A microfluidic chip based on SERS and a preparation method and application thereof

Abstract

The application discloses a microfluidic chip based on SERS and a preparation method and application thereof. The microfluidic chip comprises a chip body made of PDMS and a glass slide. The main structure of the chip comprises a plurality of inlet ports of reaction reagents, a lump impurity blocking area, a circulating tumor cell capture and detection area, a solution mixing area of a herringbone channel, a positioning area of a magnet disc, an immunomagnetic sandwich structure enrichment and detection area and a waste liquid outlet port. The inlet port and the outlet port penetrate through the chip body. The lump impurity blocking area comprises a cuboid microcolumn array. The plurality of functional areas are arranged at the bottom of the chip body. The plurality of functional areas, the inlet port and the outlet port are connected through channels and are irreversibly sealed with the glass slide. The application has the advantages of simple operation, low manufacturing cost, short detection time and high sensitivity. The application has important significance for multiple detection of cancer markers.

Description

TECHNICAL FIELD

[0001] The present application relates to surface enhanced Raman spectroscopy detection method, and particularly to a preparation of a microfluidic chip based on SERS and its application in prostate cancer detection. BACKGROUND

[0002] One technology that is developing very rapidly is the development of microfluidic devices, in which microscale precision manipulation of fluid flow is used for a variety of applications, including biomedical, tissue engineering and many applications that are being explored. The development of microfluidic devices provides strong impetus for microfluidic research, which makes traditional biochemical experimental schemes small or even micro-sized, i.e. highly integrated sample preparation, reaction, separation and detection functions on a micron-scale chip, making it a micro-channel networking system, and therefore, microfluidic chip is also called Lab on a chip. In recent years, with the rapid development of micro-nano technology, microfluidic technology shows great potential in the research of cells and molecules, which also means that microfluidic chip shows great prospects in cancer diagnosis and management. Analysis of urine, blood and other tissue fluids on a microfluidic scale has led to the detection of cancer-specific biomarkers including circulating tumor cells, exosomes, proteins, and allows growth and research of tissues at the single-cell level.

[0003] In recent years, surface Raman scattering (SERS) technology as a fast vibrational spectroscopy technology has been widely used in the analysis of various substances in the environment, biology, chemistry and biology, so that various biochemical components can be detected with ultra-sensitivity. SERS technology provides fingerprint information of target molecules in very small detection volume. Compared with fluorescence detection method, SERS technology avoids the influence of self-photobleaching interference, and can give non-overlapping Raman characteristic peaks in multi-component detection, showing great application potential in biomolecule detection. Studies have found that when the analyte is adsorbed on the rough metal surface, especially when the metal nanoparticles are aggregated, the intensity of the Raman signal can be greatly enhanced, so that ultra-sensitive detection of low concentration analyte can be realized. Therefore, in order to use SERS to realize sensitive detection of target analyte, it is necessary to select appropriate nanomaterials, so as to realize ultra-sensitive trace detection of the analyte.

[0004] Prostate cancer is the second most common cause of male cancer deaths, and in China, the incidence of prostate cancer has not been significantly reduced compared to lung cancer in recent years, which is closely related to cancer screening and treatment practices. The current gold standard for prostate cancer diagnosis is an invasive detection method of transrectal ultrasound-guided biopsy. It is well known that early prostate cancer diagnosis is crucial for ensuring improved survival through simple and effective treatment, and there have been a lot of research and discussion on the international screening process for prostate cancer. However, the biomarkers of prostate cancer can be non-specific. A sensitive microfluidic detection system can detect certain markers at clinically significant levels with a small amount of liquid, plus a small size point-of-care test, providing instant cancer diagnosis, thus completely changing cancer screening. The microfluidic system can prevent patients from increasing the incidence due to prostate biopsy and allow for proper diagnosis of "liquid" with great potential for downstream research to assist in cancer management. In addition, in the high prevalence stage, microfluidic instant detection equipment that avoids biopsy and minimizes patient contact can provide help.

[0005] Prostate-specific antigen (PSA) has been widely used for early diagnosis of prostate cancer, leading to a sharp decline in the number of deaths related to prostate cancer. However, the sensitivity and specificity of PSA testing are low, which can lead to over-diagnosis and over-treatment. In the PSA gray zone, PSA values from 4-10 ng mL -1 , PSA values have been reported to be closely related to some non-malignant diseases such as benign prostatic hyperplasia and prostatitis. In this case, in order to determine the diagnosis of prostate cancer, invasive prostate biopsy is usually required. However, biopsy can cause pain, bleeding, and potential risk of tumor metastasis. Therefore, especially in the PSA gray zone, there is an urgent need to develop a highly sensitive and non-invasive alternative method for early diagnosis of prostate cancer. Circulating tumor cells as specific markers carrying all tumor "genetic factor" information, PSA detection in conjunction with them may be an effective means to improve the accuracy of prostate cancer detection, cancer grading, and determine whether subsequent biopsy is necessary.

[0006] In 2014, Goodman et al. presented a case of a 54-year-old man diagnosed with metastatic prostate cancer. The patient's disease status was monitored by imaging and serial determination of serum prostate-specific antigen concentration, performance status, and CTC count. At multiple visits, the disease status was unclear due to inconsistencies between standard assessments. The supplemental information provided by CTC testing helped to accurately determine the disease status and support treatment decisions.

[0007] In 2017, Lung et al. proposed to place the prepared biochip of three modified antibodies into the detection well of a microfluidic device, and measure the signal change through ultraviolet-visible spectrum to quantify the concentrations of PSA, VEGF and PC3 cells in a PCa sample. Although the detection result obtained has no significant difference compared with ELISA, and has the advantages of high specificity, no labeling, high speed and stability, the design is only simple integration, and the function of the microfluidic chip is not fully played.

[0008] Wang et al. proposed a hierarchical micro / nano-pleated CTC chip based on evaporation-induced graphene oxide (RGO) coating, which helps to make highly sensitive and non-invasive diagnosis of prostate cancer in the PSA gray area by combining CTC detection and PSA-based machine learning testing, and further improves the diagnostic sensitivity from 58.3% of the conventional PSA test to 91.7%. However, the method of relying on specific antibody modified substrate for static cultivation capture of CTC causes the adhesion of biomolecules on the surface of cancer cells, which is not conducive to the downstream analysis of CTC, and the designed chip only realizes the function of CTC capture, and the detection of PSA is not carried out at the same time

[0009] At present, the detection of prostate cancer circulating tumor cells (CTC) and PSA mainly has the following two ways: single PSA or multiple serum marker detection including PSA; single CTC detection, or after the design of micro device for CTC detection, the detection data of PSA is combined to realize the comprehensive consideration of two marker data. The serum marker detection of the former lacks tumor specificity, and the latter still lacks a certain accuracy for the detection data of CTC due to the rarity of CTC, and the processing mode of combining with PSA data has the problems of complex operation, high cost, large difference between samples, insufficient sensitivity and the like. SUMMARY

[0010] The purpose of the present application is to overcome the shortcomings of the existing prostate cancer detection technology, and a simple operation, low manufacturing cost, short detection time and high sensitivity SERS-based microfluidic chip device is developed.

[0011] In order to achieve the purpose of the present application, the present application proposes the following technical solutions:

[0012] A microfluidic chip based on SERS, the chip body is provided with liquid inlet 1, block impurity blocking area 2, circulating tumor cell capture detection area 3, solution mixing area 4 of herringbone channel, positioning area 5 of magnet disc, immune magnetic "sandwich" structure enrichment detection area 6 and waste liquid outlet 7 connected by connecting channel 8 in turn; the liquid inlet 1 is at least five, the block impurity blocking area 2 includes cuboid microcolumn array, at least one liquid inlet 1 through the chip body is connected with the inlet end of the block impurity blocking area 2, the outlet end of the block impurity blocking area 2 is connected with at least two liquid inlets 1 through the connecting channel 8, and then the outlet end of the block impurity blocking area 2 is connected with the inlet end of the circulating tumor cell capture detection area 3; the outlet end of the circulating tumor cell capture detection area 3 is connected with at least two liquid inlets 1 through the connecting channel 8, and then the outlet end of the circulating tumor cell capture detection area 3 is connected with the inlet end of the solution mixing area 4 of the herringbone channel; the outlet end of the solution mixing area 4 of the herringbone channel is connected with the inlet end of the immune magnetic "sandwich" structure enrichment detection area 6, the side of the immune magnetic "sandwich" structure enrichment detection area 6 is the magnet disc positioning area 5, and a magnet is placed in the magnet disc positioning area 5 to collect immune magnetic complexes; the outlet end of the immune magnetic "sandwich" structure enrichment detection area 6 is connected with the waste liquid outlet 7.

[0013] The block impurity blocking area 2, the circulating tumor cell capture detection area 3, the solution mixing area (4) of the herringbone channel, the positioning area 5 of the magnet disc, the immune magnetic "sandwich" structure enrichment detection area 6 and the connecting channel 8 are all at the bottom of the chip body, are connected with the liquid inlet 1 and the waste liquid outlet 7 through fluid channels, and are tightly sealed by a glass slide.

[0014] The further preferred technical scheme provided by the application is that the height of the chip body is 3mm-5mm, the length of the chip body is 40mm, the width of the chip body is 18mm, the diameter of the connecting channel 8 and the solution mixing area 4 of the herringbone mixing channel is 200um-400um, the diameter of the circulating tumor cell capture detection area 3 is 400um-725um, the circulating tumor cell capture detection area 3 is a three-stage pine tree type capture detection area with gradually widened overall size in series, and the inside is a mango pair type capture unit with gradually smaller spacing size of 30um-20um, the internal gap of the capture unit is 8um, the size of the immune magnetic "sandwich" structure enrichment detection area 6 is 1.4mm, and the diameter of the liquid inlet 1 and the waste liquid outlet 7 is 1.8mm.

[0015] The further preferred technical scheme provided by the application is that the liquid inlet 1, the impurity blocking area 2 and the circulating tumor cell capture detection area 3 are close to the front part of the chip body, the herringbone mixing channel 4 is in the middle part of the chip body, and the immune magnetic "sandwich" structure enrichment detection area 6 and the waste liquid outlet 7 are in the rear part of the chip body.

[0016] The further preferred technical solutions provided by the application are characterized in that the two SERS nano-detection probes used in the circulating tumor cell capture detection area 3 and the immunomagnetic "sandwich" structure enrichment detection area 6 are PSA-SERS nano-probes and CTC-SERS nano-probes; the preparation steps include:

[0017] Step A, first, sodium citrate is used to reduce gold hydrochloride to obtain 0.1 nM of gold sol, wherein the diameter of the gold nanoparticles is about 40 nm;

[0018] Step B, take 4 1.5 mL centrifuge tubes, and add 1 mL of the above gold sol into each tube, wherein 100 uL of 1 uM of apt-PSA aptamer is added into two tubes, and 100 uL of 1 uM of wy5a aptamer is added into the other two tubes; after the addition is completed, the four mixed solutions are incubated in a 50℃ metal bath for 48 hours, and 10 uL of 1×10 -5 M of Raman reporter molecule MGITC is introduced in the last two hours, and 50X PBS solution and sodium chloride solution are added multiple times during the incubation;

[0019] The apt-PSA DNA sequence is 5'-dithiol-TTTTTTTTTTA TTAAAGCTCGC CATCAAATAG CTGC-3';

[0020] The wy5a DNA sequence is: 5'-dithiol-TGCCACTACAG CTGGTTTGGT TTGGTG

[0021] ACTTCGTTCT TTGCTTAGTG GC-3'.

[0022] The application further provides a preparation method of the above-mentioned SERS-based microfluidic chip, characterized by comprising the following steps in sequence:

[0023] Step S1, first draw a chip design graph on Auto CAD, and then print it on a transparent film plate;

[0024] Step S2, use the film plate in step S1 to transfer the pattern to the silicon wafer through ultraviolet lithography, and fix the patterned silicon wafer in a culture dish;

[0025] Step S3, place the magnet disc on the magnet disc positioning area on the silicon wafer, and place a flat iron sheet under the culture dish to prevent the magnet discs from attracting each other, since multiple chip design graphs are engraved on one silicon wafer;

[0026] Step S4, the PDMS monomer and the curing agent are mixed in a mass ratio of 10:1, and after the bubbles are removed, they are poured on the silicon wafer in step S2; if the magnet disc is offset due to the pouring of the PDMS, the magnet disc is gently moved to the original position by using tweezers, and the bubbles generated are broken by blowing the ear ball in a directional manner;

[0027] Step S5, the culture dish is placed in an oven, and is baked at 75 DEG C for two hours to be cured;

[0028] Step S6, after curing, the liquid PDMS is converted into an elastomer, the culture dish is placed in a room temperature condition and is cooled for 5-10 min, then the PDMS chip region with a pattern is peeled off from the silicon wafer template;

[0029] Step S7, the inlet and outlet of the microfluidic chip are punched by using a suitable puncher, and in subsequent experiments, the punched chip can be connected with a hose so that sample liquid can flow through the whole chip system;

[0030] Step S8, the punched chip and the glass sheet are soaked in ethanol and are ultrasonically cleaned for 10 min, and after being taken out, the chip is dried on a clean hot plate;

[0031] Step S9, the chip after drying is gently adhered to the surface of the contaminant by using a medical adhesive tape, the glass sheet is gently wiped by using a dust-free cloth, the chip and the glass sheet are kept clean, and then the treated chip and the glass sheet are sealed by using oxygen plasma.

[0032] The application further provides an application of the SERS-based microfluidic chip in detection of a prostate cancer marker.

[0033] The application further provides a step of the application of the SERS-based microfluidic chip in detection of a prostate cancer marker.

[0034] Firstly, a certain amount of a sample to be measured and corresponding reagents are sucked by using a syringe, and are connected with the chip through a syringe pump and a hose, under the power of the syringe pump, the sample to be measured passes through the circulating tumor cell capture detection area 3, and PC3 cells are captured by the mango pair type capture unit due to physical size and deformability;

[0035] The sample to be measured continues to flow downwards, and the left and right two inlets 1 at the outlet end of the circulating tumor cell capture detection area 3 are connected with the specific biomolecule modified micrometer magnetic beads and the PSA-SERS nanoprobes respectively, the remaining sample to be measured is in full contact with the two in the herringbone-shaped mixing channel 4 to form an immunomagnetic complex, and the immunomagnetic “sandwich” structure enrichment detection area 6 is attracted and enriched by magnetic force.

[0036] Second, the liquid inlet 1 at the left side of the inlet end of the circulating tumor cell capture detection area 3 is connected with a buffer solution, and the non-specific adsorption molecules of the circulating tumor cell capture detection area 3 and the immunomagnetic sandwich structure enrichment detection area 6 are washed away.

[0037] Then, the microfluidic chip with the immunomagnetic sandwich structure enrichment detection area 6 enriched with the immunomagnetic complex is placed on a Raman detection platform for Raman single-point testing, and the Raman characteristic peak displacement of the PSA detection probe labeled by the Raman reporter molecule MGITC is 1614cm -1 , and the PSA is qualitatively and quantitatively detected by detecting the peak intensity at 1614cm -1 ;

[0038] The Raman spectrum is collected by a Virsa TM Raman analyzer system, the power of the helium-neon laser is 10mW, the selected laser wavelength is 632.8nm, the center of the detected Raman displacement range is set to 1200cm -1 , the laser power is 10%, the integration time is 10 seconds, and the integration number is 3 times.

[0039] Finally, the liquid inlet 1 at the right side of the inlet end of the circulating tumor cell capture detection area 3 is connected with PC3-SERS nano probes, and after standing and cultivating for 5 minutes, the liquid inlet 1 at the left side of the inlet end of the circulating tumor cell capture detection area 3 is connected with a buffer solution to remove the unbound SERS nano probes, and then Raman mapping detection is carried out: the detection range is selected, the laser wavelength is 632.8nm, the center of the detected Raman displacement range is set to 1200cm -1 , the laser power is 1%, the integration is 1 second, and the integration is 1 time. If the detected mapping image is nearly circular and the Raman signal is strong, it indicates that the PC3 cells are captured, otherwise, it is white blood cells and other molecules.

[0040] The advantages and beneficial effects of the present application are as follows:

[0041] 1. The designed microfluidic chip has low manufacturing cost and simple detection operation process.

[0042] 2. The SERS detection realizes high sensitivity, short detection time and low detection lower limit.

[0043] 3. The designed microfluidic chip can realize the combined detection of two biomarkers of prostate cancer, wherein the physical method is used to capture the circulating tumor cells PC3, so as to avoid the surface adhesion of other biological molecules and facilitate the downstream analysis of CTC; and the immunomagnetic capture is used to capture PSA, so as to improve the sensitivity and accuracy of the current detection technology. BRIEF DESCRIPTION OF DRAWINGS

[0044] In order to more clearly illustrate the technical solutions of the embodiments of the present application or the prior art, the drawings needed to be used in the embodiments or the prior art description will be briefly introduced. Obviously, the drawings in the following description are only some embodiments of the present application, and other drawings can be obtained by those skilled in the art without creative labor on the basis of these drawings.

[0045] Figure 1 is a schematic diagram of the microfluidic chip based on SERS of the present application;

[0046] Figure 2 is a schematic diagram of each region of Figure 1

[0047] Figure 3 is a schematic diagram of the circulating tumor cell capture structure region of the present application;

[0048] Figure 4 is a microscopic diagram of the immunomagnetic complex enrichment of the present application. DETAILED DESCRIPTION

[0049] The examples of the present application will be described in detail below with reference to the drawings, and the specific implementation schemes and operation processes of the examples are given with respect to the technical solutions of the present application, but the protection scope of the present application is not limited to the examples.

[0050] Referring to Figures 1-4 ​The example discloses a SERS-based microfluidic chip, which comprises a chip body and a glass slide in close sealing connection with the chip body, and the chip body is provided with a liquid inlet 1, a lump impurity blocking area 2, a circulating tumor cell capture detection area 3, a mixed solution mixing area 4 of a herringbone channel, a magnet disc positioning area 5, an immunomagnetic "sandwich" structure enrichment detection area 6 and a waste liquid outlet 7 connected through a connecting channel 8; the liquid inlet 1 is at least five, the lump impurity blocking area 2 comprises a cuboid microcolumn array, at least one liquid inlet 1 penetrating through the chip body is connected with an inlet end of the lump impurity blocking area 2, an outlet end of the lump impurity blocking area 2 is connected with at least two liquid inlets 1 through the connecting channel 8, and then the outlet end is connected with an inlet end of the circulating tumor cell capture detection area 3 after being converged; an outlet end of the circulating tumor cell capture detection area 3 is connected with at least two liquid inlets 1 through the connecting channel 8, is converged, and then is connected with an inlet end of the mixed solution mixing area 4 of the herringbone channel; an outlet end of the mixed solution mixing area 4 of the herringbone channel is connected with an inlet end of the immunomagnetic "sandwich" structure enrichment detection area 6, a magnet disc positioning area 5 is arranged on a side of the immunomagnetic "sandwich" structure enrichment detection area 6, and a magnet is arranged in the magnet disc positioning area 5 to collect an immunomagnetic complex; and an outlet end of the immunomagnetic "sandwich" structure enrichment detection area 6 is connected with the waste liquid outlet 7.

[0051] The lump impurity blocking area 2, the circulating tumor cell capture detection area 3, the mixed solution mixing area (4) of the herringbone channel, the magnet disc positioning area 5, the immunomagnetic "sandwich" structure enrichment detection area 6 and the connecting channel 8 are all arranged at the bottom of the chip body, are connected with the liquid inlets 1 and the waste liquid outlet 7 through fluid channels, and are in close sealing connection with the glass slide.

[0052] The chip body has a height of 3mm-5mm, a length of 40mm and a width of 18mm, the connecting channel 8 and the mixed solution mixing area 4 of the herringbone channel have a diameter of 200um-400um, the circulating tumor cell capture detection area 3 has a diameter of 400mm-725mm, the circulating tumor cell capture detection area 3 is a three-stage pine tree type capture detection area with gradually widened overall sizes in series, and the inside of the capture detection area is a mango pair type capture unit with gradually smaller interval sizes of 30um-20um, the inside gap of the capture unit is 8um, the immunomagnetic "sandwich" structure enrichment detection area 6 has a size of 1.4mm, and the liquid inlets 1 and the waste liquid outlet 7 have a diameter of 1.8mm.

[0053] The liquid inlets 1, the impurity blocking area 2 and the circulating tumor cell capture detection area 3 are close to the front part of the chip body, the herringbone mixing channel 4 is in the middle part of the chip body, and the immunomagnetic "sandwich" structure enrichment detection area 6 and the waste liquid outlet 7 are in the rear part of the chip body.

[0054] The circulating tumor cell capture detection zone 3 and the two SERS nano-detection probes used in the immunomagnetic "sandwich" structure enrichment detection zone 6 are PSA-SERS nano-probes and CTC-SERS nano-probes; the preparation steps include:

[0055] Step A, first reduce sodium citrate to obtain 0.1 nM of gold sol, wherein the gold nanoparticles have a diameter of about 40 nm;

[0056] Step B, take 4 1.5 mL centrifuge tubes, and add 1 mL of the above gold sol, wherein 100 uL of 1 uM of apt-PSA aptamer is added to two tubes, and 100 uL of 1 uM of wy5a aptamer is added to the other two tubes; after the addition is completed, the four tube mixtures are incubated together in a 50°C metal bath for 48 hours, and 10 uL of 1 x 10 -5 M of Raman reporter molecule MGITC is introduced during the incubation, and 50X PBS solution and sodium chloride solution are added multiple times;

[0057] The apt-PSA DNA sequence is 5'-dithiol-TTTTTTTTTTA TTAAAGCTCGCCA TCAAATAGCTGC-3';

[0058] The wy5a DNA sequence is: 5'-dithiol-TGC CAC TAC AGC TGG TTC GGT TTG GTG

[0059] ACT TCG TTC GTT GTG CTT AGT GGC-3'.

[0060] The embodiment specifically illustrates the preparation method of the above-mentioned SERS-based microfluidic chip, which comprises the following steps in sequence:

[0061] Step S1, first draw a chip design on Auto CAD, and then print it on a transparent film plate;

[0062] Step S2, use the film plate in step S1 to transfer the pattern to the silicon wafer by ultraviolet lithography, and fix the patterned silicon wafer in a culture dish;

[0063] Step S3, place the magnet disc on the magnet disc positioning area on the silicon wafer, and place a flat iron sheet under the culture dish to prevent the magnet discs from attracting each other, since multiple chip designs are engraved on one silicon wafer;

[0064] Step S4, the PDMS monomer and the curing agent are mixed in a mass ratio of 10:1, and after the bubbles are removed, they are poured on the silicon wafer described in step S2; if the magnet disc is offset due to pouring of the PDMS, it is gently moved back into place with tweezers, and the bubbles generated are broken by directional blowing with an ear bulb;

[0065] Step S5, the culture dish is placed in an oven and baked at 75 DEG C for two hours for curing;

[0066] Step S6, after curing, the liquid PDMS is converted into an elastomer, the culture dish is placed in a room temperature condition for cooling for 5-10 min, and then the PDMS chip region with a pattern is peeled off from the silicon wafer template;

[0067] Step S7, the sample inlet and the sample outlet on the microfluidic chip are punched by using an appropriate puncher, and in subsequent experiments, the punched chip can be connected with a hose so that sample liquid can flow through the whole chip system;

[0068] Step S8, the punched chip and the glass sheet are soaked in ethanol and ultrasonically cleaned for 10 min, and then taken out and placed on a clean hot plate for drying;

[0069] Step S9, the dried chip is gently adhered to the surface of the contaminant by using a medical adhesive tape, the glass sheet is gently wiped by using a dust-free cloth, the two are kept clean, and then the treated chip and the glass sheet are subjected to oxygen plasma sealing.

[0070] The microfluidic chip based on SERS can be applied in detection of a prostate cancer marker, and the specific detection steps are as follows:

[0071] Firstly, a certain amount of a sample to be detected and corresponding reagents are sucked by using a needle tube, and are connected with the chip through a syringe pump and a hose; under the power of the syringe pump, the sample to be detected passes through the circulating tumor cell capture detection area 3, and PC3 cells are captured by the mango-shaped structure due to physical size and deformability;

[0072] The sample to be detected continues to flow downward, and the left and right two liquid inlets 1 at the outlet end of the circulating tumor cell capture detection area 3 are connected with the specific biomolecule modified micrometer magnetic beads and the PSA-SERS nanoprobes respectively, the remaining sample to be detected is fully contacted with the two in the herringbone-shaped mixing channel 4 to form an immunomagnetic complex, and the immunomagnetic sandwich structure enrichment detection area 6 is attracted and enriched by magnetic force.

[0073] Secondly, the buffer solution is introduced into the left liquid inlet 1 at the inlet end of the circulating tumor cell capture detection area 3, and the non-specific adsorption molecules of the circulating tumor cell capture detection area 3 and the immunomagnetic sandwich structure enrichment detection area 6 are washed away.

[0074] Then, the microfluidic chip with the immunomagnetic "sandwich" structure enrichment detection area 6 enriched with the immunomagnetic complex is placed on the Raman detection table for Raman single-point testing, and the Raman characteristic peak of the PSA detection probe labeled by the Raman reporter molecule MGITC is shifted to 1614cm -1 , and the PSA is qualitatively and quantitatively detected by detecting the peak intensity at 1614cm -1 ;

[0075] The Raman spectrum is collected by a Virsa TM Raman analyzer system, the power of the helium-neon laser is 10mW, the selected laser wavelength is 632.8nm, the center of the detected Raman shift range is set to 1200cm -1 , the laser power is 10%, the integration time is 10 seconds, and the integration number is 3 times. The concentrations of the PSA to be tested are 0.01ng / mL, 0.1ng / mL, 1ng / mL, 5ng / mL, 10ng / mL, 50ng / mL and 100ng / mL.

[0076] Finally, the PC3-SERS nanoprobes are introduced into the liquid inlet 1 at the right side of the inlet end of the circulating tumor cell capture detection area 3, and after standing and cultivating for 5 minutes, the buffer is introduced into the liquid inlet 1 at the left side of the inlet end of the circulating tumor cell capture detection area 3 to remove the unbound SERS nanoprobes, and then Raman mapping detection is performed: the detection range is selected, the laser wavelength is 632.8nm, the center of the detected Raman shift range is set to 1200cm -1 , the laser power is 1%, the integration is 1 second, and the integration is 1 time. If the detected mapping image is nearly circular and the Raman signal is strong, it indicates that the PC3 cells are captured there, and otherwise, it is leukocytes or other molecules.

[0077] The above realizes the preparation of the microfluidic chip based on SERS and its example application in prostate cancer detection, wherein the circulating tumor cells PC3 are captured by a physical method, the surface adhesion of other biological molecules is avoided, and the downstream analysis of CTCs is facilitated; meanwhile, the immunomagnetic capture of PSA is realized, and the two cooperate to improve the sensitivity and accuracy of the current detection technology.

[0078] The above-described spectrum data and the equal changes and modifications made according to the patent application scope are covered by the scope of the claims of the present application.

Claims

1. A SERS-based microfluidic chip, characterized by: The chip body is provided with liquid inlet (1), lump impurity blocking area (2), circulating tumor cell capture detection area (3), herringbone channel solution mixing area (4), magnet disc positioning area (5), immunomagnetic "sandwich" structure enrichment detection area (6) and waste liquid outlet (7) connected by connecting channel (8) in turn; the liquid inlet (1) has at least five, the lump impurity blocking area (2) includes cuboid microcolumn array, at least one liquid inlet (1) through the chip body is connected with the inlet end of the lump impurity blocking area (2), the outlet end of the lump impurity blocking area (2) is connected with at least two liquid inlets (1) through the connecting channel (8), and then the outlet end of the lump impurity blocking area (2) is connected with the inlet end of the circulating tumor cell capture detection area (3); the outlet end of the circulating tumor cell capture detection area (3) is connected with at least two liquid inlets (1) through the connecting channel (8), and then the outlet end of the circulating tumor cell capture detection area (3) is connected with the inlet end of the herringbone channel solution mixing area (4); the outlet end of the herringbone channel solution mixing area (4) is connected with the inlet end of the immunomagnetic "sandwich" structure enrichment detection area (6), and the immunomagnetic "sandwich" structure enrichment detection area (6) is provided with the magnet disc positioning area (5) on the side, and a magnet is placed in the magnet disc positioning area (5) to collect immunomagnetic complex; the outlet end of the immunomagnetic "sandwich" structure enrichment detection area (6) is connected with the waste liquid outlet (7); The lump impurity blocking area (2), the circulating tumor cell capture detection area (3), the herringbone channel solution mixing area (4), the magnet disc positioning area (5), the immunomagnetic "sandwich" structure enrichment detection area (6) and the connecting channel (8) are all arranged on the bottom of the chip body, are connected with the liquid inlet (1) and the waste liquid outlet (7) through fluid channels, and are tightly sealed by a glass slide; The height of the chip body is 3mm-5mm, the length of the chip body is 40mm, the width of the chip body is 18mm, the diameter of the connecting channel (8) and the herringbone channel solution mixing area (4) is 200um-400um, the diameter of the circulating tumor cell capture detection area (3) is 400um-725um, the circulating tumor cell capture detection area (3) is a three-stage pine tree type capture detection area with gradually widened overall size in series, and the inside is a mango pair type capture unit with gradually smaller interval size of 30um-20um, the internal gap of the capture unit is 8um, the size of the immunomagnetic "sandwich" structure enrichment detection area (6) is 1.4mm, and the diameter of the liquid inlet (1) and the waste liquid outlet (7) is 1.8mm.

2. The SERS-based microfluidic chip of claim 1, wherein: The liquid inlet (1) and the circulating tumor cell capture detection area (3) are close to the front part of the chip body, the herringbone channel solution mixing area (4) is in the middle part of the chip body, and the immunomagnetic "sandwich" structure enrichment detection area (6) and the waste liquid outlet (7) are in the rear part of the chip body.

3. The SERS-based microfluidic chip of claim 1, wherein, The circulating tumor cell capture detection zone (3) and the two SERS nano-detection probes used in the immunomagnetic "sandwich" structure enrichment detection zone (6) are PSA-SERS nano-probes and CTC-SERS nano-probes; The preparation steps include: Step A, first reduce sodium citrate to hypochlorite gold acid to obtain 0.1 nM gold sol, wherein the gold nanoparticles have a diameter of 40 nm; Step B, take 4 1.5 mL centrifuge tubes, add 1 mL of the above gold sol into each, add 100 uL of apt-PSA aptamer with a concentration of 1 uM after thiol activation into two of the tubes, and add 100 uL of wy5a aptamer with a concentration of 1 uM after thiol activation into the other two tubes, after adding, incubate the four mixtures in a 50°C metal bath for 48 hours, introduce 10 uL of 1 x 10 -5 Raman reporter molecule MGITC of M, multiple times of 50X PBS solution and sodium chloride solution are added during the period Wherein the apt-PSA DNA sequence is 5'-dithiol-TTTTTTTTTTA TTAAAGCTCGCCA TCAAA TAGCTGC-3'; The wy5a DNA sequence is: 5'-dithiol-TGC CAC TAC AGC TGG TTC GGT TTG GTG ACT TCGTTCGTT GTG CTT AGT GGC-3'.

4. A method for preparing a SERS-based microfluidic chip according to any one of claims 1-3, characterized in that, The following steps are sequentially performed: Step S1, first draw a chip design on Auto CAD, and then print it on a transparent film plate; Step S2, use the film plate in step S1 to transfer the pattern to the silicon wafer by ultraviolet lithography, and fix the patterned silicon wafer in a culture dish; Step S3, place the magnet disc on the magnet disc positioning area on the silicon wafer, and place a flat iron sheet under the culture dish to prevent the magnet discs from attracting each other, since multiple chip designs are engraved on one silicon wafer; Step S4, mix the PDMS monomer and the curing agent in a mass ratio of 10:1, and pour them on the silicon wafer described in step S2 after removing the bubbles; if the magnet disc is shifted due to the pouring of PDMS, gently move it back to its original position with tweezers, and blow the bubbles generated by blowing the ear ball in a directional manner; Step S5, place the culture dish in an oven and bake it at 75°C for two hours for curing; Step S6, after curing, the liquid PDMS is converted into an elastomer, and the culture dish is placed in a room temperature environment for 5-10 min, and then the patterned PDMS chip area is peeled off from the silicon wafer template; Step S7, use an appropriate punch to punch the inlet and outlet of the microfluidic chip, which can be connected to a hose in subsequent experiments to allow the sample liquid to flow through the entire chip system; Step S8, soak the punched chip and glass sheet in ethanol and ultrasonically clean them for 10 min, and then place them on a clean hot plate for drying; Step S9, gently remove the surface contaminants from the dried chip with medical tape, and gently wipe the glass sheet with a dust-free cloth to keep them clean, and then seal the treated chip and glass sheet with oxygen plasma.

5. The application of a SERS-based microfluidic chip according to any one of claims 1-3 in prostate cancer marker detection.

6. The use of a SERS-based microfluidic chip in the detection of prostate cancer markers according to claim 5, characterized in that, Specifically includes the following steps: First, a certain amount of the sample to be tested and the corresponding reagent are sucked by a needle tube and connected with the chip through a syringe pump and a hose. Under the power of the syringe pump, when the sample to be tested passes through the circulating tumor cell capture detection area (3), PC3 cells are captured by the mango pair type capture unit due to physical size and deformability; The sample to be tested continues to flow down and meets the left and right two liquid inlets (1) at the outlet end of the circulating tumor cell capture detection area (3), respectively, and the specific biomolecule modified micromagnetic beads and PSA-SERS nanoprobes are introduced, and the remaining sample to be tested is fully contacted with the two in the herringbone channel solution mixing area (4) to form an immunomagnetic complex, which is attracted and enriched by magnetic force when flowing through the immunomagnetic "sandwich" structure enrichment detection area (6); Secondly, the buffer is introduced into the liquid inlet (1) on the left side of the inlet end of the circulating tumor cell capture detection area (3), so as to wash away the non-specific adsorption molecules of the circulating tumor cell capture detection area (3) and the immunomagnetic "sandwich" structure enrichment detection area (6); Then, the microfluidic chip with the immunomagnetic "sandwich" structure enrichment detection area (6) enriched with the immunomagnetic complex is placed on the Raman detection table for Raman single-point testing. The Raman characteristic peak of the PSA detection probe labeled by the Raman reporter molecule MGITC is shifted to 1614 cm -1 , and the PSA is qualitatively and quantitatively detected by detecting the peak intensity at 1614 cm -1 . Raman spectra were collected by Virsa™ Raman Analyzer System, with a helium-neon laser power of 10 mW, a selected laser wavelength of 632.8 nm, a set center of the Raman shift range for detection of 1200 cm -1 , a laser power of 10%, an integration time of 10 seconds, and an integration number of 3 times; the PSA concentrations to be tested were 0.01 ng / mL, 0.1 ng / mL, 1 ng / mL, 5 ng / mL, 10 ng / mL, 50 ng / mL, and 100 ng / mL, respectively. Finally, the liquid inlet (1) on the right side of the entrance of the circulating tumor cell capture detection zone (3) is connected to the PC3-SERS nanoprobes, and after standing for 5 minutes, the liquid inlet (1) on the left side of the entrance of the circulating tumor cell capture detection zone (3) is connected to the buffer to remove the unbound SERS nanoprobes, and then the Raman mapping detection is carried out: selecting the detection range, the laser wavelength is 632.8 nm, the center of the detected Raman shift range is set to 1200 cm -1 , the laser power is 1%, the integration is 1 second, and the integration is 1 time; if the detected mapping image is nearly circular and the Raman signal is strong, it indicates that PC3 cells are captured at this position, otherwise it is leukocytes or other molecules.

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