Preparation method of pump-free SERS (Surface Enhanced Raman Scattering) micro-fluidic chip for detecting liver cancer related miRNA (Micro Ribonucleic Acid) and pump-free SERS micro-fluidic chip

Abstract

The invention provides a preparation method of a pump-free SERS (Surface Enhanced Raman Scattering) micro-fluidic chip for detecting mi RNA (Ribonucleic Acid) related to liver cancer and the pump-free SERS micro-fluidic chip. The method comprises the following steps: 1) preparing 20nm gold particles (AuNPs) by adopting a sodium citrate reduction method; 2) respectively modifying Raman signal molecules DTNB and 4-MBA and nucleic acid complementary chains H1 and H2 on AuNPs to prepare AuNPs (at) DTNB (at) H1 and AuNPs (at) 4-MBA (at) H2; (3) modifying the surface of the magnetic core Fe3O4 with nucleic acid aptamers cDNA (complementary deoxyribonucleic acid) 1 and cDNA 2 of the mi R-21 and the mi R-155 to obtain Fe3O4 (at) cDNA 1 and Fe3O4 (at) cDNA 2 (Fe3O4 (at) cDNA); (4) the AuNPs (at) DTNB (at) H1 and the AuNPs (at) 4-MBA (at) H2 prepared in the step (2) are coupled with the Fe3O4 (at) cDNA prepared in the step (3), and Fe3O4 (at) cDNA-AuNPs (at) Raman report (at) H is obtained; and 5) mixing polydimethylsiloxane (PDMS) with a curing agent, adding the mixture into the template to prepare a micro-fluidic chip, and carrying out hydrophilic treatment to construct the pump-free SERS micro-fluidic chip. The method has the advantages of high sensitivity, high specificity, high detection speed and the like.

Description

Technical Field

[0001] The invention relates to the technical field of materials, and in particular to a preparation method of a pump-free SERS microfluidic chip for detecting liver cancer-related miRNA and a pump-free SERS microfluidic chip. Background Art

[0002] Liver cancer is one of the most common malignant tumors and the fourth leading cause of cancer-related death in the world. It has a high mortality rate and a five-year survival rate of about 15-17%. Liver cancer includes multiple subtypes such as hepatocellular carcinoma and cholangiocarcinoma. Since liver cancer has no obvious symptoms in the early stages, most patients are already in the late stages when they are diagnosed with liver cancer. Although the diagnosis and treatment of liver cancer have made great progress, its mortality rate is still rising rapidly. Therefore, early diagnosis of liver cancer is crucial, which can provide patients with better treatment and obtain higher survival rates.

[0003] In recent years, the use of microRNA (miRNA) as a biomarker for early diagnosis of gastric cancer has shown great potential in clinical applications. miRNA is a class of small endogenous non-coding RNAs with a length of 22-25 nt. It is one of the gene regulatory molecules of multicellular organisms. It has an important influence on many protein-coding genes and is involved in the proliferation, differentiation, metabolism and other processes of tumor cells. It plays an important role in the occurrence and development of tumors. Studies have shown that the expression levels of miR-21 and miR-155 in liver cancer tissues are significantly increased compared with normal liver tissues, which can promote the occurrence and development of tumors. Therefore, miR-21 and miR-155 may be prospective biomarkers for early detection of liver cancer. At present, the main methods for detecting miRNA include PCR, northern blotting, electrochemical detection and microarray. However, these methods have limitations in practical application, such as complex operation, high cost and low sensitivity. In addition, in order to improve the accuracy of detection and reduce the misdiagnosis rate, simultaneous detection of multiple miRNAs is a promising method, which can provide more comprehensive and accurate diagnostic information.

[0004] Surface enhanced Raman scattering (SERS) is a new spectral enhancement technology that improves the Raman signal of molecules. It can provide unique molecular fingerprint information, with sensitivity reaching the single-molecule level, and can reflect subtle changes in tissue biochemistry. It has a wide range of applications in biochemistry, clinical diagnosis and other fields. The localized surface plasmon resonance (LSPR) effect on the surface of noble metal nanomaterials is the main source of SERS enhancement. The electromagnetic field areas generated by these plasma resonances are called "hot spots", and their enhancement effect is closely related to the structure and elemental composition of the nanomaterials. However, due to complex processes and high costs, SERS is still lacking in real-time detection, and its sensitivity and specificity to miRNA are insufficient. Therefore, a simple, fast, and ultra-sensitive miRNA detection method is urgently needed.

[0005] The SERS technology for detecting protein biomarkers still faces many challenges to be solved before it can be truly applied clinically. The primary problem is to prepare a SERS sensor with excellent surface enhancement performance, ease of use, easy preparation, good repeatability, and good biocompatibility. 3 O 4 @cDNA-AuNPs@Raman reporter@H has good dispersion, Fe 3 O 4 The strong enrichment ability of the Au particles and the many nanogaps formed by the Au particles on the magnetic core can form more hot spots, which can significantly improve the SERS signal and ensure the stability and repeatability of the detection. Another key issue is to explore a fast, stable and efficient detection strategy. The current detection methods for miRNA have limitations such as complex operation, high cost and low sensitivity. Summary of the invention

[0006] 1. Technical issues to be resolved

[0007] In view of the deficiencies in the prior art, the present invention provides a method for preparing a pump-free SERS microfluidic chip for detecting liver cancer-related miRNA and a pump-free SERS microfluidic chip, which solves the problems raised in the background technology.

[0008] (II) Technical solution

[0009] To achieve the above objectives, the present invention is implemented by the following technical scheme: A method for preparing a pump-free SERS microfluidic chip for detecting liver cancer-related miRNA comprises the following steps:

[0010] 1) Preparation of 20 nm AuNPs by sodium citrate reduction method;

[0011] 2) Raman signal molecules DTNB, 4-MBA and nucleic acid complementary chain H 1 , H2 AuNPs@DTNB@H 1 and AuNPs@4-MBA@H 2 ;

[0012] 3) Modify the miR-21 and miR-155 nucleic acid aptamers cDNA 1 and cDNA 2 to the magnetic core Fe 3 O 4 Surface preparation of Fe 3 O 4 @cDNA1 and Fe 3 O 4 @cDNA2;

[0013] 4) The AuNPs@DTNB@H prepared in step 2) 1 、AuNPs@4-MBA@H 2 and the Fe prepared in step 3) 3 O 4 @cDNA1, Fe 3 O 4 @cDNA2 coupling to obtain Fe 3 O 4 @cDNA-AuNPs@Raman reporter@H;

[0014] 5) After mixing polydimethylsiloxane and a curing agent, the mixture is added to a template to prepare a microfluidic chip and perform a hydrophilic treatment.

[0015] Preferably, the specific implementation of step 1) is:

[0016] 1.1) Take 100 mL of 0.01 wt% HAuCl 4 The solution was heated to boiling on a magnetic stirrer;

[0017] 1.2) Rapidly add 3 mL of 1 wt% sodium citrate solution under vigorous stirring;

[0018] 1.3) After reacting for 15 min, heating was stopped and stirring was continued until the solution cooled to room temperature and then water was added to make the volume to 100 mL to prepare 20 nm AuNPs.

[0019] Preferably, the specific implementation of step 2) is:

[0020] 2.1) Add 200uL of 10mM DTNB solution to 10mL of AuNPs solution and stir vigorously at room temperature for 2h;

[0021] 2.2) The mixed solution prepared in step 2.1) was centrifuged at 10000 rpm for 10 min, and after removing the supernatant, the DTNB-labeled AuNPs, i.e., AuNPs@DTNB, were resuspended in 10 mL of PBS by ultrasonic and oscillation treatment;

[0022] 2.3) Activate H with 20 mL of 1 M TCEP 1 Added into AuNPs@DTNB solution and incubated at 37 °C for 2 h;

[0023] 2.4) The mixed solution prepared in step 2.3) was added to 100 mL of 1 wt% BSA solution, reacted for 2 h, and then centrifuged at 8000 rpm for 15 min. The supernatant was removed to prepare AuNPs@DTNB@H 1 AuNPs@4-MBA@H were prepared by the same method. 2 .

[0024] Preferably, the specific implementation of step 3) is:

[0025] 3.1) Incubate cDNA 1 and cDNA 2 at 95°C for 5 min and gradually cool to room temperature after 15 min;

[0026] 3.2) Take 10 mL of activated cDNA 1 and cDNA 2 respectively and add them to the streptavidin-modified Fe 3 O 4 The nanoparticle solution was shaken at room temperature for 30 min; the concentrations of cDNA 1 and cDNA 2 were 100 mM, and the streptavidin-modified Fe 3 O 4 The concentration of the nanoparticle solution was 1 mg / mL;

[0027] 3.3) After magnetic separation using a magnet, ultrapure water was added to wash twice to remove excess cDNA 1 and cDNA 2 to obtain Fe 3 O 4 @cDNA 1 and Fe 3 O 4 @cDNA 2, mix them in a volume ratio of 1:1 to obtain Fe 3 O 4 @cDNA.

[0028] Preferably, the specific implementation of step 4) is:

[0029] 4.1) Take the same volume of AuNPs@DTNB@H 1 and AuNPs@4-MBA@H 2 After mixing with the Fe prepared in step 3.3)3 O 4 @cDNA was mixed and incubated at 37°C; the AuNPs@DTNB@H 1 、AuNPs@4-MBA@H 2 with Fe 3 O 4 @cDNA mixing ratio was 2.5:1, and the incubation time was 80 min;

[0030] 4.2) Wash twice with PBS to remove excess labeled probes and prepare Fe 3 O 4 @cDNA-AuNPs@Ramanreporter@H.

[0031] Preferably, the specific implementation of step 5) is:

[0032] 5.1) PDMS and curing agent were uniformly mixed in a mass ratio of 10:1, and then placed in a vacuum chamber for 30 minutes to remove bubbles;

[0033] 5.2) pouring the mixed solution prepared in step 5.1) onto the prepared template, curing it on a heating plate at 100°C for 1h, and removing the PDMS layer from the template after cooling to room temperature to obtain a microfluidic chip; the microfluidic chip mainly consists of a sample addition area, a sample mixing area, an enrichment and detection area, and a comb-shaped capillary pump; the sample addition area consists of two 3mm circular chambers, the sample mixing area consists of a 2mm circular chamber and a serpentine channel, the enrichment and detection area consists of a 1.8mm long rectangular chamber and a 4mm circular magnet, the channel of the comb-shaped capillary pump is 200mm wide, and the remaining channels are 300mm wide and 120mm high;

[0034] 5.3) The microfluidic chip prepared in step 5.2) was immersed in 95% ethanol and cleaned in an ultrasonic environment for 25 minutes, dried with nitrogen, immersed in a PEG solution, and heated on a heating plate at 150° C. for 30 minutes;

[0035] 5.4) After heating, the excess PEG was washed away with isopropanol solution and placed at 4°C for 1 hour to obtain a hydrophilic treated microfluidic chip.

[0036] Based on the method for preparing a pump-free SERS microfluidic chip for detecting liver cancer-related miRNA as described above, a pump-free SERS microfluidic chip for detecting liver cancer-related microRNA is prepared by this method.

[0037] A method for detecting miR-21 and miR-155 in serum samples using a pump-free SERS microfluidic chip for detecting liver cancer-related microRNAs, the detection method comprising the following steps:

[0038] 1) Prepare different aptamer targets: miR-21, miR-155, single base mismatch sequences (MT1-1, MT1-2), three base mismatch sequences (MT3-1, MT3-2) and random sequences, and combine them with Fe 3 O 4 @cDNA-AuNPs@Ramanreporter@H were simultaneously added to the pump-free SERS microfluidic chip for detecting liver cancer-related microRNA, and the reaction was carried out in a 37°C constant temperature box. After the pump-free SERS microfluidic chip was taken out, SERS detection was performed. In the presence of miR-21 and miR-155, the signal intensity at the characteristic peaks of 1080cm-1 and 1330cm-1 was significantly reduced.

[0039] 2) Different concentrations of miR-21 and miR-155 were dispersed into serum and compared with Fe 3 O 4 @cDNA-AuNPs@Ramanreporter@H were simultaneously dripped into a pump-free SERS microfluidic chip for detecting liver cancer-related microRNAs, and reacted in a 37°C incubator to obtain SERS spectra of different concentrations of miR-21 and miR-155 in serum. The analysis showed that the logarithm of the miR-21 concentration was linearly related to the intensity of the characteristic peak at 1330 cm-1, and the logarithm of the miR-155 concentration was linearly related to the intensity of the characteristic peak at 1080 cm-1, and finally their linear equations were obtained.

[0040] 3) The collected serum samples were mixed with Fe 3 O 4 @cDNA-AuNPs@Raman reporter@H were simultaneously added to the pump-free SERS microfluidic chip for detecting liver cancer-related microRNA, reacted in a 37°C incubator, and the pump-free SERS microfluidic chip was taken out for SERS testing to detect the SERS signal of the serum sample.

[0041] 4) Substituting the SERS signal of the serum sample detected in step 3) into the linear equation determined in step 2) to determine the expression levels of miR-21 and miR-155 in the serum sample.

[0042] Preferably, the reaction time in a 37° C. incubator is 5 minutes.

[0043] (III) Beneficial effects

[0044] The present invention provides a method for preparing a pump-free SERS microfluidic chip for detecting liver cancer-related miRNA and a pump-free SERS microfluidic chip, which has the following beneficial effects:

[0045] 1. The present invention provides a method for preparing a pump-free SERS microfluidic chip for detecting liver cancer-related miRNA and a pump-free SERS microfluidic chip. The pump-free SERS microfluidic chip has the advantages of high sensitivity, strong specificity, excellent repeatability, and fast detection speed. By measuring the signal intensity corresponding to different concentrations of miR-21 and miR-155 in serum, a quantitative relationship between the concentration of miR-21 and miR-155 and the SERS signal intensity is established. The concentrations of miR-21 and miR-155 in serum samples were detected based on the quantitative relationship, and the accuracy was very high. The results show that the pump-free SERS microfluidic chip of the present invention can be used for ultra-sensitive simultaneous detection of liver cancer-related miR-21 and miR-155 in serum samples, providing technical support for the development of SERS for wide application in the diagnosis and screening of liver cancer.

[0046] 2. The preparation method of the pump-free SERS microfluidic chip provided by the present invention is simple, highly operable, and does not require special instruments and equipment. The experimental cost is low, and the required reagents are all commonly used chemical reagents. The pump-free SERS microfluidic chip prepared by the present invention has good repeatability and can be prepared on a large scale. The recognition competition strategy proposed by the present invention has the advantages of being simple, fast, low in dosage, and high in throughput. 3 O 4 It has good enrichment ability, can capture more targets, and separate them from complex environments under the action of magnets. AuNPs are coupled to Fe-modified aptamers through nucleic acid complementary chains. 3 O 4 On the surface, abundant “hot spots” are formed, which greatly enhances the SERS signal. 3 O 4@cDNA-AuNPs@Raman reporter@H takes advantage of the strong specificity between nucleic acid aptamers and miR-21 and miR-155, with higher accuracy and sensitivity, which is superior to other detection strategies. The SERS microfluidic chip designed with a capillary pump gets rid of the need for an external heavy pump, and can quickly and sensitively complete the detection of miR-21 and miR-155 in a short time. Studies have shown that miR-21 is associated with the staging and poor prognosis of liver cirrhosis and liver cancer. Compared with normal cells, the expression level of miR-21 in cancer cells is significantly upregulated, which can promote tumor growth and proliferation. MiR-155 also plays an important regulatory role in tumor development. Studies have found that miR-155 expression is upregulated in hepatocellular carcinoma cells. MiR-155 promotes tumor progression by regulating the expression of proteins such as H3F3A, and can also promote the activity of hepatocellular carcinoma cells by inhibiting the PTEN / PI3K-AKT pathway. Therefore, miR-21 and miR-155 may be prospective biomarkers for early detection of HCC. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Figure 1A The Fe prepared by the present invention 3 O 4 SEM photos of

[0048] Figure 1B The Fe prepared by the present invention 3 O 4 TEM photos of

[0049] Figure 1C The Fe prepared by the present invention 3 O 4 UV-vis spectrum;

[0050] Figure 1D is a SEM photo of AuNPs prepared by the present invention;

[0051] Figure 1E is a TEM photo of AuNPs prepared by the present invention;

[0052] Figure 1F is the UV-vis spectrum of AuNPs prepared by the present invention;

[0053] Figure 1G The Fe prepared in the present invention 3 O 4 SEM image of @cDNA-AuNPs@Raman reporter@H;

[0054] Figure 1H The Fe prepared in the present invention 3 O 4TEM image of @cDNA-AuNPs@Raman reporter@H;

[0055] Fig. 1I The Fe prepared by the present invention 3 O 4 HRTEM image of @cDNA-AuNPs@Raman reporter@H;

[0056] Figure 1J The Fe prepared by the present invention 3 O 4 Electron diffraction image of @cDNA-AuNPs@Raman reporter@H;

[0057] Figure 1K The Fe prepared by the present invention 3 O 4 Elemental mapping image of @cDNA-AuNPs@Raman reporter@H;

[0058] Figure 1L The Fe prepared by the present invention 3 O 4 Elemental mapping image of @cDNA-AuNPs@Raman reporter@H;

[0059] Figure 1M The Fe prepared by the present invention 3 O 4 Elemental mapping image of @cDNA-AuNPs@Raman reporter@H;

[0060] Figure 1N The Fe prepared by the present invention 3 O 4 Elemental mapping image of @cDNA-AuNPs@Raman reporter@H;

[0061] Figure 2A It is a schematic diagram of the preparation process of the pump-free SERS microfluidic chip synthesized by the present invention and the process of using the pump-free SERS microfluidic chip to detect liver cancer-related microRNAs;

[0062] Figure 3A It is Fe 3 O 4 With AuNPs@DTNB@H 1 and AuNPs@4-MBA@H 2 The SERS signal intensity when the mixture is hybridized at different ratios;

[0063] Figure 3B It is Fe3 O 4 With AuNPs@DTNB@H 1 and AuNPs@4-MBA@H 2 The SERS signal intensity bar graph when the mixed solution is hybridized at different ratios;

[0064] Figure 3C is Fe at different temperatures 3 O 4 With AuNPs@DTNB@H 1 and AuNPs@4-MBA@H 2 The SERS signal intensity during hybridization of the mixed solution;

[0065] Figure 3D is Fe at different temperatures 3 O 4 With AuNPs@DTNB@H 1 and AuNPs@4-MBA@H 2 The SERS signal intensity bar graph of the mixed solution during hybridization;

[0066] Figure 3E Fe at different times 3 O 4 With AuNPs@DTNB@H 1 and AuNPs@4-MBA@H 2 The SERS signal intensity during hybridization of the mixed solution;

[0067] Figure 3F Fe at different times 3 O 4 With AuNPs@DTNB@H 1 and AuNPs@4-MBA@H 2 The SERS signal intensity bar graph of the mixed solution during hybridization;

[0068] Figure 4A It is the inks of different colors that flow freely in the channels of the pump-free SERS microfluidic chip;

[0069] Figure 4B are micrographs of two areas in the microfluidic chip;

[0070] Figure 4C These are the corresponding SERS spectra of regions I and II;

[0071] Figure 4D is the intensity change of the characteristic peaks of the SERS signal at 1080 cm-1 and 1330 cm-1 in the rectangular cavity;

[0072] Figure 5AIt is the SERS spectra of pump-free SERS microfluidic chips prepared in different batches;

[0073] Figure 5B It is a dot-line graph of the characteristic peak intensity at 1080cm-1 and 1330cm-1;

[0074] Figure 5C It is the SERS spectra of the pump-free SERS microfluidic chip stored at room temperature for different time periods;

[0075] Figure 5D It is a dot-line graph of the characteristic peak intensity at 1080cm-1 and 1330cm-1;

[0076] Figure 5E This is the specific SERS spectrum of the pump-free SERS microfluidic chip;

[0077] Fig. 5F It is a bar graph of the intensity of the characteristic peaks at 1080cm-1 and 1330cm-1;

[0078] Fig. 6A are the SERS spectra of different concentrations of miR-21 and miR-155 (10 pM-10 mM) in serum;

[0079] Figure 6B is the calibration curve of the characteristic peak intensity at 1080 cm-1 and the logarithm of miR-155 concentration;

[0080] Figure 6C is the calibration curve of the characteristic peak intensity at 1330 cm-1 and the logarithm of miR-21 concentration;

[0081] Fig. 7A It is fluorescent in vivo imaging of nude mice;

[0082] Figure 7B HE staining of nude mouse liver tissue;

[0083] Figure 7C is the average weight of nude mice in each group;

[0084] Fig. 8A is the serum SERS spectra of nude mice in the control group on days 0, 10, 20, and 30;

[0085] Figure 8B is the characteristic peak intensity at 1080 cm-1 and 1330 cm-1 of nude mice in the control group;

[0086] Figure 8C The serum SERS spectra of nude mice with orthotopic liver cancer on days 0, 10, 20, and 30;

[0087] Fig.8DIt is the characteristic peak intensity of nude mice with in situ liver cancer at 1080cm-1 and 1330cm-1. DETAILED DESCRIPTION

[0088] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0089] The instruments and test conditions used in the present invention are as follows:

[0090] The ultraviolet-visible-near-infrared (UV-vis-NIR) absorption spectra were measured using a UNICO 2100PC UV-Vis spectrophotometer (Japan).

[0091] Scanning electron microscopy (SEM) images were taken using a S-4800Ⅱ field emission scanning electron microscope.

[0092] Transmission electron microscopy (TEM) images were taken using a Tecnai 12 transmission electron microscope (120 KV).

[0093] High-resolution images and elemental mapping were obtained using a Tecnai G2 F30 field emission transmission electron microscope.

[0094] All SERS spectra were obtained using an inVia Raman spectrometer (UK). The test conditions were a laser wavelength of 785 nm, an exposure time of 10 s, a laser intensity of 50 mW, and a 50× objective lens. Spectral processing was performed using Renishaw software (WiRETM) for operation and data acquisition, and baseline correction was performed to suppress background noise.

[0095] Example 1: Fe 3 O 4 Preparation and characterization of @cDNA-AuNPs@Raman reporter@H

[0096] 1) Take 100 mL of HAuCl 4 The solution (0.01 wt%) was heated to boiling on a magnetic stirrer;

[0097] 2) quickly adding 3 mL of 1 wt % sodium citrate solution (TSC) under vigorous stirring;

[0098] 3) After 15 min of reaction, heating was stopped, and stirring was continued until the solution cooled to room temperature, and then water was added to make the volume 100 mL to prepare 20 nm AuNPs;

[0099] 4) Add 200uL of 10mM DTNB solution to 10mL of AuNPs solution and stir vigorously at room temperature for 2h;

[0100] 5) The mixed solution prepared in step 4) was centrifuged at 10000 rpm for 10 min, the supernatant was removed, and the DTNB-labeled AuNPs (AuNPs@DTNB) were resuspended in 10 mL of PBS by ultrasonic and oscillation treatment;

[0101] 6) Activate H with 20 mL of 1 M TCEP 1 (0.1 mM) was added to the AuNPs@DTNB solution and incubated at 37 °C for 2 h;

[0102] 7) The mixed solution prepared in step 6) was added to 100 mL of BSA (1 wt%) solution, reacted for 2 h, and then centrifuged at 8000 rpm for 15 min. The supernatant was removed to prepare AuNPs@DTNB@H 1 AuNPs@4-MBA@H were prepared by the same method. 2 ;

[0103] 8) Incubate cDNA 1 and cDNA 2 at 95°C for 5 min and gradually cool to room temperature after 15 min;

[0104] 9) Take 10 mL of activated cDNA 1 and cDNA 2 respectively and add them to the streptavidin-modified Fe 3 O 4 The nanoparticle solution was shaken at room temperature for 30 min;

[0105] 10) After magnetic separation using a magnet, wash twice with ultrapure water to remove excess cDNA 1 and cDNA 2 to obtain Fe 3 O 4 @cDNA 1 and Fe 3 O 4 @cDNA 2, mix them in a volume ratio of 1:1 to obtain Fe 3 O 4 @cDNA;

[0106] 11) Take the same volume of AuNPs@DTNB@H 1 and AuNPs@4-MBA@H 2 After mixing, it was mixed with Fe3O4@cDNA and incubated at 37 °C;

[0107] 12) Wash twice with PBS to remove excess labeled probes and prepare Fe 3 O 4@cDNA-AuNPs@Ramanreporter@H;

[0108] 13)Fe 3 O 4 Morphological characterization of @cDNA-AuNPs@Raman reporter@H.

[0109] Figure 1A and Figure 1B Fe 3 O 4 SEM and TEM images of the magnetic core: Fe 3 O 4 The core is spherical in shape, with an average particle size of about 100 nm. 3 O 4 The magnetic cores are evenly distributed, which shows that they have good dispersion. Figure 1C It is Fe 3 O 4 The UV spectrum of Fe 3 O 4 The absorption peak is at 413nm. Figure 1D and Figure 1E The SEM and TEM images of AuNPs are shown respectively. It can be seen from the images that AuNPs have a spherical morphology with an average particle size of about 20 nm. The UV spectrum is as follows Figure 1F As shown, the absorption peak of AuNPs is at 522 nm, and it can be seen that AuNPs have uniform morphology and size. Figure 1G , Figure 1H , Fig. 1I Fe 3 O 4 SEM, TEM and HRTEM imaging were performed on @cDNA-AuNPs@Ramanreporter@H. It can be seen from the images that Fe 3 O 4 @cDNA-AuNPs@Ramanreporter@H has a diameter of about 140nm and good dispersibility. 20nm AuNPs are evenly distributed on the Fe 3 O 4 surface. Figure 1J It is the lattice image of AuNPs, showing that its fringe spacing is 0.243nm, which is a polycrystalline structure. Figure 1K to Figure 1N It is Fe 3 O 4 Elemental mapping image of @cDNA-AuNPs@Raman reporter@H, showing that Fe 3 O 4 The main components of @cDNA-AuNPs@Raman reporter@H are Fe and Au.

[0110] Example 2: Optimizing experimental conditions

[0111] 1) Take the same volume of AuNPs@DTNB@H 1 and AuNPs@4-MBA@H 2 After mixing, according to different volumes, with Fe 3 O 4 @cDNA mixed to form different ratios (Fe 3 O 4 @cDNA: mixed solution = 1:1, 1:1.5, 1:2, 1:2.5, 1:3, 1:3.5), and incubated at 37°C for 1.5 h respectively; the AuNPs@DTNB@H 1 and AuNPs@4-MBA@H 2 The nucleotide sequence can bind to Fe 3 O 4 @cDNA nucleotide sequence-specific complementary pairing to obtain Fe 3 O 4 @cDNA-AuNPs@Raman reporter@H;

[0112] 2) The Fe prepared in different volume ratios 3 O 4 @cDNA-AuNPs@Raman reporter@H for SERS detection, such as Figure 3A and Figure 3B As shown, when Fe 3 O 4 @cDNA and AuNPs@DTNB@H 1 and AuNPs@4-MBA@H 2 When the ratio of the mixed solution is 1:2.5, the characteristic peak intensities at 1080cm-1 and 1330cm-1 reach the highest. When the volume of the signal probe continues to increase, the signal intensities of these two characteristic peaks change very little, indicating that the coupled signal probe on the capture probe has been saturated. This indicates that when Fe 3 O 4 The volume ratio of @cDNA and AuNPs@H is 1:2.5, which is the optimal ratio for constructing SERS sensors. Figure 3C and Figure 3D It shows that the intensity of the characteristic peaks at 1080cm-1 and 1330cm-1 increases with time. When the time reaches 80min, the SERS signal intensity does not change significantly, and the growth trend almost disappears. 3 O 4The optimal incubation time of @cDNA and AuNPs@H is 80min. They are incubated at different temperatures, and the measured SERS signal intensity is as follows Figure 3E and Figure 3F As shown in the figure, the intensity of the characteristic peaks at 1080 cm-1 and 1330 cm-1 gradually increases with the increase of temperature until it stabilizes at 37°C. Therefore, 37°C was selected as the experimental temperature.

[0113] Example 3: Characterization of the pump-free SERS microfluidic chip

[0114] 1) PDMS and curing agent were uniformly mixed in a mass ratio of 10:1, and then placed in a vacuum chamber for 30 minutes to remove bubbles;

[0115] 2) pouring the prepared mixed solution onto the prepared template, curing it on a hot plate at 100° C. for 1 h, and removing the PDMS layer from the template after cooling to room temperature to obtain a microfluidic chip;

[0116] 3) The prepared microfluidic chip was immersed in 95% ethanol and cleaned in an ultrasonic environment for 25 minutes, dried with nitrogen, immersed in a PEG solution, and heated on a heating plate at 150° C. for 30 minutes;

[0117] 4) After heating, excess PEG was washed away with isopropanol solution and placed at 4° C. for 1 hour to obtain a hydrophilic treated microfluidic chip.

[0118] The microfluidic chip is mainly composed of a sample loading area (I), a sample mixing area (II), an enrichment and detection area (III), and a comb-shaped capillary pump (IV). The sample loading area consists of two 3mm circular chambers, the sample mixing area consists of a 2mm circular chamber and a serpentine channel, the enrichment and detection area consists of a 1.8mm long rectangular chamber and a 4mm circular magnet, the comb-shaped capillary pump channel is 200mm wide, and the remaining channels are 300mm wide and 120mm high.

[0119] Figure 4A This is an image of inks of different colors flowing freely in the channel of the pump-free SERS microfluidic chip. As shown in the figure, under the action of the comb capillary pump, the ink can flow freely in the channel and fill the entire channel in 60 seconds without any leakage, indicating that the prepared pump-free SERS microfluidic chip has good hydrophilicity and sealing properties. Figure 4B These are micrographs of two regions in the pump-free SERS microfluidic chip. Figure 4CThe corresponding SERS spectra of these two regions are shown in Figure 2. Since there are no Raman signal molecules in region I, the Raman signal in this region is completely generated by PDMS. The characteristic peak intensity of region I at 1080cm-1 and 1330cm-1 is significantly lower than that of region II. Therefore, it is confirmed that the microfluidic chip made of PDMS will not affect the detection of miR-21 and miR-155. In order to find the best measurement time for the pump-free SERS microfluidic chip, we measured the SERS signal in the rectangular cavity at different times. Figure 4D The intensity changes of the characteristic peaks of the SERS signal at 1080cm-1 and 1330cm-1 in the rectangular cavity are shown. The results show that the SERS intensity decreases with time and hardly changes at 5 minutes. It can be seen that the optimal measurement time of the prepared pump-free SERS microfluidic chip is 5 minutes.

[0120] Example 4: Analysis of Stability, Repeatability and Specificity of Pump-free SERS Microfluidic Chip

[0121] 1) Prepare Fe in the same manner as in Examples 1 and 3 3 O 4 @cDNA-AuNPs@Raman reporter@H and microfluidic chip, Fe 3 O 4 @cDNA-AuNPs@Raman reporter@H was added to the microfluidic chip for detection;

[0122] 2) Five different batches of SERS microfluidic chips were prepared and their SERS spectra were measured. Figure 5A These are the SERS spectra of pump-free SERS microfluidic chips prepared in different batches. Figure 5B It is a dot-line graph of the characteristic peak intensity at 1080cm-1 and 1330cm-1 of pump-free SERS microfluidic chips prepared in different batches. As can be seen from the figure, there is no obvious difference in the SERS spectra of the five different batches of pump-free SERS microfluidic chips prepared. The relative standard deviation (RSD) of the Raman intensity at the characteristic peaks of 1080cm-1 and 1330cm-1 is calculated to be 5.68% and 5.63%, respectively, indicating that the prepared pump-free SERS microfluidic chip has good repeatability.

[0123] 3) The pump-free SERS microfluidic chips were stored at room temperature for different time periods (1d, 5d, 10d, 15d, 20d) and their SERS signal intensities were measured. Figure 5C It can be seen that the morphology of the spectrum has not changed significantly. Figure 5DThe changes in the SERS signal intensity at the characteristic peaks of 1080cm-1 and 1330cm-1 from 1 to 20 days are shown, and their RSD values ​​are calculated to be 5.64% and 5.72%, respectively, indicating that the prepared pump-free SERS microfluidic chip has good stability.

[0124] 4) Prepare different aptamer targets: miR-21, miR-155, single-base mismatch sequences (MT1-1, MT1-2), three-base mismatch sequences (MT3-1, MT3-2) and random sequences, and add them into the pump-free SERS microfluidic chip in different combinations for detection. Figure 5E The specific SERS spectra of the pump-free SERS microfluidic chip are shown. It can be seen that when the target miR-21 and miR-155 are present, the corresponding characteristic peaks are significantly reduced, and the SERS signal intensity change caused by the target miRNA is significantly higher than that of the interferent. Fig. 5F The bar graph showing the intensity of the characteristic peaks at 1080cm-1 and 1330cm-1 shows that the changes in the characteristic peaks at 1080cm-1 and 1330cm-1 represented by the two target miRNAs are stronger than those of the interferents. It can be seen that the prepared pump-free SERS microfluidic chip is highly sensitive to miR-21 and miR-155, and can accurately distinguish miR-21, miR-155 and interferents, with good specificity.

[0125] Example 5: Quantification of miR-21 and miR-155

[0126] 1) Prepared in the same manner as in Examples 1 and 3, Fe 3 O 4 @cDNA-AuNPs@Raman reporter@H and microfluidic chip;

[0127] 2) Disperse a mixture of miR-21 and miR-155 (miR-21:miR-155=1:1) at different concentrations (10pM to 10mM) into serum, and mix the mixture with Fe 3 O 4 @cDNA-AuNPs@Raman reporter@H was simultaneously added to the pump-free SERS microfluidic chip for SERS detection. Fig. 6A As shown in Figure 2, as the concentration of target miRNA in serum increases, the SERS signal gradually decreases. Figure 6B and Figure 6CThe SERS signals at 1080 cm-1 and 1330 cm-1 showed a linear relationship with the logarithm of the target miRNA concentration in serum in the range of 10 pM to 10 mM, and the corresponding regression equations were y = -2221.525x-6883.168 (R2 = 0.996) and y = -2613.809x-8185.615 (R2 = 0.996). The LODs of miR-21 and miR-155 were calculated to be 2.39 pM and 2.49 pM, respectively. Compared with other detection strategies reported previously (Table 1), the detection results of the pump-free SERS microfluidic chip prepared by us are similar to them, but the detection time is greatly shortened, which is better than most reported methods.

[0128] Table 1 Comparison of the pump-free SERS microfluidic chip with existing detection methods

[0129]

[0130] Example 6: Characterization of Hepatocellular Carcinoma in Mice

[0131] 1) Use the small animal in vivo imaging system to photograph nude mice, observe and compare the growth of in situ tumors at different time points, such as Fig. 7A As shown in the figure, the fluorescence intensity of the nude mouse liver cancer in situ increases with time. HE staining shows that the tumor cells of the nude mouse liver cancer are arranged in disorder, of different sizes, with dark-stained nuclei and pathological divisions ( Figure 7B ). The nude mice were weighed simultaneously when the serum was collected. Figure 7C It can be seen that the body weight of nude mice in the liver cancer model continued to decrease compared with the normal control group.

[0132] Example 7: Detection of miR-21 and miR-155 in serum samples of liver cancer mice using a pump-free SERS microfluidic chip

[0133] 1) Prepare Fe in the same manner as in Examples 1 and 3 3 O 4 @cDNA-AuNPs@Raman reporter@H and microfluidic chip;

[0134] 2) Collect serum samples and Fe 3 O 4 @cDNA-AuNPs@Raman reporter@H was added to the pump-free SERS microfluidic chip at the same time, and the reaction was carried out in a constant temperature box at 37°C. After 5 minutes, it was taken out for SERS testing, and the SERS signal of the serum sample was detected. Fig. 8A and Figure 8BThe average SERS spectrum of nude mice at 0d (control group) and the SERS intensity at the characteristic peaks of 1080cm-1 and 1330cm-1 are shown. It can be seen that the serum SERS intensity of nude mice in the control group did not change significantly over time. Figure 8C The average SERS spectrum of nude mice with orthotopic liver cancer decreased significantly with the growth of the tumor, and the corresponding SERS intensities at the characteristic peaks of 1080 cm-1 and 1330 cm-1 were as follows: Fig.8D As shown. Obviously, the signals of miR-21 and miR-155 in nude mice with orthotopic liver cancer were higher than those in the control group, and the expression levels continued to increase with the development of the tumor. The results of the pump-free SERS microfluidic chip for detecting miR-21 and miR-155 were compared with the results of qRT-PCR as shown in Table 2, which showed a high consistency. This shows that the pump-free SERS microfluidic chip proposed in this study has a high accuracy rate for the simultaneous and ultrasensitive detection of miR-21 and miR-155, and has a good application prospect.

[0135] Table 2 Comparison of SERS and qRT-PCR detection results

[0136]

[0137] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for preparing a pump-free SERS microfluidic chip for detecting liver cancer-related miRNA, characterized in that: The following steps are involved: 1) Preparation of 20 nm AuNPs by sodium citrate reduction method; 2) Raman signal molecules DTNB, 4-MBA and nucleic acid complementary chains H1 and H2 were modified on AuNPs to prepare AuNPs@DTNB@H1 and AuNPs@4-MBA@H2; 3) The nucleic acid aptamers cDNA 1 and cDNA 2 of miR-21 and miR-155 were modified onto the surface of the magnetic core Fe3O4 to prepare Fe3O4@cDNA1 and Fe3O4@cDNA2; 4) AuNPs@DTNB@H1 and AuNPs@4-MBA@H2 prepared in step 2) and Fe3O4@cDNA1 and Fe3O4@cDNA2 prepared in step 3) are coupled to obtain Fe3O4@cDNA-AuNPs@Raman reporter@H; 5) After mixing polydimethylsiloxane and a curing agent, the mixture is added to a template to prepare a microfluidic chip and perform a hydrophilic treatment.

2. The method for preparing a pump-free SERS microfluidic chip for detecting liver cancer-related miRNA according to claim 1, characterized in that: The specific implementation method of step 1) is: 1.1) Take 100 mL of 0.01 wt% HAuCl4 solution and place it on a magnetic stirrer and heat to boil; 1.2) Rapidly add 3 mL of 1 wt% sodium citrate solution under vigorous stirring; 1.3) After reacting for 15 min, heating was stopped and stirring was continued until the solution cooled to room temperature and then water was added to make the volume to 100 mL to prepare 20 nm AuNPs.

3. The method for preparing a pump-free SERS microfluidic chip for detecting liver cancer-related miRNA according to claim 2, characterized in that: The specific implementation method of step 2) is: 2.1) Add 200uL of 10mM DTNB solution to 10mL of AuNPs solution and stir vigorously at room temperature for 2h; 2.2) The mixed solution prepared in step 2.1) was centrifuged at 10000 rpm for 10 min, and after removing the supernatant, the DTNB-labeled AuNPs, i.e., AuNPs@DTNB, were resuspended in 10 mL of PBS by ultrasonic and oscillation treatment; 2.3) Add 20 mL of 1 M TCEP-activated H1 into the AuNPs@DTNB solution and incubate at 37 °C for 2 h; 2.4) The mixed solution prepared in step 2.3) was added to 100 mL of 1 wt% BSA solution, reacted for 2 h, and then centrifuged at 8000 rpm for 15 min. The supernatant was removed to prepare AuNPs@DTNB@H1. AuNPs@4-MBA@H2 was prepared in the same manner.

4. The method for preparing a pump-free SERS microfluidic chip for detecting liver cancer-related miRNA according to claim 3, characterized in that: The specific implementation method of step 3) is: 3.1) Incubate cDNA 1 and cDNA 2 at 95°C for 5 min and gradually cool to room temperature after 15 min; 3.2) 10 mL of each of the activated cDNA 1 and cDNA 2 were added to the streptavidin-modified Fe3O4 nanoparticle solution, and shaken at room temperature for 30 min; the concentrations of the cDNA 1 and cDNA 2 were 100 mM, and the concentration of the streptavidin-modified Fe3O4 nanoparticle solution was 1 mg / mL; 3.3) After magnetic separation using a magnet, ultrapure water was added to wash twice to remove excess cDNA 1 and cDNA 2 to obtain Fe3O4@cDNA 1 and Fe3O4@cDNA 2, which were mixed at a volume ratio of 1:1 to obtain Fe3O4@cDNA.

5. The method for preparing a pump-free SERS microfluidic chip for detecting liver cancer-related miRNA according to claim 4, characterized in that: The specific implementation method of step 4) is: 4.1) taking the same volume of AuNPs@DTNB@H1 and AuNPs@4-MBA@H2 and mixing them with the Fe3O4@cDNA prepared in step 3.3), and incubating at 37°C; the mixing ratio of AuNPs@DTNB@H1, AuNPs@4-MBA@H2 and Fe3O4@cDNA is 2.5:1, and the incubation time is 80 min; 4.2) Wash twice with PBS to remove excess labeled probes to prepare Fe3O4@cDNA-AuNPs@Ramanreporter@H.

6. The method for preparing a pump-free SERS microfluidic chip for detecting liver cancer-related miRNA according to claim 5, characterized in that: The specific implementation of step 5) is: 5.1) PDMS and curing agent were uniformly mixed in a mass ratio of 10:1, and then placed in a vacuum chamber for 30 minutes to remove bubbles; 5.2) pouring the mixed solution prepared in step 5.1) onto the prepared template, curing it on a heating plate at 100°C for 1h, and removing the PDMS layer from the template after cooling to room temperature to obtain a microfluidic chip; the microfluidic chip mainly consists of a sample addition area, a sample mixing area, an enrichment and detection area, and a comb-shaped capillary pump; the sample addition area consists of two 3mm circular chambers, the sample mixing area consists of a 2mm circular chamber and a serpentine channel, the enrichment and detection area consists of a 1.8mm long rectangular chamber and a 4mm circular magnet, the channel of the comb-shaped capillary pump is 200mm wide, and the remaining channels are 300mm wide and 120mm high; 5.3) The microfluidic chip prepared in step 5.2) was immersed in 95% ethanol and cleaned in an ultrasonic environment for 25 minutes, dried with nitrogen, immersed in a PEG solution, and heated on a heating plate at 150° C. for 30 minutes; 5.4) After heating, the excess PEG was washed away with isopropanol solution and placed at 4°C for 1 hour to obtain a hydrophilic treated microfluidic chip.

7. The method for preparing a pump-free SERS microfluidic chip for detecting liver cancer-related miRNA according to claim 6, characterized in that: A pump-free SERS microfluidic chip for detecting liver cancer-related miR-2 and miR-155 was prepared by this method.

8. A method for detecting miR-21 and miR-155 in serum samples using a pump-free SERS microfluidic chip for detecting liver cancer-related microRNAs, characterized in that: The detection method comprises the following steps: 1) preparing different aptamer targets: miR-21, miR-155, single base mismatch sequences (MT1-1, MT1-2), three base mismatch sequences (MT3-1, MT3-2) and random sequences, adding them in different combinations and Fe3O4@cDNA-AuNPs@Ramanreporter@H to the pump-free SERS microfluidic chip for detecting liver cancer-related microRNA prepared as claimed in claim 7, and reacting in a 37°C constant temperature box; taking out the pump-free SERS microfluidic chip and performing SERS detection; in the presence of miR-21 and miR-155, the signal intensity at the characteristic peaks of 1080cm-1 and 1330cm-1 was significantly reduced; 2) Dispersing different concentrations of miR-21 and miR-155 into serum, and simultaneously dropping them into the pump-free SERS microfluidic chip for detecting liver cancer-related microRNA prepared as claimed in claim 7 together with Fe3O4@cDNA-AuNPs@Ramanreporter@H, reacting in a 37°C incubator, and obtaining SERS spectra of different concentrations of miR-21 and miR-155 in serum; analyzing and obtaining that the logarithm of the miR-21 concentration is linearly related to the intensity of the characteristic peak at 1330 cm-1, and the logarithm of the miR-155 concentration is linearly related to the intensity of the characteristic peak at 1080 cm-1, and finally obtaining their linear equations; 3) adding the collected serum sample and Fe3O4@cDNA-AuNPs@Raman reporter@H simultaneously to the pump-free SERS microfluidic chip for detecting liver cancer-related microRNA prepared as claimed in claim 7, reacting in a 37° C. incubator, taking out the pump-free SERS microfluidic chip for SERS testing, and detecting the SERS signal of the serum sample; 4) Substituting the SERS signal of the serum sample detected in step 3) into the linear equation determined in step 2) to determine the expression levels of miR-21 and miR-155 in the serum sample.

9. The method for detecting miR-21 and miR-155 in serum samples using a pump-free SERS microfluidic chip for detecting liver cancer-related microRNA according to claim 8, characterized in that: The reaction time was 5 minutes in a 37°C incubator.

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