Controllable nanorod array SERS (Surface Enhanced Raman Scattering) chip-based digital detection device for exosome phenotype of serous ovarian cancer as well as detection method and application thereof

Abstract

The invention discloses a controllable nanorod array SERS (Surface Enhanced Raman Scattering) chip-based digital detection device for a serous ovarian cancer exosome phenotype as well as a detection method and application thereof, and relates to the technical field of biological detection. The device comprises a controllable nanorod array module, a high-sensitivity SERS probe module, an integrated microfluidic platform module and a digital analysis module. The controllable nanorod array module is used for accurately capturing a single exosome; the SERS probe module realizes specific recognition of multiple biomarkers on the surface of the exosome by conjugating a specific antibody and a Raman reporter molecule; the micro-fluidic platform module integrates filtering, reaction and imaging functions, so that high-throughput and automatic detection is realized; and the digital analysis module completes qualitative and quantitative analysis of exosome phenotypes. The problems that a traditional detection technology cannot analyze the phenotype difference of single exosomes, the sensitivity is low and the flux is insufficient are solved, early accurate diagnosis of the serous ovarian cancer can be achieved, and the kit has wide clinical application prospects.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological detection, in particular to a digital detection device for exosome phenotypes of serous ovarian cancer based on a controllable nano-pillar array SERS chip and a detection method and application thereof. BACKGROUND

[0002] Serous ovarian cancer is the most common and highest malignant subtype of epithelial ovarian cancer. Its early symptoms are occult, and most patients are diagnosed at an advanced stage, with a 5-year survival rate of less than 30%. The 5-year survival rate of patients in stage I / II is more than 80%, so early diagnosis is the key to improving the prognosis of patients. The current clinical biomarker detection method relies on serum CA125 and HE4, but CA125 also increases in diseases such as endometrial heterotopy and inflammation, and has insufficient specificity, and HE4 has low sensitivity, which makes it difficult to meet the needs of early diagnosis.

[0003] Exosomes, as lipid membrane vesicles with a diameter of 30-200 nm, are widely present in body fluids such as blood, carrying specific protein markers (such as CA125, EpCAM, CD9, etc.) of the parent cell, and the detection sensitivity of exosome CA125 is higher than that of serum CA125, becoming an ideal biomarker carrier for early diagnosis of serous ovarian cancer. However, traditional exosome detection techniques (such as ELISA and flow cytometry) have the following defects: 1) relying on batch analysis, unable to capture the phenotypic heterogeneity of individual exosomes, masking the signal of low-abundance specific exosomes; 2) requiring a large amount of samples and cumbersome labeling steps, long detection time and low throughput; 3) signal reading based on absorbance or fluorescence intensity, poor quantitative stability, and sensitivity difficult to meet the needs of early diagnosis.

[0004] Surface-enhanced Raman scattering (SERS) technology has the advantages of significant signal enhancement effect, narrow spectral peak, and resistance to photobleaching, and can realize simultaneous detection of multiple components; nano-pillar array (NPAs) can achieve precise capture of individual exosomes through size matching; microfluidic technology has the characteristics of low sample consumption, high automation, and high-throughput detection. Integrating the three can solve the pain points of traditional techniques, but the current related techniques still have the following shortcomings: 1) poor uniformity of nano-pillar array, leading to large fluctuations in SERS signal; 2) limited multiplexing capability of SERS probes, making it difficult to achieve simultaneous detection of multiple biomarkers; 3) low integration of microfluidic platform and SERS detection, prone to non-specific adsorption interference with detection results.

[0005] Therefore, it is of great significance to develop a high-uniformity, high-sensitivity, and high-throughput exosome phenotype digital detection device for early diagnosis of serous ovarian cancer. SUMMARY

[0006] In view of the defects in the prior art, the application provides a kind of based on controllable nano pillar array SERS chip serous ovarian cancer exosome phenotype digital detection device and its detection method and application, by optimizing nano pillar array preparation process, SERS probe design and microfluidic platform integration, can realize the accurate capture of single exosome and the digital analysis of multiple biomarkers, meet the clinical needs of early diagnosis of serous ovarian cancer.

[0007] In order to achieve the above technical purpose, the application mainly adopts the following technical solutions:

[0008] In a first aspect, the application discloses a kind of based on controllable nano pillar array SERS chip serous ovarian cancer exosome phenotype digital detection device, including sequentially cooperative controllable nano pillar array module, high-sensitivity SERS probe module, integrated microfluidic platform module and digital analysis module:

[0009] The controllable nano pillar array module includes a substrate and a gold-plated nano pillar array grown on the surface of the substrate, the substrate is a silicon wafer, the gold-plated nano pillar array is a cubic array or a cylindrical array, the silicon wafer is composed of multiple chips of an integrated circuit, each chip contains multiple nano pillars, and the surface of the nano pillars is modified with exosome capture antibodies;

[0010] The high-sensitivity SERS probe module includes a variety of SERS nano probes, the SERS nano probes use gold-silver core-shell nanospheres as carriers, and the surface is conjugated with Raman reporter molecules and serous ovarian cancer exosome specific recognition antibodies;

[0011] The integrated microfluidic platform module includes a double-layer chip structure, the upper layer of the double-layer chip structure is a fluid layer, and the lower layer is a controllable nano pillar array module, the fluid layer is provided with an exosome sample inlet, a SERS nano probe inlet and a PBS buffer inlet, the three inlets are connected to a multi-parallel chamber microarray imaging module through a total sample inlet, the other end of the multi-parallel chamber microarray imaging module is connected to a solution outlet, and the multi-parallel chamber microarray imaging module and the nano pillar array region of the lower controllable nano pillar array module correspond one-to-one, the microarray imaging module is sealed by oxygen plasma cleaning bonding, and a filter module is arranged between the exosome sample inlet and the total sample inlet;

[0012] The digital analysis module comprises a Raman confocal microscope, an image acquisition unit and a data processing unit, the Raman confocal microscope is used for acquiring the Raman signal of the SERS nano probe, the image acquisition unit is used for capturing the SERS imaging graph of the exosome, the data processing unit is based on the position and intensity of the Raman signal peak to qualitatively analyze the biomarker, based on the signal bright spot count to quantitatively analyze, and draw the receiver operating characteristic curve (ROC), calculate the area under the curve (AUC) to evaluate the detection effectiveness.

[0013] In the preferred embodiment of the present application, the exosome capture antibody is a CD63 antibody, and the Raman reporter molecule is selected from any one or several of Rhodamine B, RBITC, MGITC, Cy5, and DTDC; the specific recognition antibody is selected from any one or several of CD24, VCAN, EpCAM, TNC, and HE4.

[0014] In the preferred embodiment of the present application, the preparation method of the controllable nano pillar array module is a silicon wafer ion beam etching method, comprising the following steps:

[0015] S31: first clean the silicon wafer and then dehydrate and bake, then coat photoresist on the cleaned wafer, and bake the wafer on a hot plate;

[0016] S32: transfer the mask pattern to the wafer surface, expose, after exposure, develop the wafer in a developing solution, fix with deionized water, and then dry with N2;

[0017] S33: first remove the residual photoresist bottom film on the wafer using O2 plasma, and then sputter titanium and gold on the photoresist;

[0018] S34: after thinning and polishing the wafer, use a UV film to stick the wafer, and perform wafer cutting to obtain multiple chips.

[0019] In the preferred embodiment of the present application, the preparation method of the SERS nano probe comprises the following steps:

[0020] S41: synthesis of gold-silver core-shell nanosphere carrier;

[0021] S42: dissolve the Raman reporter molecule in the gold-silver core-shell nanosphere solution, mix with the nano carrier, incubate at room temperature, add HS-PEGCOOH, and centrifuge to remove unbound Raman reporter molecules and HS-PEGCOOH;

[0022] S43: EDC and NHS are added to activate the carboxyl group on the surface of the nanocarrier, and then a specific recognition antibody is added and incubated overnight. Bovine serum albumin (BSA) is used to block the unbound sites, and the SERS nanoprobes are obtained by centrifugal purification.

[0023] In a preferred embodiment of the present application, the preparation method of the integrated microfluidic platform module comprises the following steps:

[0024] S51: The channel structure of the chip is drawn using computer-aided design (CAD) and a mask is made. The silicon wafer is spin-coated with photoresist, the mask is covered, and after ultraviolet light irradiation and curing, the photoresist is cleaned to obtain a silicon wafer template.

[0025] S52: Mix the polydimethylsiloxane (PDMS) oligomer with a curing agent, vacuum degas, pour into the silicon wafer template, heat and cure, remove the PDMS layer and punch holes. Spin-coat a thin layer of PDMS on the unetched silicon wafer of the lower chip, place the gold-plated nanocolumn array, heat and cure, then align the upper and lower chips according to the channel position, clean with oxygen plasma and heat to permanently bond, obtaining the integrated microfluidic platform.

[0026] In a second aspect, the present application discloses a method for digital detection of exosome phenotypes of serous ovarian cancer based on the detection device of the first aspect, comprising the following steps:

[0027] S61 sample pretreatment: collect blood samples and cell culture liquid samples from serous ovarian cancer patients, benign ovarian tumor patients or healthy people, obtain exosome precipitate by ultracentrifugation, and resuspend in PBS for standby;

[0028] S62 chip functionalization modification: embed the controllable nanocolumn array module into the lower array layer of the integrated microfluidic platform. First, add SH-PEGCOOH to the channel and incubate, remove the SH-PEGCOOH, repeatedly aspirate PBS, then add EDC and NHS and react completely, remove NHS and EDC, repeatedly aspirate PBS, then add CD63 antibody and incubate, repeatedly aspirate PBS, remove the unbound antibody, then add BSA for blocking, and place the functionalized chip at 4℃ for standby;

[0029] S63 exosome capture and SERS probe incubation: introduce the sample to be detected from the exosome sample inlet of the integrated microfluidic platform, incubate at room temperature, then introduce PBS from the PBS buffer inlet to wash away the unbound exosomes; introduce the mixture of 5 kinds of SERS nanoprobes from the SERS nanoprobe inlet, incubate at room temperature, and wash away the unbound probes with PBS;

[0030] S64 SERS signal acquisition and digital analysis: start the Raman confocal microscope, scan the nanorod array region in the microchannel, obtain the Raman spectrum and imaging graph; use the data processing unit to identify the characteristic peaks of different Raman reporter molecules, qualitatively determine the surface biomarkers of exosomes; count the number of signal bright spots, calculate the concentration of exosomes combined with the density of the nanorod array; compare the detection results of different samples, draw the ROC curve and calculate the AUC value, and evaluate the sensitivity and specificity of the detection;

[0031] S65 Clinical verification: blind test on unknown clinical samples, compare the SERS detection results with the PET / CT, HE staining results and chemiluminescence immunoassay results, and verify the clinical applicability of the detection device.

[0032] In the preferred embodiment of the present application, in step S62, the concentration of the exosome capture antibody CD63 is 10-20 μg / mL, and the incubation time is 2h; in step S63, the dilution multiple of the sample to be detected is 10 2 -10 6 exosome samples.

[0033] In the preferred embodiment of the present application, in step S64, the Raman confocal microscope uses an excitation wavelength of 633 nm, a laser power of 35 mW, a grating of 600 g mm-1, a spectral resolution of 1.390-2.114 cm-1, an integration time of 0.1 s-1s, a scanning area size of 60 μm x 48 μm, and a system frequency of point-by-point scanning of the nanorod array region to obtain the Raman spectrum and imaging graph; the data processing unit uses ImageJ software to count the bright spots of the imaging graph, and uses SPSS software to analyze the ROC curve and calculate the AUC value, and when the AUC value is greater than or equal to 0.9, the detection result is determined to be valid.

[0034] In a third aspect, the present application discloses an application of the detection device as described in the first aspect in the preparation of a device for early diagnosis of serous ovarian cancer.

[0035] In the preferred embodiment of the present application, the detection device distinguishes between patients with stage I-IV serous ovarian cancer and healthy people by detecting the content of at least four biomarkers on the surface of exosomes.

[0036] Compared with the prior art, the present application has the following beneficial effects:

[0037] The application provides a device and method for digital detection of serous ovarian cancer exosome phenotype based on a controllable nano-pillar array SERS chip. BRIEF DESCRIPTION OF DRAWINGS

[0038] Figure 1 The preparation process of the SERS probe and the characteristic Raman peak of the Raman reporter molecule in 5;

[0039] Figure 2 The transmission electron microscope image of Au@AgNPs, the scale is 50 nm, and the X-ray spectrum (EDS);

[0040] Figure 3 The preparation process of the upper microfluidic chip (fluid layer) and the model diagram of the entire microfluidic chip; in the diagram, ① SERS nano-probe inlet; ② filtering module; ③ exosome sample inlet; ④ PBS buffer inlet; ⑤ microarray imaging module; ⑥ solution outlet; a fluid layer; b controllable nano-pillar array module; c glass layer;

[0041] Figure 4 The exosome detection process diagram of the microfluidic chip;

[0042] Figure 5 The SERS imaging diagram of the microfluidic chip for detecting exosomes. DETAILED DESCRIPTION

[0043] The application will be further described below in combination with the drawings and examples.

[0044] Example 1 Preparation and characterization of controllable nano-pillar array

[0045] A cubic gold-plated nano-pillar array was prepared by photo-etching ion sputtering:

[0046] 1) The chip is made of a sensing array with a size of 1 mm x 1 mm, which is composed of 250,000 individual pillars. Each pillar is 1 μm wide, 1 μm long, and 1 μm high. The pillars are uniformly spaced 1 μm apart from one pillar to the next.

[0047] 2) First, the silicon wafer was cleaned by ultrasonic treatment in acetone and isopropyl alcohol for 2 minutes each, and then dehydrated at 180°C for 2 minutes with deionized H2O.

[0048] 3) First, sputter 10 nm Titanium and 200 nm Gold on the surface of silicon wafer using a magnetron sputtering (FHR Germany).

[0049] 4) Then, coat the cleaned wafer with photoresist (AZ 6112 series, AZ Electronic Materials, Germany) using an automatic spin coater (SUSS Coater) at 3000 rpm for 30 seconds. After coating, immediately bake the wafer on a hot plate at 100 °C for 2 min.

[0050] 5) The photoresist thickness is 1 um. Use a stepper photolithography machine (NiKon NSR Nikon, Japan) with I-line light source (365 nm wavelength) and 5:1 reduction projection lens, exposure intensity: 450 mW / cm2, exposure time 50 ms, use high-precision projection lens imaging and high-precision interferometer to control the stepping motion of the workbench, and transfer the mask pattern to the wafer surface for exposure.

[0051] 6) After exposure, develop the wafer in RZX3038 developer, fix it with deionized water, and then dry it with N2. Then the wafer is further subjected to O2 plasma to remove the residual photoresist bottom film for 3 minutes at 200 W (plasma stripper PVA TePla).

[0052] 7) First, remove the sputtered 10 nm Titanium and 200 nm Gold on the surface using an IBE ion beam etching machine (SCIA, Switzerland), and then apply SPTS deep silicon etching machine (KLA Corporation, USA) for anisotropic etching of silicon to create a column height (i.e., 1 um).

[0053] 8) Therefore, the deposited gold acts as a mask to protect the underlying silicon while removing the unmasked silicon. Then clean the residual photoresist with ultrasonic treatment in acetone, isopropyl alcohol, and then thin and polish the wafer to 100 um by thinning (AM HRG-150) and polishing (AM AP-380F).

[0054] 9) Finally, use a uv film to stick the wafer, cut the wafer using a disco cutter (DAD 3350, Japan), and finally use a uv film to remove the glue based on the curing effect of ultraviolet light. After these ultraviolet light energy is absorbed, it will trigger the chemical reaction of photosensitive substances (also known as photoinitiator), thereby hardening the glue on the UV film to complete the degumming, making it easy to take out the array.

[0055] Example 2 Preparation and characterization of SERS nanoprobes

[0056] Preparation of gold-silver core-shell nanospheres:

[0057] 1) SERS with gold nanoparticles as substrate, preparation of gold nanoparticles: gold nanoparticles were synthesized by seed-mediated growth method.

[0058] 2) First, 75 mL of 2.2 mM sodium citrate solution was added to a round-bottom three-necked flask, heated and stirred vigorously, while opening the condenser. After the solution boiled, 0.5 mL of 25 mM chloroauric acid (HAuCl4) solution was quickly added, and the heating and stirring were continued, and the solution color gradually changed from bright yellow to pink within 20 min.

[0059] 3) Then, the solution temperature was reduced to 90 o C. At this time, a 60 mM sodium citrate solution and a 25 mM chloroauric acid solution were prepared, and 0.5 mL of sodium citrate solution and an equal volume of chloroauric acid solution were added in turn, and mixed thoroughly after each addition. This process was repeated 12 times, with an interval of 2 min between each operation. After each addition, the color of the solution gradually changed from the initial pink to deep burgundy. The reaction solution was kept at 90 o C, and stirring was continued for 30 min.

[0060] 4) Finally, the heating device was turned off, and after the solution cooled to room temperature, it was transferred to a 4 o C refrigerator for standby.

[0061] Preparation of SERS probe:

[0062] 1) First, add Raman reporter molecules MGITC (2 μL, 0.1 mM), RBITC (17 μL, 0.1 mM), DTDC (17 μL, 0.1 mM), Cy5 (3.5 μL, 0.1 mM), Rhodamine (1 μL, 1 mM) to the diluted AuNPs solution, respectively, and stir vigorously at room temperature for 30 min.

[0063] 2) Add silver nitrate (300 μL, 10 mM) and ascorbic acid (300 μL, 10 mM), and stir vigorously at room temperature for 1 h.

[0064] 3) Then add MGITC (2 μL, 0.1 mM), RBITC (17 μL, 0.1 mM), DTDC (17 μL, 0.1 mM), Cy5 (3.5 μL, 0.1 mM), Rhodamine (1 μL, 1 mM) and stir vigorously at room temperature for 30 min.

[0065] 4) Add HS-PEGCOOH (25 μL, 1 mM) and stir the reaction vigorously for 3 h to form PEG layer coated Au@Ag shell structure to improve its surface stability.

[0066] 5) Subsequently, add 5 μL, 5 mM of EDC (N-ethyl-N'-(3-(dimethylamino)propyl)- carbodiimide) and 5 μL, 5 mM of NHS (N-hydroxysuccinimide) solution to activate the carboxyl group on HS-PEGCOOH at room temperature for 20 min.

[0067] 6) After the reaction is complete, centrifuge (7000 g, 10 min) twice to remove unbound EDC, NHS and HS-PEGCOOH, and resuspend the nanoparticles with ultrapure water.

[0068] 7) Next, add 15 μL of antibodies Cluster of Differentiation 24 (CD24), Versican (VCAN), Epithelial cell adhesion molecule (EpCAM), The Nature Conservancy (TNC) and human epididymal protein 4 (HE4) at a concentration of 0.1 mg / mL, respectively, to the PEG-coated Au@Ag nanostructure with activated -COOH end at room temperature for 2 h, and then incubate overnight at 4 o C.

[0069] 8) After that, add ethanolamine (1 μL, 5 mM) solution to block unreacted binding sites.

[0070] 9) Finally, centrifuge (7000 g, 10 min) to remove unbound antibodies, and resuspend with ultrapure water to obtain five SESR probes, AuNPs-RBITC@Ag-RBITC-VCAN, AuNPs-MGITC@Ag-MGITC-CD24, AuNPs-Rhodamine@Ag-Rhodamine-HE4, AuNPs-Cy5@Ag-Cy5-TNC, AuNPs-DTDC@Ag-DTDC-EpCAM. The characteristic peaks at RBITC, MGITC, Rhodamine, Cy5, DTDC are 1646, 1616, 1405, 1264, 1133 cm -1 .

[0071] Example 3 Preparation and performance test of integrated microfluidic platform

[0072] Preparation of double-layer PDMS microfluidic platform:

[0073] 1) Silicon wafer template preparation: CAD design 4 sets of parallel chamber microchannels (each chamber size 5x5mm), mask preparation; silicon wafer spin coating SU-8 photoresist (thickness 50um), UV light irradiation curing, after development to get silicon wafer template;

[0074] 2) PDMS upper layer preparation: PDMS oligomer and curing agent are mixed at 10:1, vacuum degassing for 30min, poured into the silicon wafer template, baked at 80℃ for 2h, remove the PDMS layer, and punch holes (diameter 1mm) at the sample inlet, buffer inlet, probe inlet and outlet with a puncher;

[0075] 3) PDMS lower layer preparation: spin coating PDMS (thickness 100um) on unetched silicon wafer, bake at 80℃ for 1h, paste the nanorod array prepared in example 1 at the corresponding position of the PDMS lower layer;

[0076] 4) Bonding: upper and lower PDMS chips are cleaned by oxygen plasma (power 50W, time 30s), aligned and baked at 80℃ for 1h to achieve permanent bonding.

[0077] 5) Performance test: 0.1% sodium fluorescein solution is introduced, flow rate 5ul / min, observed by fluorescence microscope (model: FV3000), the fluid flows uniformly in the microchannel without leakage, and the fluorescence intensity of the parallel chamber is uniform (coefficient of variation ≤5%), indicating that the platform has stable performance.

[0078] Example 4 Digital detection device for exosome phenotype of serous ovarian cancer based on controllable nanorod array SERS chip

[0079] The digital detection device for exosome phenotype of serous ovarian cancer based on controllable nanorod array SERS chip of the application comprises a controllable nanorod array (NPAs) module, a high-sensitivity SERS probe module, an integrated microfluidic platform module and a digital analysis module, which work cooperatively to realize the capture, identification, signal collection and data analysis of exosomes.

[0080] 1. Controllable nanorod array (NPAs) module:

[0081] The controllable nanorod array module comprises a substrate and a gold-plated nanorod array grown on the surface of the substrate, the substrate is a silicon wafer, and the gold-plated nanorod array is a cubic array or a cylindrical array, wherein the silicon wafer is composed of multiple chips of integrated circuits, each chip contains multiple nanorods, and the surface of the nanorods is modified with exosome capture antibodies;

[0082] The module is the core of single exosome capture, using a silicon wafer as the substrate, and gold-coated nanorod arrays as the detection site. The size and spacing of the nanorods are both controlled at 1 µm, ensuring that a single nanorod captures a single exosome; a single chip contains 250,000 nanorods, allowing simultaneous detection of a large number of exosomes and improving throughput. The nanorod surface is modified with CD63 antibodies (a general marker antibody for exosomes), which achieve precise capture of exosomes through immunospecific binding and reduce non-specific adsorption.

[0083] The nanorod array is prepared using photoetching ion sputtering and ion beam etching processes, which pattern the photoresist and deposit ions through ion sputtering, ensuring uniform nanorod morphology with a size error of ≤5%;

[0084] 2. High-sensitivity SERS probe module:

[0085] The high-sensitivity SERS probe module includes various SERS nanoprobes, with gold-silver core-shell nanospheres as the carrier, and Raman reporter molecules and serum ovarian cancer exosome-specific recognition antibodies conjugated to the surface.

[0086] This module is used for specific recognition of multiple biomarkers on the surface of exosomes, with gold-silver core-shell nanospheres as the carrier, and the surface plasmon resonance effect of gold-silver nanomaterials is used to enhance the Raman signal. The carrier surface is conjugated with two key components:

[0087] Raman reporter molecules: Rhodamine B isothiocyanate (RBITC), Malachite Green isothiocyanate (MGITC), Rhodamine, Cyanine 5 (Cy5), and 3,3'-Diethylthiadicarbocyanine iodide (DTDC) are selected, with characteristic peaks at 1646, 1616, 1405, 1264, 1133 cm -1 , which allows for simultaneous detection of multiple markers;

[0088] Specific recognition antibodies: selected from Versican (VCAN), Cluster of Differentiation 24 (CD24), The Nature Conservancy (TNC), Epithelial cell adhesion molecule (EpCAM), and human epididymal protein 4 (HE4) to form 5 detection probe combinations, improving diagnostic accuracy.

[0089] During the preparation of the SERS probe, the antibody is stably coupled to the nanocarrier through the EDC / NHS activation method, and BSA is used to block unbound sites, ensuring the specificity and stability of the probe; DLS and TEM are used to characterize the size of the probe to avoid aggregation and affect the detection effect.

[0090] 3. Integrated microfluidic platform module

[0091] The integrated microfluidic platform module comprises a double-layer chip structure, the upper layer of which is a fluid layer and the lower layer is a controllable nanocolumn array module, and the double-layer chip is located on a glass layer, wherein the fluid layer is provided with an exosome sample inlet, a SERS nanoprobe inlet and a PBS buffer inlet, the three inlets are connected with a multi-parallel chamber microarray imaging module through a total sample inlet, the other end of the multi-parallel chamber microarray imaging module is connected with a solution outlet, and the multi-parallel chamber microarray imaging module and the nanocolumn array region of the lower controllable nanocolumn array module correspond one-to-one, the microarray imaging module is sealed by oxygen plasma cleaning bonding, and a filtering module is arranged between the exosome sample inlet and the total sample inlet.

[0092] The module is a carrier for realizing automatic and high-throughput detection, and adopts a double-layer PDMS chip structure:

[0093] 1) The upper fluid layer: integrates an exosome sample inlet, a SERS nanoprobe inlet, a PBS buffer inlet, a 0.22 μm filter membrane filtering module, four groups of parallel chamber microchannels (each group contains two nanometer array detection zones) and a solution outlet. The filtering module can remove cell fragments and impurities in the blood sample, thereby improving the detection throughput.

[0094] 2) The lower nanocolumn array module: the prepared controllable nanocolumn array is embedded in a PDMS thin layer, and corresponds to the upper microchannel one-to-one, so as to ensure that the sample and the probe can precisely act on the nanocolumn array region.

[0095] 3) The microfluidic platform is sealed by oxygen plasma cleaning bonding to avoid liquid leakage; the inner surface of the channel is hydrophilic treated to ensure uniform flow of the fluid (5-10 μL / min) and reduce non-specific adsorption of exosomes; the whole platform has a small volume (2×3 cm) and low sample consumption (≤50 μL), and can realize point-of-care testing (POCT).

[0096] 4. Digital analysis module

[0097] The digital analysis module comprises a Raman confocal microscope, an image acquisition unit and a data processing unit, the Raman confocal microscope is used for acquiring the Raman signal of the SERS nanoprobe, the image acquisition unit is used for capturing the SERS imaging graph of the exosome, the data processing unit qualitatively analyzes the biomarker based on the position and intensity of the Raman signal peak, quantitatively analyzes based on the signal bright spot counting, draws a receiver operating characteristic curve ROC, and calculates the area under the curve AUC to evaluate the detection effectiveness.

[0098] The module is the core for realizing qualitative and quantitative detection results, and comprises a Raman confocal microscope, an image acquisition unit and a data processing unit:

[0099] 1) Raman confocal microscope: 633 nm excitation wavelength was used, and the laser power was controlled at 35 mW to avoid damaging the exosomes; the nanorod array region was scanned point by point to obtain Raman spectra and imaging images, the spectral resolution was 1.390-2.114 cm-1, the integration time was 0.1-1 s, and the balance detection sensitivity and efficiency were obtained;

[0100] 2) Image acquisition unit: capture SERS imaging images, and locate the position of a single exosome through signal bright spots;

[0101] 3) Data processing unit: ① qualitative analysis: according to the characteristic peak position of the Raman reporter molecule, the biomarker on the surface of the exosome is identified; ② quantitative analysis: the number of signal bright spots is counted, and the exosome concentration is calculated combined with the nanorod array density; ③ effectiveness evaluation: the detection results of patients and healthy people are compared, the ROC curve is drawn, and the AUC value is calculated, and when AUC≥0.9, it is determined that the detection result is effective; ④ clinical verification: the detection results of unknown samples are compared with the PET / CT and HE staining results to verify the clinical applicability of the device.

[0102] Example 5: Digital detection method for exosome phenotypes of serous ovarian cancer based on controllable nanorod array SERS chip

[0103] The detection method of the application is based on the above-mentioned device, which includes five core steps of sample pretreatment, chip functionalization modification, exosome capture and SERS probe incubation, SERS signal acquisition and digital analysis, and clinical verification, which are as follows:

[0104] 1. Sample pretreatment

[0105] Blood samples: collect venous blood of serous ovarian cancer patients (I-IV stage, age 45-65 years old), benign ovarian tumor patients or healthy people (age 45-65 years old), use blood collection tubes without anticoagulant to obtain serum (3000 rpm centrifugation for 10 min), dilute the extracted clinical serum in PBS, and then filter it with a 0.22 μm filter membrane, and the filtered serum is centrifuged at 150,000 g, 4 o C for 8 h. Remove the supernatant, add 8 mL of PBS for resuspension, centrifuge at 150,000 g, 4 o C for 2 h, remove the supernatant, and collect the exosome precipitate

[0106] Cell culture solution sample: after culturing serous ovarian cancer cell lines (OVCAR3, SKOV3, CAOV3) and normal ovarian epithelial cells in serum-free medium for 72 h, collect the culture solution from three or more T175 cm 2The supernatant was collected in a culture flask and the extracellular vesicles were extracted using a high-speed refrigerated centrifuge. The specific operation steps are as follows:

[0107] 1) The cell culture supernatant was centrifuged at 300 x g, 4 o C for 10 min to remove live cells.

[0108] 2) The supernatant was centrifuged again at 2,000 x g, 4 o C for 10 min to remove dead cells.

[0109] 3) Continue to centrifuge at 10,000 x g, 4 o C for 30 min to remove cell debris.

[0110] 4) Filter the culture supernatant through a 0.22 μm filter to remove larger particulate impurities.

[0111] 5) Use Macrosep ultrafiltration tubes to concentrate the sample at 5,000 x g, 4 o C for 30 min.

[0112] 6) After centrifugation, take the supernatant and add it to the ultrafiltration tube. Centrifuge at 100,000 x g (about 39,000 rpm), 4 o C for 70 min, remove the supernatant, collect the extracellular vesicle precipitate, and remove the residual liquid.

[0113] First, add SH-PEGCOOH (10 μL, 1 mM) to the channel for 1 h, remove SH-PEGCOOH, PBS repeatedly rinse 3 times, then add EDC (N-ethyl-N'-(3 (dimethylamino) propyl)-carbodiimide, 5 μL, 1 mM) and NHS (N-hydroxysuccinimide) for 30 min, remove NHS and EDC, PBS repeatedly rinse 3 times, then add CD63 (25 mg / mL, 25 μL) antibody for 2 h, PBS repeatedly suction 3 times, remove unbound antibody, then add 2.5% BSA for blocking for 30 min, and the functionalized chip is ready for use. o C.

[0114] 3. Exosome capture and SERS probe incubation

[0115] 1) Exosome capture: from the exosome sample inlet, pre-treat and dilute 10 2 -10 6After the serum sample, ensure the appropriate concentration of exosomes, achieve monodisperse capture, control the flow rate 5-10 μL / min, incubate at room temperature for 60 min, and the exosomes are specifically bound to the nanorod surface by CD63 antibody;

[0116] 2) Cleaning: flush the microchannel with PBS buffer to remove unbound exosomes;

[0117] 3) SERS probe incubation: introduce the mixed solution of 5 kinds of SERS nanoprobes (AuNPs-RBITC@Ag-RBITC-VCAN, AuNPs-MGITC@Ag-MGITC-CD24, AuNPs-Rhodamine@Ag-Rhodamine-HE4, AuNPs-Cy5@Ag-Cy5-TNC, AuNPs-DTDC@Ag-DTDC-EpCAM, each concentration 30 nM, each probe volume ratio 1:1:1:1:1) from the SERS probe inlet, incubate at room temperature for 60 min, and the probes are bound to the surface biomarkers of exosomes through specific antibodies;

[0118] 4) Secondary cleaning: flush the microchannel with PBS buffer to remove unbound probes and avoid false positive signals.

[0119] 4. SERS signal acquisition and digital analysis

[0120] 1) Signal acquisition: start the Raman confocal microscope (model: inViaTM QontorTM), set the excitation wavelength to 633 nm, the laser power to 35 mw, the grating to 600 mm −1 , the spectral resolution to 1.390-2.114 cm −1 , the integration time to 0.1 s-1 s, and the scanning area size to 60 μm × 48 μm, and then scan the nanorod array region in the microchannel point by point to obtain the Raman spectrum of each detection point and the SERS imaging map of the entire region;

[0121] 2) Qualitative analysis: the data processing unit identifies the characteristic peaks in the Raman spectrum, such as RBITC corresponding to VCAN, MGITC corresponding to CD24, Rhodamine corresponding to HE4, Cy5 corresponding to TNC, and DTDC corresponding to EpCAM, and determines the biomarkers expressed on the surface of exosomes;

[0122] 3) Quantitative analysis: ImageJ software counts the number of signal bright spots in the SERS imaging map, assuming that a single bright spot corresponds to a single exosome, and calculates the concentration of exosomes combined with the area of the nanorod array;

[0123] 4) Effectiveness evaluation: detect I-IV stage serous ovarian cancer patients and healthy people samples, draw ROC curve, calculate AUC value, the results show that: the exosome concentration of patient group is significantly higher than that of healthy group, AUC=0.94, sensitivity 88%, specificity 92%, indicating that the detection device can effectively distinguish patients from healthy people.

[0124] Example 6 Clinical blind test verification

[0125] Select 10 cases of unknown clinical blood samples, detect according to the method of example 5, get exosome phenotype data; At the same time, detect serum CA125 and HE4 by chemiluminescence immunoassay, combined with PET / CT, HE staining results to determine the diagnosis conclusion: 6 cases are I / II stage serous ovarian cancer patients, 4 cases are healthy people.

[0126] The coincidence rate of SERS detection results and clinical conclusions is 100% (6 patients are all detected positive, 4 healthy people are all detected negative), indicating that the clinical applicability of the device is good.

[0127] The above only describes some examples of the present application, not all examples. The detailed description of the present application is not intended to limit the scope of the claimed application, but only to represent selected examples of the present application. Based on the examples of the present application, all other examples obtained by ordinary skilled in the art without creative labor belong to the scope of protection of the present application.

Claims

1. A digital detection device for exosome phenotypes in serous ovarian cancer based on a controllable nanopillar array (SERS) chip, characterized in that, It includes a controllable nanopillar array module that works in sequence and in concert, a highly sensitive SERS probe module, an integrated microfluidic platform module, and a digital analysis module: The controllable nanopillar array module includes a substrate and a gold-plated nanopillar array grown on the substrate surface. The substrate is a silicon wafer, and the gold-plated nanopillar array is a cubic array or a cylindrical array. The silicon wafer is composed of multiple chips of an integrated circuit, and a single chip contains multiple nanopillars. The surface of the nanopillars is modified with exosome-capturing antibodies. The highly sensitive SERS probe module includes a variety of SERS nanoprobes, which use gold and silver core-shell nanospheres as carriers and have Raman reporter molecules and antibodies that specifically recognize serous ovarian cancer exosomes conjugated on their surfaces. The integrated microfluidic platform module includes a dual-layer chip structure. The upper layer of the dual-layer chip structure is a fluid layer, and the lower layer is a controllable nanopillar array module. The fluid layer is provided with an exosome sample inlet, a SERS nanoprobe inlet, and a PBS buffer inlet. The three inlets are connected to a multi-parallel chamber microarray imaging module through a total inlet. The other end of the multi-parallel chamber microarray imaging module is connected to a solution outlet. The multi-parallel chamber microarray imaging module corresponds one-to-one with the nanopillar array region of the lower controllable nanopillar array module. The microarray imaging module is sealed by oxygen plasma cleaning bonding. A filter module is provided between the exosome sample inlet and the total inlet. The digital analysis module includes a Raman confocal microscope, an image acquisition unit, and a data processing unit. The Raman confocal microscope is used to acquire the Raman signal of the SERS nanoprobe. The image acquisition unit is used to capture the SERS image of exosomes. The data processing unit performs qualitative analysis of biomarkers based on the position and intensity of the Raman signal peaks, performs quantitative analysis based on the number of signal bright spots, plots the receiver operating characteristic (ROC) curve, and calculates the area under the curve (AUC) to assess the effectiveness of the detection.

2. The detection device according to claim 1, characterized in that, The exosome capture antibody is a CD63 antibody; the Raman reporter molecule is selected from any one or more of Rhodamine B, RBITC isothiocyanate, MGITC isothiocyanate, Cy5 anthocyanin, and DTDC 3,3'-diethylthiodic dicarbonyl iodide; and the specific recognition antibody is selected from any one or more of CD24, VCAN, EpCAM, TNC, and HE4.

3. The detection device according to claim 1, characterized in that, The controllable nanopillar array module is fabricated using silicon wafer ion beam etching, and includes the following steps: S31: First, the silicon wafer is cleaned, dehydrated, and baked. Then, photoresist is applied to the cleaned wafer, and the wafer is baked on a hot plate. S32: Transfer the mask pattern to the wafer surface, expose it, develop the wafer in a developer, fix it with deionized water, and then dry it with N2. S33: First, use O2 plasma to remove the residual photoresist base film on the wafer, and then sputter titanium and gold onto the photoresist; S34: After thinning and polishing the wafer, a UV film is used to adhere the wafer, and the wafer is cut to obtain multiple chips.

4. The detection device according to claim 1, characterized in that, The preparation method of the SERS nanoprobe includes the following steps: S41: Synthetic gold-silver core-shell nanosphere carrier; S42: Dissolve the Raman reporter molecule in a solution of gold and silver core-shell nanospheres, mix it with the nanocarrier, incubate at room temperature, add HS-PEGCOOH, and centrifuge to remove unbound Raman reporter molecules and HS-PEGCOOH; S43: Add EDC and NHS to activate the carboxyl groups on the surface of the nanocarrier, then add a specific recognition antibody, incubate overnight, block unbound sites with bovine serum albumin (BSA), and centrifuge to purify and obtain the SERS nanoprobe.

5. The detection device according to claim 1, characterized in that, The preparation method of the integrated microfluidic platform module includes the following steps: S51: The chip channel structure is drawn using computer-aided design (CAD) and a mask is fabricated. Photoresist is spin-coated onto the silicon wafer, covering the mask. After curing by ultraviolet light, the photoresist is cleaned to obtain the silicon wafer template. S52: Mix polydimethylsiloxane (PDMS) oligomer with a curing agent, degas under vacuum, pour into a silicon wafer template, heat to cure, remove the PDMS layer and drill holes; spin-coat a thin layer of PDMS onto an unetched silicon wafer for the lower chip, place a gold-plated nanopillar array, heat to cure, then align the upper and lower chips according to the channel positions, clean with oxygen plasma, and heat to permanently bond, thus obtaining an integrated microfluidic platform.

6. A digital detection method for the exosome phenotype of serous ovarian cancer based on the detection device according to any one of claims 1-5, characterized in that, Includes the following steps: S61 Sample Pretreatment: Blood samples and cell culture medium samples were collected from patients with serous ovarian cancer, patients with benign ovarian tumors, or healthy individuals. Exosomes were obtained by ultracentrifugation and resuspended in PBS for later use. S62 chip functionalization modification: The controllable nanopillar array module is embedded into the lower array layer of the integrated microfluidic platform. First, SH-PEGCOOH is added to the channel for incubation. After removing SH-PEGCOOH, PBS is repeatedly aspirated. Then, EDC and NHS are added and reacted completely. After removing NHS and EDC, PBS is repeatedly aspirated. Then, CD63 antibody is added for incubation. PBS is repeatedly aspirated to remove unbound antibody. Then, BSA is added for blocking. The functionalized chip is placed at 4°C for later use. S63 Exosome Capture and SERS Probe Incubation: The sample to be tested was introduced through the exosome sample inlet of the integrated microfluidic platform and incubated at room temperature. Then, PBS was introduced through the PBS buffer inlet to rinse and remove unbound exosomes. A mixture of five SERS nanoprobes was introduced through the SERS nanoprobe inlet and incubated at room temperature. Unbound probes were then rinsed with PBS. S64 SERS Signal Acquisition and Digital Analysis: The Raman confocal microscope is activated to scan the nanopillar array region within the microchannel, acquiring Raman spectra and images; the data processing unit is used to identify the characteristic peaks of different Raman reporter molecules, qualitatively identifying exosome surface biomarkers; the number of signal bright spots is counted, and the exosome concentration is calculated based on the nanopillar array density; the detection results of different samples are compared, ROC curves are plotted, and AUC values ​​are calculated to evaluate detection sensitivity and specificity; S65 Clinical Validation: Blind testing of unknown clinical samples is conducted, and the SERS test results are compared with PET / CT, HE staining results, and chemiluminescent immunoassay results to verify the clinical applicability of the detection device.

7. The detection method according to claim 6, characterized in that, In step S62, the concentration of the exosome-capturing antibody CD63 is 10-20 μg / mL, and the incubation time is 2 h; in step S63, the dilution factor of the sample to be tested is 10. 2 -10 6 Exosome samples.

8. The detection method according to claim 6, characterized in that, In step S64, the Raman confocal microscope uses an excitation wavelength of 633 nm, a laser power of 35 mW, and a grating of 600 g mm. −1 ; Spectral resolution: 1.390–2.114 cm⁻¹ −1 Integration time: 0.1 s-1 s; Scanning area size: 60 μm × 48 μm; System frequency: Scanning the nanopillar array region point by point to obtain Raman spectra and imaging images; The data processing unit uses ImageJ software to count bright spots in the imaging images and uses SPSS software to perform ROC curve analysis and AUC value calculation. When the AUC value is ≥0.9, the detection result is considered valid.

9. The application of the detection device according to any one of claims 1-5 in the preparation of an early diagnostic device for serous ovarian cancer.

10. The application according to claim 9, characterized in that, The detection device distinguishes patients with stage I-IV serous ovarian cancer from healthy individuals by detecting the levels of at least four biomarkers on the surface of exosomes.

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