent to the 3D flow focusing part (2), 3D focused into a single column at the center of the 3D flow focusing part (2), and finally flows through the laser area of ​​the single cell SERS detection part (3), and its SERS spectrum is collected for detection.

2. A dynamic liquid integrated single cell surface enhanced Raman scattering detection platform according to claim 1, characterized in that: The twisted mixing part (1), the 3D flow focusing part (2) and the single cell SERS detection part (3) are connected in sequence through a soft silicone tube (4); the twisted mixing part (1) comprises a spirally twisted mixing microfluidic chip (1-1); a synchronously spirally twisted hollow channel (1-2) is arranged in the middle of the spirally twisted mixing microfluidic chip (1-1); a first inlet end (1-3) for injecting a cell solution and a second inlet end (1-4) for injecting a multifunctional modified nanoprobe solution are respectively arranged on both sides of the spirally twisted mixing microfluidic chip (1-1); the first inlet end (1-3) and the second inlet end (1-4) merge into the hollow channel (1-2); and a rectangular observation area (1-5) is arranged at the outlet end of the hollow channel (1-2) for taking a fluorescent image and observing the state of the mixed solution.

3. A dynamic liquid integrated single cell surface enhanced Raman scattering detection platform according to claim 2, characterized in that: The cross section of the hollow channel (1-2) of the spirally twisted hybrid microfluidic chip (1-1) is rectangular, trapezoidal, right-angled triangle or isosceles triangle, and the spiral twist angle is 30° to 90°.

4. The dynamic liquid integrated single cell surface enhanced Raman scattering detection platform according to claim 3, characterized in that: The optimal cross-section of the hollow channel (1-2) of the spirally twisted hybrid microfluidic chip (1-1) is an isosceles triangle, and the optimal twisting angle is 30°.

5. The dynamic liquid integrated single cell surface enhanced Raman scattering detection platform according to claim 2, characterized in that: The spirally twisted hybrid microfluidic chip (1-1) is divided into n units, each unit is a complete spiral, and the length of each unit is 0.5-3 mm; the length of the spirally twisted hybrid microfluidic chip (1-1) is 3-24 mm.

6. The dynamic liquid integrated single cell surface enhanced Raman scattering detection platform according to claim 1, characterized in that: The multifunctional modified nanoprobe is a Ag NSs surface modified with PEG and 3COOH (Ag NSs@PEG@3COOH), that is, the PEG and 3COOH molecules replace the CIT molecules on the Ag NSs surface.

7. The dynamic liquid integrated single cell surface enhanced Raman scattering detection platform according to claim 6, characterized in that: The specific process of modifying PEG and 3COOH on the surface of Ag NSs is as follows: In the first step, the surface of Ag NSs was modified with long PEG chain 5KDa PEG; 1.1) Concentrating Ag NSs to 1.8-2 a.u.: Centrifuge the Ag NSs solution at 2500-3500 rpm for 13-15 minutes, remove the supernatant, and then measure the concentration of the Ag NSs solution using an ultraviolet-visible spectrometer (UV-Vis) until the absorbance of the solution reaches 1.8-2.2 au. 1.2) preparing a 5-10 mM 5KDa PEG aqueous solution: taking 8-8.89 mL of the Ag NSs solution after the concentration in step 1.1), adding 10-20 μL of a 5-10 mM 5KDa PEG aqueous solution, stirring at 600-800 rpm for 1.3-1.5 hours to react, after the reaction is completed, centrifuging at 3000-3500 rpm for 13-15 minutes, removing the supernatant, and adding an equal volume of ultrapure water to the precipitated Ag NSs as the removed supernatant to redisperse the nanoparticles, and storing the modified Ag NSs solution in a refrigerator at 3-5°C in the dark for subsequent use; In the second step, based on the modification of the Ag NSs surface with a long PEG chain of 5KDa, the Ag NSs surface was modified with a short PEG chain of 550Da; 2.1) Concentrating the modified AgNSs solution in step 1.2) to 1.8-2 a.u.: centrifuging the AgNSs solution at 3000-3500 rpm for 13-15 minutes, removing the supernatant, and then measuring the concentration of the AgNSs solution using an ultraviolet-visible spectrometer (UV-Vis) until the absorbance of the solution reaches 1.8-2 a.u.; 2.2) preparing a 0.076-0.08 mM 5KDa PEG aqueous solution and a 9.924-10 mM 550Da PEG aqueous solution; 2.2.1) Take 8-10 mL of the Ag NSs solution after the concentration in step 2.1), add 16-20 μL of 5 KDa PEG solution, and stir at 600-800 rpm for 1.3-1.5 hours to react; after the reaction is completed, centrifuge at 3000-3500 rpm for 13-15 minutes, remove the supernatant, and set the concentration of the obtained reactant to 1.8-2 a.u.; 2.2.2) Short-chain PEG modification: 8-10 mL of the reaction product concentrated in step 2.2.1) was added with 16-20 μL of 550 Da PEG solution, and the mixture was stirred at 600-800 rpm for 1.3-1.5 hours for reaction; after the reaction, the mixture was centrifuged at 3000-3500 rpm for 13-15 minutes, the supernatant was removed to remove unreacted PEG molecules, and an equal volume of ultrapure water was added to the removed supernatant to disperse the Ag NSs@PEG solution, which was then stored in a refrigerator at 3-5°C away from light for subsequent use; The third step is to modify the Ag NSs@PEG obtained in the second step with 3COOH to obtain Ag NSs@PEG@3COOH; The concentration of the Ag NSs@PEG solution obtained in step 2.2.2) was measured using an ultraviolet-visible spectrometer (UV-Vis) until the absorbance of the solution reached 1.8-2 a.u.; 7.2-8 mL of the concentrated Ag NSs@PEG solution was taken into a glass bottle, 20-22 μL of 3COOH aqueous solution was added, and the mixture was stirred at 600-800 rpm for 25-30 minutes for reaction; after the reaction, the mixture was centrifuged at 3000-3500 rpm for 13-15 minutes, the supernatant was removed, and an equal volume of ultrapure water was added to the removed supernatant to obtain an Ag NSs@PEG@3COOH solution, which was stored in a refrigerator at 3-5°C away from light.

8. The dynamic liquid integrated single cell surface enhanced Raman scattering detection platform according to claim 1, characterized in that: The 3D flow focusing part (2) comprises a coaxial needle (2-1), the coaxial needle (2-1) comprises an inner needle (2-1-1) and an outer needle (2-1-2) which are coaxially stacked, the size of the inner needle (2-1-1) is 22-30G (0.40×0.70mm-0.16×0.31mm), and the size of the outer needle (2-1-2) is 17-21G (0.10×1.50mm-0.50×0.80mm); the inlet end of the inner needle (2-1-1) The end of the hollow channel (1-2) of the spirally twisted hybrid microfluidic chip (1-1) is connected to the nuclear fluid mixed with circulating cells and nanoprobes, so that the cells are 3D focused in a single column at the center of the channel of the inner needle; two side inlets (2-1-3) are provided on both sides of the inlet end of the outer needle (2-1-2) to introduce the outer sheath fluid; the nuclear fluid is surrounded by the outer sheath fluid to form a narrow column, and the flow rate ratio of the nuclear fluid to the sheath fluid is adjusted by setting the flow rate parameters of the injection pump to achieve 3D flow focusing of each cell in the middle of the channel.

9. The dynamic liquid integrated single cell surface enhanced Raman scattering detection platform according to claim 8, characterized in that: When the flow rate ratio of the nuclear liquid to the sheath liquid is 1:1, it is a 3D flow focusing state. When focusing, the cells are restricted to a single cell narrow band, and when not focusing, the cells are dispersed in the entire channel.

10. The dynamic liquid integrated single cell surface enhanced Raman scattering detection platform according to claim 1, characterized in that: The single-cell SERS detection part (3) includes a quartz or glass capillary and a Raman spectrometer; the quartz or glass capillary is connected to the end of the coaxial probe (2-1) through a silicone hose, and the cells combined with the nanoprobe flow through the quartz or glass capillary in a single column. Whenever a cell passes through the laser area of ​​the Raman spectrometer, the Raman spectrometer will collect a cell spectrum to achieve single-cell detection.

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