A method for detecting tumor marker miRNA155 by adsorbing polyA-ssDNA with gold nanoparticles

Abstract

The present invention provides a method for detecting tumor marker miRNA155 by adsorbing polyA-ssDNA with gold nanoparticles; comprising the following steps: step 1, preparation of bismuth vanadate / gold photoanode, adsorbing polynucleotide-modified ssDNA on the bismuth vanadate / gold photoanode; step 2, dropping different concentrations of miRNA155 on the photoanode for incubation of base complementary pairing, washing away excess test substances for standby use; step 3, performing photoelectrochemical detection. The present invention adopts a one-step detection method, which not only greatly reduces the operation steps, but also reduces the time and operation required for extraction and amplification, and realizes sensitive detection in a complex matrix.

Description

Technical Field

[0001] The present invention relates to the field of detecting tumor marker miRNA155, and in particular to a method for detecting tumor marker miRNA155 by adsorbing polyA-ssDNA with gold nanoparticles. Background Art

[0002] New methods for accurate diagnosis before tumor metastasis are currently being developed. Highly stable circulating microRNAs (miRNAs) in the blood are considered promising and minimally invasive cancer biomarker candidates. They are short non-coding RNA molecules (-22nt) that inhibit or degrade messenger RNAs (mRNAs) through complementary base pair binding, and regulate the level of mRNA after transcription. Their abnormal expression is often observed in various cancers. A large number of research results have shown that miRNA155 has a wide range of functions. It is involved in many biological processes such as the development and differentiation of hematopoietic cells, the development and differentiation of immune cells, inflammatory response, immune response, muscle development, and fat differentiation. It is highly expressed in various cancer tissues or cell lines such as liver cancer, lymphoma, breast cancer, pancreatic cancer, and lung cancer. It is closely related to the occurrence, invasion, and metastasis of tumors. Therefore, the detection of its concentration is necessary and important.

[0003] Currently, the quantification of miRNAs is mostly carried out using real-time reverse transcription quantitative PCR (RT-qPCR)16, Northern blotting, microarray hybridization and other technologies. However, these methods have many limitations, such as insufficient specificity and sensitivity due to cross-hybridization and contamination, and complex and time-consuming operation procedures. In contrast, photoelectrochemical biosensors react at body temperature and do not require complex thermal cycles to mediate denaturation, annealing and subsequent extension; secondly, they use light as an excitation signal and collect the electrical signal generated by the transducer, which can avoid the noise caused by the excitation signal. In recent years, it has become a research hotspot. Summary of the invention

[0004] The purpose of the present invention is to provide a method for detecting tumor marker miRNA155 by adsorbing polyA-ssDNA with gold nanoparticles.

[0005] The present invention is achieved through the following technical solutions:

[0006] The present invention relates to a method for detecting tumor marker miRNA155 by adsorbing polyA-ssDNA with gold nanoparticles, comprising the following steps:

[0007] Step 1, preparation of bismuth vanadate / gold photoanode, adsorbing polynucleotide-modified ssDNA on the bismuth vanadate / gold photoanode;

[0008] Step 2, applying different concentrations of miRNA155 on the photoanode for incubation of base complementary pairing, washing away the excess analyte for later use;

[0009] Step 3, perform photoelectrochemical detection.

[0010] Preferably, in step 1, the specific steps are as follows:

[0011] A uniform and stable bismuth vanadate (BVO) film was prepared by a typical electrodeposition bismuth oxyiodide method and converted into bismuth vanadate. 3mmol bismuth nitrate pentahydrate and 40mmol sodium iodide were dissolved in 100mL ultrapure water, and the pH value of the solution was adjusted to 1.2 with nitric acid to obtain solution A; 0.0135mmol p-benzoquinone was added to 45mL ethanol, ultrasonically dispersed, and stirred to obtain solution B; solution A was mixed with solution B to obtain an electrodeposition solution, and an orange-yellow bismuth oxyiodide film was obtained; gold was magnetron sputtered on the deposited bismuth oxyiodide film at room temperature for 2s, 7s and 15s, and then 40μL 0.2M acetylacetonate vanadium solution was drop-coated, dried, annealed, and cooled, and then excess vanadium pentoxide was removed in 1M sodium hydroxide solution to obtain a BVO / Au photoanode film.

[0012] Preferably, the electrodeposition solution is subjected to a three-electrode system for 40 seconds at a deposition potential of -0.3 V vs. Ag / AgCl, wherein the working electrode, the counter electrode and the reference electrode are FTO, a Pt sheet and an Ag / AgCl reference electrode, respectively.

[0013] Preferably, the drying temperature is 60° C.; the annealing temperature is 450° C., and the time is 60 min.

[0014] Preferably, in step 2, the incubation condition for base complementary pairing is: at 37° C. for 1 hour.

[0015] Preferably, in step 2, the specific steps are as follows:

[0016] ssDNA was diluted to a concentration of 1uM with 1M tris at pH 7.4 and then a 10uL / cm 2 A drop of ssDNA was applied on the above-mentioned photoanode and incubated at 37°C for 12 h to ensure that polyadenine and gold nanoparticles can be fully adsorbed. The excess ssDNA was rinsed with ultrapure water and dried for later use.

[0017] Preferably, in step 3, the detection conditions are as follows:

[0018] The electrolyte was 0.1 M phosphate buffer solution, pH = 7, using AM 1.5G 100mW / cm 2The solar simulated light source (Xe light) was used for linear sweep voltammetry (LSV), chronoamperometry (IT) and photoelectrochemical impedance spectroscopy (PEIS) tests; the frequency range of the photoelectrochemical impedance spectroscopy test was 100KHz-100mHz, and the amplitude was 10mV;

[0019] The controller intensity modulation spectroscopy (CIMPS / CIMVS) was tested under a 360 nm light source (LS360, zhner, Germany) with a frequency range of 100 KHz-100 MHz.

[0020] The present invention has the following advantages:

[0021] (1) The present invention performs early blood detection of miRNA155 based on the photoelectrochemical (PEC) performance of bismuth vanadate photoanode and polyadenylation; the present invention adopts a one-step detection method, which not only greatly reduces the operation steps, but also reduces the time and operation required for extraction and amplification, and realizes sensitive detection in a complex matrix.

[0022] (2) The method of the present invention adsorbs the polynucleotide-modified ssDNA onto a bismuth vanadate / gold photoanode; secondly, different concentrations of miRNA155 are dropped onto the photoanode with fixed complementary chains and incubated at body temperature to perform base complementary pairing; finally, the excess analyte is washed away and then photoelectrochemical detection is performed; due to the change in the photocurrent of the photoanode caused by the binding of complementary sequences, ultrasensitive detection of circulating miRNA155 in the sample is achieved.

[0023] (3) The method of the present invention proposes a new quantitative detection scheme for cell-free miRNA extraction and amplification, opening up a new way for early diagnosis of cancer that is time-saving, simple to operate, and ultra-sensitive. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 is the detection image of BVO photoanode; Figure 1 (A) is a scanning electron microscope test image; Figure 1 (B) Transmission electron microscope test image; Figure 1 (C) is the X-ray diffraction test pattern; Figure 1 (D) is a UV-visible spectrophotometry test chart;

[0025] Figure 2 This is a diagram of detecting miRNAs of different concentrations using photoanodes with different magnetron sputtering times of gold nanoparticles; Figure 2 (A) is the linear detection curve of the 2s gold nanoparticle deposition photoanode; Figure 2 (B) is the linear detection curve of the 7s gold nanoparticle deposition photoanode; Figure 2(C) is the linear detection curve of the gold nanoparticle deposition photoanode for 15s; Figure 2 (D) Specific distribution diagram of the photoanode. DETAILED DESCRIPTION

[0026] The present invention is described in detail below in conjunction with specific embodiments. It should be noted that the following embodiments are only further descriptions of the present invention, but the protection scope of the present invention is not limited to the following embodiments.

[0027] Example

[0028] This embodiment relates to a method for detecting tumor marker miRNA155 by adsorbing polyA-ssDNA with gold nanoparticles, comprising the following steps:

[0029] Step 1, preparation of bismuth vanadate / gold photoanode, adsorbing polynucleotide-modified ssDNA on the bismuth vanadate / gold photoanode: a uniform and stable bismuth vanadate (BVO) film is prepared by a typical electrodeposition bismuth oxyiodide method, and converted into bismuth vanadate, 3mmol bismuth nitrate pentahydrate and 40mmol sodium iodide are dissolved in 100mL ultrapure water, and the pH value of the solution is adjusted to 1.2 with nitric acid to obtain solution A; 0.0135mmol p-benzoquinone is added to 45mL ethanol, ultrasonically dispersed, and stirred to obtain solution B; solution A is mixed with solution B to obtain an electrodeposition solution, and the electrodeposition solution is at a deposition potential of -0.3V vs.Ag / AgCl conditions, a three-electrode system was carried out for 40s, with the working electrode, counter electrode and reference electrode being FTO, Pt sheet and Ag / AgCl reference electrode respectively, to obtain an orange-yellow iodine bismuth oxide film; gold was magnetron sputtered on the deposited iodine bismuth oxide film at room temperature for 2s, 7s and 15s, followed by a drop coating of 40μL of 0.2M acetylacetonate vanadium solution, drying at 60℃, annealing at 450℃ in a muffle furnace for 60min, and after cooling, excess vanadium pentoxide was removed in 1M sodium hydroxide solution to obtain a BVO / Au photoanode film.

[0030] Step 2: dilute the ssDNA to a concentration of 1uM with 1M tris (tris(hydroxymethyl)aminomethane) at pH 7.4 and use 10uL / cm 2 The ssDNA was dropped onto the photoanode and incubated at 37°C for 12 h to ensure that polyadenine and gold nanoparticles can be fully adsorbed. The excess ssDNA was rinsed with ultrapure water and dried for later use.

[0031] Step 3, perform photoelectrochemical detection, the detection conditions are as follows:

[0032] The electrolyte was 0.1 M phosphate buffer solution, pH = 7, using AM 1.5G 100mW / cm 2Linear sweep voltammetry (LSV), chronoamperometry (IT) and photoelectrochemical impedance spectroscopy (PEIS) tests were carried out using a solar simulated light source (Xe light); the frequency range of the photoelectrochemical impedance spectroscopy test was 100KHz-100mHz, and the amplitude was 10mV; the controller intensity modulation spectroscopy (CIMPS / CIMVS) was tested under a 360nm light source (LS360, zhner, Germany) with a frequency range of 100KHz-100mHz.

[0033] The microstructure of the BVO / Au photoanode prepared in this example was observed using a scanning electron microscope (SEM). Figure 1 As shown in (A), the pure BVO electrode exhibits a nanoworm structure, and the deposition of gold nanoparticles makes the nanoworm structure of pure BVO rough. As the gold deposition time increases, the morphology of the photoanode gradually changes into a nanosheet structure.

[0034] The presence of gold nanoparticles was confirmed using transmission electron microscopy (TEM). Figure 1 As shown in (B), the number of gold nanoparticles increases with the increase of magnetron sputtering time. After treatment at 450°C, nanoparticles with a diameter of about 30 nm were obtained, and the lattice spacing of 0.235 nm represents the crystal plane of gold (labeled 111 in the figure).

[0035] like Figure 1 As shown in (C), the XRD test of the photoanode confirmed that the diffraction peaks of the electrodeposited bismuth vanadate corresponded one to one with the PDF#48–0744 card, and the deposition of gold nanoparticles did not change its lattice structure.

[0036] like Figure 1 As shown in (D), the light absorption range of the photoanode was characterized by UV-visible diffuse reflectance spectroscopy. The absorption band edge of bismuth vanadate is around 510nm. After the deposition of gold nanoparticles, a new absorption peak appeared around 625nm and the light absorption in the entire range also increased, which also confirmed the growth of gold nanoparticles.

[0037] Different concentrations of miRNA were detected using photoanodes with different magnetron sputtering times of gold nanoparticles. The test results are as follows: Figure 2 As shown by Figure 2 The results show that, Figure 2 (A) It can be seen that BVO / Au 2s can only be detected linearly in the range of -13M to -9M. Figure 2 (B) Figure 2(C) It can be seen that BVO / Au 7s and 15s can be detected in the range of -15 to -9M; based on the results shown above, it is speculated that magnetron sputtering 2s produces too few gold nanoparticles and the fixed ssDNA is also less than the other two groups, so the linear detection area is narrow. However, if the magnetron sputtering time is too long, the light absorption of the photoanode is reduced, and the size of the gold nanoparticles is not uniform, which reduces the sensitivity of the photoanode. Figure 2 (B) It can be seen that the gold nanoparticles deposited for 7 s have the most suitable detection range.

[0038] The BVO / Au7s photoanode was further specifically tested. Figure 2 As shown in (D), it can be seen that the photoanode is specific for miRNA21, non-miRNA155, single-miRNA155, and CEA and AFP tumor markers.

[0039] In summary, the present invention uses the semiconductor material bismuth vanadate photoanode that responds to visible light as a transducer, and magnetron sputtering grows gold nanoparticles of about 30 nm on the photoanode to adsorb ssDNA and perform base pairing with miRNA155 to detect the concentration of miRNA. The detection limit obtained by the direct photoelectrochemical detection method using bismuth vanadate photoanode is 3*10e -16 mol / L. The linear detection zone equation is y=1.3611e -6X +2.6165e -5 The method of the present invention provides a new solution for sensitive biomolecule detection in complex biological environments and shows great hope for future clinical cancer diagnosis.

[0040] The above describes the specific embodiments of the present invention. It should be understood that the present invention is not limited to the above specific embodiments, and those skilled in the art may make various modifications or variations within the scope of the claims, which will not affect the essence of the present invention.

Claims

1. A method for detecting tumor marker miRNA155 by adsorbing polyA-ssDNA with gold nanoparticles, characterized in that: The following steps are involved: Step 1, preparation of the photoanode: 3mmol bismuth nitrate pentahydrate and 40mmol sodium iodide are dissolved in 100mL ultrapure water, and the pH value of the solution is adjusted to 1.2 with nitric acid to obtain solution A; 0.0135mmol p-benzoquinone is added to 45mL ethanol, ultrasonically dispersed, and stirred to obtain solution B; solution A is mixed with solution B to obtain an electrodeposition solution, and an orange-yellow iodine oxybismuth film is obtained by electrodeposition; gold is magnetron sputtered on the deposited iodine oxybismuth film at room temperature for 2s, 7s or 15s, and then 40μL of 0.2M acetylacetone vanadium solution is drop-coated, dried, annealed, and after cooling, excess vanadium pentoxide is removed in 1M sodium hydroxide solution to obtain a bismuth vanadate / gold photoanode, and polyadenine-modified ssDNA is adsorbed on the bismuth vanadate / gold photoanode; Step 2, applying different concentrations of miRNA155 on the photoanode for incubation of base complementary pairing, washing away the excess analyte for later use; Step 3, perform photoelectrochemical detection.

2. The method for detecting tumor marker miRNA155 by adsorbing polyA-ssDNA with gold nanoparticles according to claim 1, characterized in that: The electrodeposition solution was electrodeposited for 40 seconds under the condition of a deposition potential of -0.3 V, using a three-electrode system, wherein the working electrode, the counter electrode and the reference electrode were FTO, a Pt sheet and an Ag / AgCl reference electrode, respectively.

3. The method for detecting tumor marker miRNA155 by adsorbing polyA-ssDNA with gold nanoparticles according to claim 1, characterized in that: The drying temperature is 60° C.; the annealing temperature is 450° C. and the annealing time is 60 min.

4. The method for detecting tumor marker miRNA155 by adsorbing polyA-ssDNA with gold nanoparticles according to claim 1, characterized in that: In step 2, the incubation condition for base complementary pairing is: at 37° C. for 1 hour.

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