Preparation method of a pump-free SERS microfluidic chip for detecting liver cancer-related miRNA and the pump-free SERS microfluidic chip

Abstract

The present invention provides a preparation method of a pump-free SERS microfluidic chip for detecting liver cancer-related miRNAs and the pump-free SERS microfluidic chip. The method includes: 1) preparing 20-nm gold nanoparticles (AuNPs) by the sodium citrate reduction method; 2) modifying Raman signal molecules DTNB, 4-MBA, and nucleic acid complementary strands H1 and H2 on the AuNPs respectively to obtain AuNPs@DTNB@H1 and AuNPs@4-MBA@H2; 3) modifying nucleic acid aptamers cDNA 1 and cDNA 2 of miR-21 and miR-155 on the surface of magnetic core Fe3O4 to obtain Fe3O4@cDNA 1 and Fe3O4@cDNA 2 (Fe3O4@cDNA); 4) coupling AuNPs@DTNB@H1 and AuNPs@4-MBA@H2 prepared in step 2) with Fe3O4@cDNA prepared in step 3) to obtain Fe3O4@cDNA-AuNPs@Raman reporter@H; 5) mixing polydimethylsiloxane (PDMS) with a curing agent and adding a template, preparing a microfluidic chip and performing hydrophilic treatment to construct a pump-free SERS microfluidic chip. The present invention has the advantages of high sensitivity, strong specificity, fast detection speed, etc.

Description

Technical Field

[0001] The present invention relates to the technical field of materials, and specifically to a preparation method of a pump-free SERS microfluidic chip for detecting liver cancer-related miRNAs 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 globally, with a relatively high mortality rate. Its five-year survival rate is approximately 15-17%. Liver cancer includes various subtypes such as hepatocellular carcinoma and cholangiocarcinoma. Since liver cancer has no obvious symptoms in the early stage, most patients are already in the advanced stage when diagnosed with liver cancer. Although significant progress has been made in the diagnosis and treatment of liver cancer, its mortality rate is still rising rapidly. Therefore, early diagnosis of liver cancer is crucial, which can provide better treatment for patients and higher survival rates.

[0003] In recent years, the use of MicroRNA (miRNA) as a biomarker for the 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 and is one of the gene regulatory molecules in multicellular organisms. It has an important impact on many protein-coding genes and is involved in processes such as the proliferation, differentiation, and metabolism of tumor cells, playing an important role in the occurrence and development of tumors. Some studies have shown that the expression levels of miR-21 and miR-155 in liver cancer tissues are significantly higher than those in normal liver tissues, and they can promote the occurrence and development of tumors. Therefore, miR-21 and miR-155 may be prospective biomarkers for the early detection of liver cancer. Currently, the main detection methods for miRNAs include PCR, northern blotting, electrochemical detection, and microarrays. However, these methods have limitations such as complex operation, high cost, and low sensitivity in actual applications. Moreover, in order to improve the detection accuracy and reduce the misdiagnosis rate, detecting multiple miRNAs simultaneously is a promising method, which can provide more comprehensive and accurate diagnostic information.

[0004] Surface-enhanced Raman scattering (SERS) is a new type of spectral enhancement technology that improves the Raman signal of molecules. It can provide unique molecular fingerprint information, with a sensitivity that can reach the single-molecule level, and can reflect subtle changes in tissue biochemistry. It has a wide range of applications in the fields of biochemistry, clinical diagnosis, etc. The local surface plasmon resonance (LSPR) effect on the surface of noble metal nanomaterials is the main source of SERS enhancement. The electromagnetic field regions generated by these plasmon resonances are called "hot spots", and the enhancement effect is closely related to the structure and elemental composition of the nanomaterials. However, due to reasons such as complex processes and high costs, SERS still lacks in point-of-care testing and has insufficient sensitivity and specificity for miRNA. Therefore, there is an urgent need for a simple, rapid, and ultrasensitive miRNA detection method.

[0005] The technology of SERS detecting protein biomarkers still faces many problems that need to be solved before truly entering clinical applications. The primary problem is to prepare SERS sensors with excellent surface-enhanced performance, easy to use, easy to prepare, good reproducibility, and good biocompatibility. The Fe3O4@cDNA-AuNPs@Raman reporter@H innovatively prepared in this invention has good dispersibility. The strong enrichment ability of Fe3O4 and the many nanogaps formed by the aggregation of Au particles on the magnetic core can form more hot spots, which can significantly improve the SERS signal and ensure the stability and reproducibility of the detection. Another key issue is to explore fast, stable, and efficient detection strategies. Currently, the detection methods for miRNA have limitations such as complex operations, high costs, and low sensitivity. Summary of the Invention

[0006] (1) Technical Problems to be Solved

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

[0008] (2) Technical Solutions

[0009] To achieve the above objectives, the present invention is realized through the following technical solutions: A preparation method of a pump-free SERS microfluidic chip for detecting liver cancer-related miRNA, comprising the following steps:

[0010] 1) Prepare 20nm AuNPs by the sodium citrate reduction method;

[0011] 2) Modify the Raman signal molecules DTNB, 4-MBA and nucleic acid complementary strands H1, H2 on the AuNPs respectively to obtain AuNPs@DTNB@H1 and AuNPs@4-MBA@H2;

[0012] 3) Modify the nucleic acid aptamer cDNA 1 and cDNA 2 of miR-21 and miR-155 onto the surface of the magnetic core Fe3O4 to prepare Fe3O4@cDNA1 and Fe3O4@cDNA2;

[0013] 4) Couple the AuNPs@DTNB@H1 and AuNPs@4-MBA@H2 prepared in step 2) with the Fe3O4@cDNA1 and Fe3O4@cDNA2 prepared in step 3) to obtain Fe3O4@cDNA-AuNPs@Raman reporter@H;

[0014] 5) Mix polydimethylsiloxane with a curing agent and then add a template to prepare a microfluidic chip and perform hydrophilic treatment.

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

[0016] 1.1) Take 100 mL of 0.01 wt% HAuCl4 solution and place it on a magnetic stirrer to heat and boil;

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

[0018] 1.3) After reacting for 15 min, stop heating, continue stirring until the solution cools to room temperature, and then add water to make up the volume to 100 mL to prepare 20 nm AuNPs.

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

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

[0021] 2.2) Centrifuge the mixed solution prepared in step 2.1) at 10000 rpm for 10 min, remove the supernatant, and then resuspend the DTNB-labeled AuNPs, namely AuNPs@DTNB, in 10 mL of PBS by ultrasonic and oscillating treatment;

[0022] 2.3) Add H1 activated by 20 mL of 1 M TCEP to the AuNPs@DTNB solution and incubate at 37 °C for 2 h;

[0023] 2.4) Add the mixed solution prepared in step 2.3) to 100 mL of 1 wt% BSA solution, react for 2 h, then centrifuge at 8000 rpm for 15 min, remove the supernatant, and prepare AuNPs@DTNB@H1. Prepare AuNPs@4-MBA@H2 in the same way.

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

[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 μL of each of the activated cDNA 1 and cDNA 2 and add them to a streptavidin-modified Fe3O4 nanoparticle solution, and shake at room temperature for 30 min; the concentrations of the cDNA 1 and cDNA 2 are 100 mM, and the concentration of the streptavidin-modified Fe3O4 nanoparticle solution is 1 mg / mL;

[0027] 3.3) After magnetic separation using a magnet, add ultrapure water and wash twice to remove the excess cDNA 1 and cDNA 2 to obtain Fe3O4@cDNA 1 and Fe3O4@cDNA 2. Mix them in a volume ratio of 1:1 to obtain Fe3O4@cDNA.

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

[0029] 4.1) Take the same volume of AuNPs@DTNB@H1 and AuNPs@4-MBA@H2, mix them with the Fe3O4@cDNA prepared in step 3.3), and incubate 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;

[0030] 4.2) Wash twice with PBS to remove the excess labeled probes to prepare Fe3O4@cDNA-AuNPs@Ramanreporter@H.

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

[0032] 5.1) Uniformly mix PDMS and the curing agent at a mass ratio of 10:1, and then place it in a vacuum chamber for 30 min to remove air bubbles;

[0033] 5.2) Pour the mixed solution prepared in step 5.1) onto the prepared template, cure it on a heating plate at 100 °C for 1 h, and after cooling to room temperature, take out the PDMS layer from the template to obtain a microfluidic chip; the microfluidic chip mainly consists of a sample loading area, a sample mixing area, an enrichment and detection area, and a comb-shaped capillary pump; the sample loading area consists of 2 circular chambers with a diameter of 3 mm, the sample mixing area consists of a circular chamber with a diameter of 2 mm and a meandering channel, the enrichment and detection area consists of a rectangular chamber with a length of 1.8 mm and a circular magnet with a diameter of 4 mm, the channels of the comb-shaped capillary pump have a width of 200 mm, and the other channels have a width of 300 mm and a height of 120 mm;

[0034] 5.3) Immerse the microfluidic chip prepared in step 5.2) in 95% ethanol, clean it in an ultrasonic environment for 25 min, dry it with nitrogen, then immerse it in a PEG solution, and heat it on a heating plate at 150 °C for 30 min;

[0035] 5.4) After heating, wash off the excess PEG with an isopropyl alcohol solution and place it at 4 °C for 1 h to obtain a microfluidic chip with hydrophilic treatment.

[0036] Based on the above preparation method of a pump-free SERS microfluidic chip for detecting liver cancer-related miRNA, 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 microRNA, the detection method includes 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 add them to the pump-free SERS microfluidic chip for detecting liver cancer-related microRNA in different combinations together with Fe3O4@cDNA-AuNPs@Ramanreporter@H, and react in an incubator at 37 °C; after taking out the pump-free SERS microfluidic chip, perform SERS detection; in the presence of miR-21 and miR-155, the signal intensities at the characteristic peaks of 1080 cm-1 and 1330 cm-1 decrease significantly;

[0039] 2) Disperse different concentrations of miR-21 and miR-155 into serum, and simultaneously drip them into a pump-free SERS microfluidic chip for detecting liver cancer-related microRNAs together with Fe3O4@cDNA-AuNPs@Ramanreporter@H. React in an incubator at 37°C to obtain the SERS spectra of different concentrations of miR-21 and miR-155 in the serum; analyze and obtain that the logarithm of the miR-21 concentration has a linear relationship with the intensity of the characteristic peak at 1330 cm-1, and the logarithm of the miR-155 concentration has a linear relationship with the intensity of the 1080 cm-1 characteristic peak, and finally obtain their linear equations;

[0040] 3) Simultaneously drip the collected serum samples and Fe3O4@cDNA-AuNPs@Raman reporter@H into a pump-free SERS microfluidic chip for detecting liver cancer-related microRNAs, react in an incubator at 37°C, take out the pump-free SERS microfluidic chip for SERS testing, and detect the SERS signals of the serum samples;

[0041] 4) Substitute the SERS signals of the serum samples detected in step 3) into the linear equations determined in step 2) to determine the expression levels of miR-21 and miR-155 in the serum samples.

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

[0043] (III) Beneficial effects

[0044] The present invention provides a preparation method of a pump-free SERS microfluidic chip for detecting liver cancer-related miRNAs and the pump-free SERS microfluidic chip. It has the following beneficial effects:

[0045] 1. The present invention provides a preparation method of a pump-free SERS microfluidic chip for detecting liver cancer-related miRNAs and the pump-free SERS microfluidic chip. This pump-free SERS microfluidic chip has the advantages of high sensitivity, strong specificity, excellent repeatability, and fast detection speed. By measuring the signal intensities corresponding to different concentrations of miR-21 and miR-155 in the serum, a quantitative relationship between the concentrations of miR-21 and miR-155 and the SERS signal intensities is established. According to the quantitative relationship, the concentrations of miR-21 and miR-155 in the serum samples are detected, and the accuracy is very high. The results show that the pump-free SERS microfluidic chip of the present invention can be used for the ultrasensitive simultaneous detection of liver cancer-related miR-21 and miR-155 in serum samples, providing technical support for the extensive application of SERS in the diagnosis and screening of liver cancer.

[0046] 2. The method for preparing the pump-free SERS microfluidic chip provided by the present invention is simple and highly operable, and does not require special instrument equipment. The experimental cost is low, and the required reagents are all common 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 simplicity, speed, small dosage, high throughput, etc. In the present invention, Fe3O4 as the magnetic core has good enrichment ability, can capture more target substances, and separate them from the complex environment under the action of a magnet. AuNPs are coupled to Fe3O4 modified with nucleic acid aptamer through nucleic acid complementary strands, forming abundant "hot spots", which greatly enhances the SERS signal. The prepared Fe3O4@cDNA-AuNPs@Raman reporter@H utilizes the strong specificity between nucleic acid aptamer and miR-21 and miR-155, with higher precision and sensitivity, superior to other detection strategies. The SERS microfluidic chip designed with a capillary pump gets rid of the need for external heavy pumps and can quickly and sensitively complete the detection of miR-21 and miR-155 in a short time. Some studies have shown that miR-21 is related to the staging and poor prognosis of liver cirrhosis and liver cancer. Compared with normal cells, the expression level of miR-21 is significantly up-regulated in cancer cells, which can promote the growth and proliferation of tumors. miR-155 also plays an important regulatory role in tumor development. It has been found that the expression of miR-155 is up-regulated 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 the early detection of liver cancer. Description of the Drawings

[0047] Figure 1A is the SEM photograph of Fe3O4 prepared by the present invention;

[0048] Figure 1B is the TEM photograph of Fe3O4 prepared by the present invention;

[0049] Figure 1C is the UV-vis spectrum of Fe3O4 prepared by the present invention;

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

[0051] Figure 1E is the TEM photograph 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 is the SEM image of Fe3O4@cDNA-AuNPs@Raman reporter@H prepared in the present invention;

[0054] Figure 1H is the TEM image of Fe3O4@cDNA-AuNPs@Raman reporter@H prepared in the present invention;

[0055] Figure 1I is the HRTEM image of Fe3O4@cDNA-AuNPs@Raman reporter@H prepared in the present invention;

[0056] Figure 1J is the electron diffraction image of Fe3O4@cDNA-AuNPs@Raman reporter@H prepared in the present invention;

[0057] Figure 1K is the elemental mapping image of Fe3O4@cDNA-AuNPs@Raman reporter@H prepared in the present invention;

[0058] Figure 1L is the elemental mapping image of Fe3O4@cDNA-AuNPs@Raman reporter@H prepared in the present invention;

[0059] Figure 1M is the elemental mapping image of Fe3O4@cDNA-AuNPs@Raman reporter@H prepared in the present invention;

[0060] Figure 1N is the elemental mapping image of Fe3O4@cDNA-AuNPs@Raman reporter@H prepared in the present invention;

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

[0062] Figure 3A is the SERS signal intensity when the mixture of Fe3O4 and AuNPs@DTNB@H1 and AuNPs@4-MBA@H2 hybridizes in different proportions;

[0063] Figure 3B is the bar chart of the SERS signal intensity when the mixture of Fe3O4 and AuNPs@DTNB@H1 and AuNPs@4-MBA@H2 hybridizes in different proportions;

[0064] Figure 3C is the SERS signal intensity when Fe3O4 hybridizes with the mixture of AuNPs@DTNB@H1 and AuNPs@4-MBA@H2 at different temperatures;

[0065] Figure 3D is a histogram of the SERS signal intensity when Fe3O4 hybridizes with the mixture of AuNPs@DTNB@H1 and AuNPs@4-MBA@H2 at different temperatures;

[0066] Figure 3E is the SERS signal intensity when Fe3O4 hybridizes with the mixture of AuNPs@DTNB@H1 and AuNPs@4-MBA@H2 at different times;

[0067] Figure 3F is a histogram of the SERS signal intensity when Fe3O4 hybridizes with the mixed solution of AuNPs@DTNB@H1 and AuNPs@4-MBA@H2 at different times;

[0068] Figure 4A Inks of different colors 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 Here 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 5A These are 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 intensities at 1080 cm-1 and 1330 cm-1;

[0074] Figure 5C The SERS spectra of the pump-free SERS microfluidic chip stored at room temperature for different times;

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

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

[0077] Figure 5F is a bar chart of the characteristic peak intensities at 1080 cm-1 and 1330 cm-1;

[0078] Figure 6A is 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 the miR-155 concentration;

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

[0081] Figure 7A is the fluorescence in vivo imaging of nude mice;

[0082] Figure 7B is the HE staining of the liver tissue of nude mice;

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

[0084] Figure 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 intensities at 1080 cm-1 and 13,30 cm-1 of nude mice in the control group;

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

[0087] Figure 8D is the characteristic peak intensities at 1080 cm-1 and 1330 cm-1 of nude mice with orthotopic liver cancer. Specific embodiments

[0088] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0089] The instrument equipment 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 microscope (SEM) images were taken using an S-4800Ⅱ field emission scanning electron microscope.

[0092] Transmission electron microscope (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. The spectra were processed using Renishaw software (WiRETM) for operation and data acquisition, and baseline correction was performed to suppress background noise.

[0095] Example 1: Preparation and Characterization of Fe3O4@cDNA-AuNPs@Raman reporter@H

[0096] 1) Take 100 mL of HAuCl4 solution (0.01 wt%) and place it on a magnetic stirrer to heat and boil.

[0097] 2) Rapidly add 3 mL of 1 wt% sodium citrate solution (TSC) under vigorous stirring.

[0098] 3) After reacting for 15 min, stop heating and continue stirring until the solution cools to room temperature, then add water to make up the volume to 100 mL to prepare 20 nm AuNPs.

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

[0100] 5) Centrifuge the mixed solution prepared in step 4) at 10000 rpm for 10 min. After removing the supernatant, redisperse the DTNB-labeled AuNPs (AuNPs@DTNB) in 10 mL of PBS by ultrasonic and shaking treatments.

[0101] 6) Add H1 (0.1 mM) activated by 20 mL of 1 M TCEP to the AuNPs@DTNB solution and incubate at 37 °C for 2 h.

[0102] 7) Add the mixed solution prepared in step 6) to a 100 mL BSA (1 wt%) solution, react for 2 h, then centrifuge at 8000 rpm for 15 min. After removing the supernatant, AuNPs@DTNB@H1 is prepared. AuNPs@4-MBA@H2 is prepared in the same way.

[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 the activated cDNA 1 and cDNA 2 respectively and add them to a streptavidin-modified Fe3O4 nanoparticle solution, and oscillate at room temperature for 30 min.

[0105] 10) After magnetic separation using a magnet, add ultrapure water to wash twice to remove the excess cDNA 1 and cDNA 2, and Fe3O4@cDNA 1 and Fe3O4@cDNA 2 are obtained. Mix them in a volume ratio of 1:1 to get Fe3O4@cDNA.

[0106] 11) Take the same volume of the mixture of AuNPs@DTNB@H1 and AuNPs@4-MBA@H2 and mix it with Fe3O4@cDNA, and incubate at 37 °C.

[0107] 12) Wash twice with PBS to remove the excess labeled probes, and Fe3O4@cDNA-AuNPs@Ramanreporter@H is prepared.

[0108] 13) Morphology characterization of Fe3O4@cDNA-AuNPs@Raman reporter@H.

[0109] Figure 1A and Figure 1B are the SEM image and TEM image of the Fe3O4 magnetic core respectively: The morphology of the Fe3O4 magnetic core is spherical, its average particle size is about 100 nm, and the Fe3O4 magnetic core is evenly distributed, indicating its good dispersibility. Figure 1C is the UV spectrum of Fe3O4, and it can be seen that the absorption peak of Fe3O4 is at 413 nm. Figure 1D and Figure 1E show the SEM image and TEM image of AuNPs respectively. From the images, it can be seen that AuNPs have a spherical morphology, with an average particle size of about 20 nm. The UV spectrum is as Figure 1F shown. The absorption peak of AuNPs is at 522 nm, indicating that AuNPs have a uniform morphology and size. Figure 1G 、 Figure 1H 、 Figure 1ISEM, TEM, and HRTEM imaging were performed on Fe3O4@cDNA-AuNPs@Ramanreporter@H respectively. From the images, it can be seen that the diameter of Fe3O4@cDNA-AuNPs@Ramanreporter@H is about 140 nm, with good dispersibility, and 20 nm AuNPs are evenly distributed on the surface of Fe3O4. Figure 1J is the lattice image of AuNPs, showing that its fringe spacing is 0.243 nm, which is a polycrystalline structure. Figures 1K to 1N is the elemental mapping image of Fe3O4@cDNA-AuNPs@Raman reporter@H, indicating that the main components of Fe3O4@cDNA-AuNPs@Raman reporter@H are Fe and Au.

[0110] Example 2: Optimization of experimental conditions

[0111] 1) After mixing the same volume of AuNPs@DTNB@H1 and AuNPs@4-MBA@H2, they were mixed with Fe3O4@cDNA in different volumes to form different ratios (Fe3O4@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 nucleotide sequences of AuNPs@DTNB@H1 and AuNPs@4-MBA@H2 can specifically complementarily pair with the nucleotide sequence of Fe3O4@cDNA to obtain Fe3O4@cDNA-AuNPs@Raman reporter@H;

[0112] 2) The Fe3O4@cDNA-AuNPs@Raman reporter@H prepared in different volume ratios was subjected to SERS detection, as Figure 3A and Figure 3B shown, when the ratio of Fe3O4@cDNA to the mixed solution of AuNPs@DTNB@H1 and AuNPs@4-MBA@H2 is 1:2.5, the characteristic peak intensities at 1080 cm-1 and 1330 cm-1 reach the highest. When the volume of the signal probe continues to increase, the signal intensities of these two characteristic peaks change little, indicating that the coupling signal probe on the capture probe has tended to be saturated, suggesting that when the volume ratio of Fe3O4@cDNA and AuNPs@H is 1:2.5, it is the optimal ratio for constructing the SERS sensor. Figure 3C and Figure 3DIt was shown that the intensities of the characteristic peaks at 1080 cm-1 and 1330 cm-1 increased with the prolongation of time. When the time reached 80 min, the SERS signal intensity did not change significantly and the growth trend almost disappeared. Therefore, the optimal incubation time of Fe3O4@cDNA and AuNPs@H was 80 min. When they were incubated at different temperatures, the measured SERS signal intensities were as Figure 3E and Figure 3F shown. With the increase of temperature, the intensities of the characteristic peaks at 1080 cm-1 and 1330 cm-1 gradually increased and tended to be stable until 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 the curing agent were uniformly mixed at a mass ratio of 10:1, and then placed in a vacuum chamber for 30 min to remove air bubbles;

[0115] 2) The prepared mixed solution was poured onto the prepared template and cured on a hot plate at 100 °C for 1 h. After cooling to room temperature, the PDMS layer was removed from the template to obtain the microfluidic chip;

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

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

[0118] The microfluidic chip mainly consists 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 2 circular chambers with a diameter of 3 mm. The sample mixing area consists of a circular chamber with a diameter of 2 mm and a meandering channel. The enrichment and detection area consists of a rectangular chamber with a length of 1.8 mm and a circular magnet with a diameter of 4 mm. The width of the comb-shaped capillary pump channel is 200 mm, and the width of the remaining channels is 300 mm, with a height of 120 mm.

[0119] Figure 4A are images of different colored inks flowing freely in the channels of the pump-free SERS microfluidic chip. As shown in the figure, under the action of the comb-shaped capillary pump, the ink can flow freely in the channels and fill the entire channel without any leakage at 60 s, indicating that the prepared pump-free SERS microfluidic chip has good hydrophilicity and sealing performance. Figure 4B are micrographs of two areas in the pump-free SERS microfluidic chip, Figure 4CThese are the corresponding SERS spectra of the two regions. Since there are no Raman signal molecules in Region I, the Raman signal in this region is completely generated by PDMS. The characteristic peak intensities of Region I at 1080 cm-1 and 1330 cm-1 are significantly lower than those of Region II. Therefore, it is confirmed that the microfluidic chip made of PDMS does not affect the detection of miR-21 and miR-155. To find the optimal measurement time for the pump-free SERS microfluidic chip, we measured the SERS signals in the rectangular cavity at different times. Figure 4D It shows the intensity changes of the characteristic peaks of the SERS signal in the rectangular cavity at 1080 cm-1 and 1330 cm-1. The results show that the SERS intensity decreases with the increase of time and hardly changes at 5 min. Thus, it can be known that the optimal measurement time for the prepared pump-free SERS microfluidic chip is 5 min.

[0120] Example 4: Analysis of the stability, repeatability and specificity of the pump-free SERS microfluidic chip

[0121] 1) Prepare Fe3O4@cDNA-AuNPs@Raman reporter@H and the microfluidic chip as in Example 1 and Example 3, and add Fe3O4@cDNA-AuNPs@Raman reporter@H to the microfluidic chip for detection;

[0122] 2) Prepare five different batches of SERS microfluidic chips and measure their SERS spectra. Figure 5A These are the SERS spectra of the pump-free SERS microfluidic chips prepared in different batches. Figure 5B This is the dot plot of the characteristic peak intensities of the pump-free SERS microfluidic chips prepared in different batches at 1080 cm-1 and 1330 cm-1. As can be seen from the figure, there are no obvious differences in the SERS spectra of the five different batches of pump-free SERS microfluidic chips prepared. The relative standard deviations (RSD) of the Raman intensities at the 1080 cm-1 and 1330 cm-1 characteristic peaks are calculated to be 5.68% and 5.63% respectively, indicating that the prepared pump-free SERS microfluidic chip has good repeatability.

[0123] 3) Measure the SERS signal intensities of the pump-free SERS microfluidic chips after storing them at room temperature for different times (1d, 5d, 10d, 15d, 20d). As Figure 5C can be seen, there are no obvious changes in the morphology of the spectra. Figure 5DThe changes in the SERS signal intensities at the characteristic peaks of 1080 cm-1 and 1330 cm-1 from 1 to 20 days were shown. The calculated RSD values were 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 to the pump-free SERS microfluidic chip for detection according to different combinations. Figure 5E The specific SERS spectrogram of the pump-free SERS microfluidic chip was shown. It can be seen that when the target substances miR-21 and miR-155 are present, the corresponding characteristic peaks are significantly reduced, and the changes in the SERS signal intensity caused by the target miRNA are significantly higher than those of the interferents. Figure 5F The bar chart of the characteristic peak intensities at 1080 cm-1 and 1330 cm-1 was shown. It can be more clearly seen that the changes at the characteristic peaks of 1080 cm-1 and 1330 cm-1 represented by the two target miRNAs are stronger than those of the interferents. Thus, it can be known that the prepared pump-free SERS microfluidic chip has high sensitivity 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) Prepare Fe3O4@cDNA-AuNPs@Raman reporter@H and the microfluidic chip in the same way as in Example 1 and Example 3.

[0127] 2) Disperse the mixed solution of different concentrations (10 pM to 10 mM) of miR-21 and miR-155 (miR-21:miR-155 = 1:1) into serum, and add the mixed solution and Fe3O4@cDNA-AuNPs@Raman reporter@H into the pump-free SERS microfluidic chip for SERS detection simultaneously. As Figure 6A shown, as the concentration of the 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. The corresponding regression equations were y = -2221.525x - 6883.168 (R2 = 0.996) and y = -2613.809x - 8185.615 (R2 = 0.996), respectively. The LODs of miR-21 and miR-155 were calculated to be 2.39 pM and 2.49 pM, respectively. Compared with other previously reported detection strategies (Table 1), the detection results of the pump-free SERS microfluidic chip prepared by us were similar to theirs, but the detection time was significantly shortened, which was better than most of the reported methods.

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

[0129]

[0130] Example 6: Characterization of liver cancer mice

[0131] 1) The nude mice were photographed using a small animal in vivo imaging system to observe and compare the growth of in-situ tumors at different time points. As Figure 7A shown, the fluorescence intensity at the site of in-situ liver cancer in nude mice increased continuously with time. HE staining showed that the tumor cells of liver cancer nude mice were arranged disorderly, with different sizes, deeply stained cell nuclei, and pathological mitosis ( Figure 7B ). The weight of the nude mice was weighed synchronously when collecting serum. As Figure 7C seen, the weight of the nude mice with liver cancer model decreased continuously compared with that of 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 Fe3O4@cDNA-AuNPs@Raman reporter@H and microfluidic chips in the same way as in Example 1 and Example 3;

[0134] 2) The collected serum samples and Fe3O4@cDNA-AuNPs@Raman reporter@H were simultaneously added to the pump-free SERS microfluidic chip, and the reaction was carried out in an incubator at 37 °C. After 5 min, it was taken out for SERS testing to detect the SERS signals of the serum samples. Figure 8A and Figure 8B show the average SERS spectra of nude mice at 0 d (control group) and the SERS intensities at the characteristic peaks of 1080 cm-1 and 1330 cm-1. It can be seen that the serum SERS intensity of the control group nude mice did not change significantly with time. At Figure 8CAmong them, the average SERS spectra of nude mice with hepatocellular carcinoma in situ decreased significantly with the growth of tumors, and the SERS intensities at the corresponding characteristic peaks of 1080 cm-1 and 1330 cm-1 were as Figure 8D shown. Obviously, the signals of miR-21 and miR-155 in nude mice with hepatocellular carcinoma in situ were higher than those in the control group of mice, and the expression levels increased continuously with the development of tumors. The results of detecting miR-21 and miR-155 by the pump-free SERS microfluidic chip were compared with those of qRT-PCR as shown in Table 2, showing high consistency. This indicates that the pump-free SERS microfluidic chip proposed in this study has high accuracy for the simultaneous and ultrasensitive detection of miR-21 and miR-155, and has good application prospects.

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

[0136]

[0137] Although the embodiments of the present invention have been shown and described, for those of ordinary skill in the art, it can be understood that various changes, modifications, substitutions and variations can be made to these embodiments without departing from the principles and spirit of the present invention. The scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A preparation method of Fe3O4@cDNA-AuNPs@Raman reporter@H for detecting liver cancer-related miRNA, characterized in that: It includes the following steps: 1) Prepare 20-nm AuNPs by the sodium citrate reduction method; 2) Modify Raman signal molecules DTNB, 4-MBA, and nucleic acid complementary strands H1 and H2 on AuNPs respectively to obtain AuNPs@DTNB@H1 and AuNPs@4-MBA@H2; 3) Modify nucleic acid aptamers cDNA 1 and cDNA 2 of miR-21 and miR-155 on the surface of magnetic core Fe3O4 to obtain Fe3O4@cDNA1 and Fe3O4@cDNA2; 4) Couple AuNPs@DTNB@H1 and AuNPs@4-MBA@H2 prepared in step 2) with Fe3O4@cDNA1 and Fe3O4@cDNA2 prepared in step 3) to obtain Fe3O4@cDNA-AuNPs@Raman reporter@H.

2. The preparation method of Fe3O4@cDNA-AuNPs@Ramanreporter@H for detecting miRNA related to liver cancer according to claim 1, which is characterized in that: The specific implementation method of step 1) is as follows: 1.1) Take 100 mL of 0.01 wt% HAuCl4 solution and place it on a magnetic stirrer to heat and boil; 1.2) Rapidly add 3 mL of 1 wt% sodium citrate solution under vigorous stirring conditions; 1.3) After reacting for 15 min, stop heating, continue stirring until the solution cools to room temperature, and then add water to make up the volume to 100 mL to prepare 20-nm AuNPs.

3. The preparation method of Fe3O4@cDNA-AuNPs@Ramanreporter@H for detecting liver cancer-related miRNA according to claim 2, characterized in that: The specific implementation method of step 2) is as follows: 2.1) Add 200 μL of 10 mM DTNB solution to 10 mL of AuNPs solution and stir vigorously at room temperature for 2 h; 2.2) Centrifuge the mixed solution prepared in step 2.1) at 10000 rpm for 10 min, remove the supernatant, and then resuspend the DTNB-labeled AuNPs, namely AuNPs@DTNB, in 10 mL of PBS by ultrasonic and oscillation treatment; 2.3) Add H1 activated by 20 mL of 1 M TCEP to the AuNPs@DTNB solution and incubate at 37 °C for 2 h; 2.4) Add the mixed solution prepared in step 2.3) to 100 mL of 1 wt% BSA solution, react for 2 h, then centrifuge at 8000 rpm for 15 min, remove the supernatant to prepare AuNPs@DTNB@H1, and prepare AuNPs@4-MBA@H2 in the same way.

4. The preparation method of Fe3O4@cDNA-AuNPs@Ramanreporter@H for detecting miRNA related to liver cancer according to claim 3, wherein: The specific implementation method of step 3) is as follows: 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) Take 10 mL of the activated cDNA 1 and cDNA 2 respectively and add them to the streptavidin-modified Fe3O4 nanoparticle solution, and oscillate at room temperature for 30 min; the concentrations of cDNA 1 and cDNA 2 are 100 mM, and the concentration of the streptavidin-modified Fe3O4 nanoparticle solution is 1 mg / mL; 3.3) After magnetic separation using a magnet, add ultrapure water and wash twice to remove excess cDNA 1 and cDNA 2, obtaining Fe3O4@cDNA 1 and Fe3O4@cDNA 2. Mix them in a volume ratio of 1:1 to obtain Fe3O4@cDNA.

5. The preparation method of Fe3O4@cDNA-AuNPs@Ramanreporter@H for detecting miRNA related to liver cancer according to claim 4, characterized in that: The specific implementation method of step 4) is as follows: 4.1) Take the same volume of AuNPs@DTNB@H1 and AuNPs@4-MBA@H2, mix them, and then mix with the Fe3O4@cDNA prepared in step 3.3), and incubate 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, and prepare Fe3O4@cDNA-AuNPs@Ramanreporter@H.

6. A Fe3O4@cDNA-AuNPs@Raman reporter@H for detecting liver cancer-related miRNA, characterized in that: Prepared by the preparation method of Fe3O4@cDNA-AuNPs@Ramanreporter@H for detecting miRNA related to liver cancer according to any one of claims 1-5.

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