WO2019241948A1 - Method for preparing electrochemical sensor for non-coding rna and use thereof - Google Patents

Method for preparing electrochemical sensor for non-coding rna and use thereof Download PDF

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WO2019241948A1
WO2019241948A1 PCT/CN2018/092124 CN2018092124W WO2019241948A1 WO 2019241948 A1 WO2019241948 A1 WO 2019241948A1 CN 2018092124 W CN2018092124 W CN 2018092124W WO 2019241948 A1 WO2019241948 A1 WO 2019241948A1
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concentration
scx8
electrochemical sensor
preparing
rgo
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French (fr)
Chinese (zh)
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赵卉
李灿鹏
张亚平
刘凤
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云南大学
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    • C—CHEMISTRY; METALLURGY
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    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
    • G01N27/28—Electrolytic cell components
    • G01N27/30—Electrodes, e.g. test electrodes; Half-cells
    • G01N27/327—Biochemical electrodes, e.g. electrical or mechanical details for in vitro measurements
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
    • G01N27/416—Systems
    • G01N27/48—Systems using polarography, i.e. measuring changes in current under a slowly-varying voltage

Definitions

  • the invention belongs to the field of electrochemical biosensing, and in particular relates to an electrochemical detection method for non-coding RNA and its application in selective polyadenylation detection.
  • Ultrasensitive nucleic acid detection technology is widely used in functional gene detection, biological function evaluation, and disease diagnosis.
  • quantitative nucleic acid detection technologies such as fluorescent quantitative PCR technology and rolling circle amplification technology are based on polymerase chain reaction and fluorescent signal detection methods to achieve quantitative nucleic acid quantification or detection.
  • these detection methods have complicated operation steps, expensive optical signal probes, and need Problems with specific testing equipment.
  • Electrochemical sensing technology has always been the most widely used biomedical and chemical analysis and detection platform, with advantages such as simplicity, portability, low cost, and high detection sensitivity.
  • Electrochemical biosensors have broad application prospects in biomedical fields such as immediate bedside diagnosis, minimally invasive detection, tumor typing, and cell differentiation.
  • Selective polyadenylation refers to the pre-mRNA maturation process, due to slight changes in the environment, the mRNA undergoes selective splicing and polyadenylation at different splicing sites Phosphorylation.
  • APA plays an important role in the formation of mRNA 3 'non-coding region (3'UTR), and it is an important part of post-transcriptional regulation.
  • the formation of APA mainly includes the selection of poly (A) sites and polyadenylation. The difference in selective polyadenylation signal sites is the key to APA formation.
  • the precursor mRNA is cut off 10-30 nt downstream of the polyadenylation signal site, and then, under the catalysis of polyadenylation polymerase, polyadenylic acid poly (A) is added at the 3 'end. tail.
  • the classical polyadenylation site is AAUAAA, and other non-classical variants include AUUAAA, AAGAAA, and UAUAAA.
  • genes, especially proto-oncogenes are more likely to use mRNA subtypes with short-chain 3'UTRs, and this subtype is closely related to the diagnosis and prognosis of tumors.
  • APA is also closely related to many physiological functions such as cell differentiation, cell localization, RNA stability, DNA methylation, gene expression and silencing, and embryonic tissue development. Therefore, APA is used in cell and biomedicine. Very important function.
  • the typical methods for detecting APA mainly include blotting hybridization, real-time quantitative PCR, gene chip, high-throughput sequencing and other methods, but there are some disadvantages in these methods, such as expensive equipment, high maintenance costs, tedious sample processing, Time-consuming, high detection cost, complicated operation, low detection sensitivity, high false positive rate, etc. have limited their application and popularity. Therefore, the development of a simple, fast, accurate, and low-cost APA detection method that does not require PCR amplification and overcomes the shortcomings of the above methods has important scientific and application value.
  • the purpose of the present invention is to provide a simple, fast, accurate and low-cost method for preparing a non-coding RNA electrochemical sensor.
  • the steps are as follows:
  • Ferric trichloride hydrate is added to ethylene glycol to form a clear solution, and sodium acetate and polyethylene glycol are added. After stirring for 30 to 60 minutes, it is placed in a hydrothermal reactor to heat the reaction. After cooling to room temperature, a black color is obtained. Precipitate, wash the precipitate with absolute ethanol, and obtain Fe 3 O 4 nanospheres after drying;
  • the mass-volume concentration of the ferric chloride in ethylene glycol is 0.2% to 0.5%.
  • the hydrothermal reaction is performed at 120 to 220 ° C for 5 to 10 hours, and the drying is performed at 60 to 100 ° C for 6 to For 10 hours, the mass ratio of sodium acetate to ferric chloride is 3 to 6: 1, and the mass ratio of polyethylene glycol to ferric chloride is 1: 2 to 3: 1;
  • step (1) Disperse the Fe 3 O 4 nano-microspheres in ultrapure water in step (1).
  • step (2) Disperse the Fe 3 O 4 nano-microspheres in ultrapure water in step (1).
  • step (3) After the ultrasonic dispersion is uniform, add polyethylene glycol 400, trisodium citrate, chloroauric acid, and ascorbic acid in this order. Stir and separate with a magnet to obtain The black precipitate was washed with anhydrous ethanol to obtain Au @ Fe 3 O 4 complex; the concentrations of the polyethylene glycol 400, trisodium citrate, chloroauric acid, and ascorbic acid in ultrapure water were 0.10 to 0.25, respectively. mg / mL, 1 ⁇ 5mg / mL, 2 ⁇ 6mg / mL, 1 ⁇ 6mg / mL;
  • Disperse the 4-sulfonic acid calix [8] arene hydrate and graphene oxide in deionized water, adjust the pH value to 7.0 ⁇ 12.0 after sonication, and after refluxing, centrifuge, discard the supernatant, and wash the solid with deionized water 3 to 4 times to obtain the reduced graphene oxide-SCX8 composite; disperse the reduced graphene oxide-SCX8 composite in deionized water, and add HAuCl 4 after homogeneous ultrasonic dispersion, stir, centrifuge and discard the supernatant
  • the solid was washed with deionized water to obtain the Au @ RGO-SCX8 complex; the Au @ RGO-SCX8 complex was ultrasonically dispersed in deionized water, then an electric signal substance was added, stirred, and centrifuged to obtain a precipitate, which was washed with deionized water Precipitation, finally the Au @ RGO-SCX8-electric signal substance complex;
  • the mass concentration of the 4-sulfonic calix [8] arene hydrate and graphite oxide in deionized water are both 0.1% to 0.5%; the mass concentration of HAuCl 4 in the reduced graphene oxide-SCX8 composite dispersion is: 1% to 5%;
  • the electric signal substance is an electroactive substance that can be recognized by a 4-sulfonic acid calix [8] arene hydrate, and 0.2 mg to 1 mg of the electric signal substance is added per 1 mL of the Au @ RGO-SCX8 complex dispersion; the electric signal substance is Toluidine blue, methylene blue or ferrocene.
  • the Au @ Fe 3 O 4 complex was ultrasonically dispersed in the buffer solution I, and the concentration of the Au @ Fe 3 O 4 complex in the buffer solution I was 0.5 to 3 mg / mL, and a capture probe was added, and the capture probe was buffered
  • the concentration in solution I is 0.5 ⁇ 10 ⁇ mol / L, and it is left for 5-20 hours at 4 °C, and the magnets are separated.
  • buffer I and hexyl mercaptan are added to the solid, and the concentration of solid in buffer I is 0.5 ⁇ 3mg / mL, the concentration of hexyl mercaptan in buffer solution I is 1-5 mmol / L.
  • Hexyl mercaptan is used to block non-specific sites. After standing at room temperature for 10 to 40 minutes, it is separated with a magnet. Buffer solution II and The target RNA at a concentration range of 10 -18 to 10 -9 mol / L. The concentration of the solid in the buffer solution II is 0.5 to 5 mg / mL after separation. After standing at room temperature for 1 to 2 hours, the magnet is separated and the solid is separated. Add the signal probe, the auxiliary probe, and the Au @ RGO-SCX8-electric signal substance complex dispersion in order, and leave it at room temperature for 1 to 2 hours. After that, the magnets are separated, the solid is dispersed in the phosphate buffer solution, and the dispersion liquid is dropped on the silk.
  • the initial concentrations of the signal probes and auxiliary probes are both 10-20 ⁇ mol / L, and 100-150 ⁇ L of signal probes and auxiliary probes are added to each 1 mg of separated solids;
  • Au @ RGO-SCX8-electrical signal substance The concentration of the complex dispersion (deionized water dispersion) is 1 to 5 mg / mL, and 1 to 1.5 mL of Au @ RGO-SCX8-electric signal substance complex dispersion is added to each 1 mg of separated solids, and the probe is diluted with water. .
  • the target RNA is added in an amount of 2 to 10 ⁇ L in a concentration range of 10 -18 to 10 -9 mol / L per 0.1 mg of solid.
  • the buffer solution I is a solution containing 10mmol / L Tris-HCl, 1mmol / L EDTA, 300mmol / L NaCl, 1mmol / L MgCl 2 ;
  • the buffer solution II is a solution containing 10mmol / L Tris-HCl, 1mmol / LEDTA, 300mmol / L L NaCl, 1 mmol / LTCEP solution.
  • the pH of the phosphate buffer was 7.
  • the target gene can produce transcripts with 3'UTRs of different lengths due to the action of APA. From these 3'UTR sequences, about 40nt long sequences are selected as target sequences and the NCBI data (https: // www. ncbi.nlm.nih.gov/) to detect the specificity of the probe; then use the 5 'end 20nt sequence as a template to obtain the capture probe (CP) recognition sequence based on the base pairing reverse complementation principle; signal
  • the probe (LP) sequence consists of two parts: the 20 nt sequence at the 3 'end is derived from the reverse complement of the 20 nt sequence at the 3' end of the target sequence, and the 15 nt sequence at the 5 'end is an arbitrary 15 nt nucleic acid sequence;
  • the needle (Auxiliary probe, AP) consists of two parts: the 15 ′ end of the 5 ′ end of the signal probe LP is the reverse complement of the 15 ′ sequence, and the 3 ′ end is
  • Another object of the present invention is to use the electrochemical sensor prepared by the above method in selective polyadenylation detection, and use electrochemical method to detect the expression amount of 3'UTR transcripts of different lengths produced by the APA phenomenon.
  • the present invention forms a supramolecular nanocomplex through a designed capture probe, a signal probe, a target RNA, and Au @ SCX8-RGO-TB, and is prepared into a biosensor for detecting RNA.
  • the present invention has the following characteristics:
  • the present invention does not require processes such as PCR amplification of nucleic acids, which can effectively save time for sample pretreatment;
  • the present invention does not require complicated labeling and immobilization of DNA probe molecules, and mainly uses a thiol group modified at the end of a capture probe molecule to coordinate with gold nanoparticles on the surface of a nanomaterial to form a stable probe;
  • the invention has no special requirements for the base sequence of the DNA probe, and therefore has universal applicability
  • the electric signal enrichment material used in the electrochemical sensor in the present invention is 4-sulfonic acid calix [8] arene hydrate, which has strong recognition ability, such as p-toluidine blue, and therefore has better electric signal amplification effect; At the same time, 4-sulfonic calix [8] arene hydrate has good chemical stability, which is beneficial to the stability of products and industrial development;
  • the target RNA detected by the electrochemical sensor in the present invention is identified by a DNA capture probe with strong specificity, and has strong specificity;
  • the electrochemical sensor has the catalytic effect of ferric oxide, the enrichment effect of 4-sulfonic acid calix [8] arene hydrate, and the conductive effect of gold nanoparticles, it can effectively amplify the signal and have a lower The detection limit reached the single cell detection level (1.76 ⁇ 10 -19 mol / L);
  • the method of the present invention has the advantages of simple controllability, low detection limit, and easy acquisition of signals, and can quickly detect the concentration of the sample to be measured with high sensitivity;
  • the electrochemical sensor in the present invention can measure the actual concentration of RNA, that is, it can be absolutely quantified
  • the electrochemical sensor in the present invention can directly measure the RNA sample without the need to reverse transcribe the RNA sample to be tested into cDNA detection.
  • FIG. 1 is a transmission electron microscope image of Fe 3 O 4 (A) and Au @ Fe 3 O 4 (B);
  • Fig. 2 is a photoelectron spectrum of Au @ Fe 3 O 4 nanocomposite
  • FIG. 7 is a schematic diagram of the design of the CCND2 capture probe CP and qPCR primers
  • Figure 8 shows the construction and working principle of an electrochemical sensor, where HT is hexyl mercaptan
  • Figure 9 is the impedance change of the electrochemical sensor after modification of different materials; impedance maps of CCND2-L (A picture) and CCND2-S (B picture);
  • Figure 10 shows the results of quantitative detection of CCND2-L by electrochemical sensors.
  • Figure A is the DPV result and
  • Figure B is the standard curve based on DPV data.
  • Figure 11 shows the results of quantitative detection of CCND2-S by electrochemical sensors.
  • Figure A is the DPV result and
  • Figure B is the standard curve based on the DPV data.
  • Figure 12 shows the specific detection results of electrochemical sensors
  • Figure A shows the specific sequence detection results of different sequences
  • Figure B shows the CCND2 gene generated from human breast cancer cells H292 and human normal lung cells Beas-2B. Results of electrochemical detection of transcripts (CCND2-L and CCND2-S);
  • FIG. 13 is a qPCR test result of the expression levels of two CCND2 transcripts (CCND2-L and CCND2-S) produced by APA regulation in human breast cancer cells H292 and human normal lung cells Beas-2B.
  • the reduced graphene oxide involved in the examples was weighed commercially available graphene oxide and dispersed in deionized water, then adjusted to pH 10.0 to 12.0 with NaOH, and then refluxed at 90 ° C for 3 to 6 hours, and centrifuged at 16,000 rpm for 20 to 40 minutes. It was made by washing with centrifugal water 2 to 4 times.
  • Example 1 The method of preparing the non-coding RNA electrochemical sensor is as follows:
  • Disperse 10 mg of Fe 3 O 4 nanospheres in step (1) in 10 mL of ultrapure water After homogeneous ultrasonic dispersion, add polyethylene glycol 400, trisodium citrate, chloroauric acid, and ascorbic acid, stir, and separate with a magnet. A black precipitate was obtained, and the Au @ Fe 3 O 4 complex was obtained after washing with absolute ethanol; the concentrations of polyethylene glycol 400, trisodium citrate, chloroauric acid, and ascorbic acid in ultrapure water were 0.10 mg, respectively. / mL, 2mg / mL, 3mg / mL, 2mg / mL;
  • the mass concentration of HAuCl 4 in the reduced graphene oxide-SCX8 composite dispersion is 3%.
  • the long 3'UTR (CCND2-L) and short 3'UTR (CCND2-S) of the CCND2 gene were used as research objects, and the quantitative detection was performed with the constructed sensor. Due to the selection of APA signal sites in the CCND2 gene Differently, CCND2-L and CCND2-S can be generated with CCND2 transcripts with long and short 3'UTR, respectively.
  • CCND2-L and CCND2-S can be generated with CCND2 transcripts with long and short 3'UTR, respectively.
  • CP is complementary to a part of the sequence of the target RNA, and the other part of the target RNA is complementary to the signal probe (Label probe, LP).
  • the signal probe LP is combined with the supramolecular composite material with the electric signal substance TB, and the auxiliary probe (Auxiliary probe (AP)) connects the supramolecular composite material with the signal probe LP in series to achieve the purpose of signal amplification ( Figure 8).
  • the invention uses a screen-printed electrode for detection; screen-printed electricity can reduce human error and reduce the background electrical signal value; all steps are completed in a 0.5 mL centrifuge tube.
  • the magnetic material Fe 3 O 4 is introduced for separation. Au @ Fe 3 O 4 as metal nanoparticles can not only increase the conductivity and surface area, but also be connected to the capture probe CP with -SH ( Figures 7 and 8).
  • RNA target RNA
  • capture probe CP-S / L
  • signal-labeled probe LP-S / L
  • Auxiliary probe AP-S / L
  • the probe is diluted with sterilized water
  • the capture probe sequence is CP-S: GACGCGTCTCTCTTTCGG- (CH 2 ) 6 -SH; CP-L: AAGGCAGCT GACTATATCAT- (CH 2 ) 6 -SH;
  • the target RNA is the long 3’UTR (CCND2-L) and the short 3’UTR (CCND2-S) of the CCND2 gene.
  • the sequence of CCND2-L is: ATGATATAGTCAGCTGCCTCTTATAGAGGTCTTATCTGTTC; the sequence of CCND2-S is CCGAAAGAGAGAGACGCGTCTCCATAATCTGGTCTCTTCTTC;
  • the signal probe (LP-S) sequence is: TACTCCCCCAGGTGCGAAGAAGAGACCAGATTATG- (CH 2 ) 6 -SH; LP-L: TACTCCCCCAGGTGCGAACAGATAAGACCTCTTAA- (CH 2 ) 6 -SH;
  • Auxiliary probe (AP-S) sequence is: GCACCTGGGGGAGTACATAATCTGGTCTCTTCTTC; AP-L: GCACCTGGGGGAGTATTAAGAGGTCTTATCTGTTC.
  • Hexyl mercaptan is used to block non-specific sites. After standing at room temperature for 30 minutes, it is separated by a magnet. Buffer is added to the solid after separation.
  • Solution II a solution containing 10mmol / L Tris-HCl, 1mmol / LEDTA, 300mmol / L NaCl, 1mmol / L TCEP
  • target RNA at different concentrations ranging from 10 -18 to 10 -9 mol / L.
  • the concentration in Buffer II is 2mg / mL; after 2 hours at room temperature, the magnets are separated, and the signal probe (LP), auxiliary probe (AP), and Au @ RGO-SCX8-TB compound are added to the separated solids in sequence.
  • the dispersion solution was left at room temperature for 2 hours, and the magnet was separated.
  • Drop on and cover the surface of the screen-printed electrode and determine the relationship between the concentration of the target RNA and the peak current by differential pulse voltammetry on the electrochemical workstation (electric signals can also be realized by cyclic voltammetry or alternating current voltammetry) And obtain a standard curve of current intensity and RNA concentration to complete the construction of the electrochemical sensor;
  • the initial concentrations of the signal probe and auxiliary probe are both 10 ⁇ mol / L, and 100 ⁇ L of signal is added to each 1 mg of separated solid Probes and auxiliary probes;
  • the concentration of Au @ RGO-SCX8-TB complex dispersion is 1mg / mL, and 1mL of Au @ RGO-SCX8-TB complex dispersion is added per 1mg of solid;
  • Figure 9 shows the impedance spectra of different modified electrodes in 0.1M KCl: 1mM K 4 [Fe (CN) 6 ] / K 3 [Fe (CN) 6 ] mixed solutions;
  • Figures 9A and B are CCND2-L and CCND2, respectively.
  • a SPCE; b: SPCE / Au @ Fe 3 O 4 ; c: SPCE / Au @ Fe 3 O 4 / CP; d: SPCE / Au @ Fe 3 O 4 / CP / HT; e : SPCE / Au @ Fe 3 O 4 / CP / HT / Target; f: SPCE / Au @ Fe 3 O 4 / CP / HT / Target / Au @ SCX8-RGO-TB-LP.
  • the impedance of b is significantly lower, because Au has a good conductivity in Au @ Fe 3 O 4 and can promote the transfer of electrons, which further proves that Au @ Fe 3 O 4 was successfully compounded.
  • the impedance of c, d, and e gradually increases.
  • the impedance of f drops significantly because Au @ SCX8-RGO-TB-LP contains a large amount of gold nanoparticles to increase the electron transfer rate. It shows that the biosensor has been successfully constructed; in this embodiment, the conductivity of the modified substance on the screen-printed electrode is consistent with the obtained impedance value, and thus it is judged that the modification of the electrode is successful.
  • Figure 11A is the DPV detection curve of CCND2-S
  • This embodiment uses the electrochemical sensor in Example 1 to detect four types of mismatch sequences: 1MT-S, 2-MT-S, 1MT-L, and 2MT-L; the concentration of the mismatch sequence is diluted to 10 -10 M for electrochemical detection. ; Refer to the method of step 4 (2) of the example to prepare a complex phosphate buffer dispersion, the difference is that the target RNA is replaced with the mismatch sequence in this example, and the prepared phosphate buffer dispersion is dropped on and covered Screen printing electrode surface, measuring DPV, DPV scanning range: 0 ⁇ -0.5V;
  • the mismatch sequences are:
  • 1MT-S CCG, AAA, GAG, CGA, GAC, GCG, CAT, AAT, CTG, GTC, TCT, TCT;
  • 2MT-S CCG, AAA, GAG, CAA, GAC, GCG, TC, CAT, AAT, CTG, GTC, TCT, TCT;
  • 1MT-L ATG, ATA, TAG, CCA, GCT, TT, TTA, AGA, GGT, CTT, ATC, TGT;
  • 2MT-L ATG, TAG, CAA, GCT, GCC, TT, TGA, AGA, GGT, CTT, ATC, TGT;
  • the results are shown in Figure 12A.
  • the results show that the target DNA was mutated by one or two bases, and the current intensity measured when the four mismatch sequences were detected was very small, which was basically about 0.15 ⁇ A, and CCND2-L ( (target-L), CCND2-S (target-S), and the CCND2-S and CCND2-L sequences in lung cancer cells H292 and human normal lung cells Beas-2B have significantly higher current intensities than the four mismatched sequences, so The results show that the electrochemical biosensor prepared in this embodiment has high specificity.
  • Example 3 Application of electrochemical sensors in the detection of CCND2-S and CCND2-L in human lung cancer cells H292 and human normal lung cells Beas-2B
  • Nanodrop 2000 was used to measure the RNA concentration and agarose gel electrophoresis was used to detect the RNA quality.
  • the complex phosphate buffer solution was prepared by using the method in step 4 (2) of Example 1, except that the target RNA was replaced with the RNA sequence extracted in step (1) of this example, and the prepared phosphate buffer solution was used.
  • the dispersed liquid droplets are covered on the surface of the screen-printed electrode, and the differential pulse voltammetry DPV is used for detection.
  • the scanning range is 0 to -0.5V.
  • the test result is that the H292L current intensity is -1.0232 ⁇ A and the H292-S current intensity is -1.2681. ⁇ A, the current intensity of Beas-2B-L is -1.0029 ⁇ A, and the current intensity of Beas-2B-S is -1.2193 ⁇ A ( Figure 12A);
  • Figure 12B is human breast cancer cell H292 and human normal lung cell Beas-2B
  • the results of electrochemical detection of the expression levels of the two CCND2 transcripts (CCND2-L and CCND2-S) in the medium the result is that the concentration of Beas-2B CCND2-L is 2.15 ⁇ 10 -14 M; the concentration of Beas-2B CCND2-S 4.52 ⁇ 10 -12 M; H292 CCND2-L concentration is 5.84 ⁇ 10 -14 M; H292 CCND2-S concentration is 1.66 ⁇ 10 -11 M; CCND2-S current in human normal cells and lung cancer cells The strength is significantly higher than CCND2-L.
  • the qPCR method was used to verify the electrochemical detection results, and the PrimerScript RT reagent kit was used to complete the qPCR experiment.
  • the specific operations are as follows:
  • cDNA was synthesized by reverse transcription of the RNA prepared in step (1) of Example 3, and the cDNA was prepared using the PrimerScript RT reagent kit.
  • the primer sequences are as follows:
  • Figure 13 is the result of qPCR relative expression analysis of CCND2 transcripts (CCND2-L and CCND2-S) in human breast cancer cell H292 and human normal lung cell Beas-2B; it can be seen that whether in human normal cells or in human normal cells In human lung cancer cells, the expression of CCND2-S is also significantly higher than that of CCND2-L; this result is similar to the electrochemical detection result.
  • the results of using the electrochemical sensor constructed by the present invention to measure RNA and APA are consistent with the results of traditional qPCR. However, qPCR is generally only used for relative quantification, and rarely used for absolute quantification.
  • the electrochemical sensor in the present invention can measure the actual concentration of RNA, that is, absolute quantification.
  • the electrochemical detection method for non-coding RNA provided by the embodiment of the present invention has higher sensitivity and can obtain more accurate results.

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Abstract

Disclosed is a method for preparing an electrochemical sensor for non-coding RNA. The method comprises the steps of preparing sulfocalix[8]arene-loaded reduced graphene oxide by a one-step process, synthesizing ferroferric oxide nanomaterials by a moist heat method, and adding gold nanoparticles to synthesize composite materials Au@SCX8-RGO and Au@Fe3O4, respectively; after confirming that the materials are successfully constructed by means of material characterization, the target is electrochemically detected using screen-printed electrodes with toluidine blue and other substances as electrical signal substances. The electrochemical sensor prepared by this method can achieve the detection of the expression quantity of different lengths of transcripts of the non-coding region 3'UTR produced by alternative cleavage and polyadenylation (APA).

Description

非编码RNA的电化学传感器的制备方法及其应用Preparation method and application of non-coding RNA electrochemical sensor 技术领域Technical field
本发明属于电化学生物传感领域，具体涉及一种非编码RNA的电化学检测方法及其在选择性多聚腺苷酸化检测中的应用。The invention belongs to the field of electrochemical biosensing, and in particular relates to an electrochemical detection method for non-coding RNA and its application in selective polyadenylation detection.
背景技术Background technique
超灵敏核酸检测技术在功能基因检测及其生物学功能评价、疾病诊断等领域应用广泛。如荧光定量PCR技术、滚环扩增技术等微量核酸检测技术基于聚合酶链式反应和荧光信号检测方法达到微量核酸定量或检测，但这些检测方法存在操作步骤复杂、光学信号探针昂贵、需要特定检测仪器等问题。电化学传感技术一直是应用最广泛的生物医学和化学分析检测平台，具有简单、便携式、低成本、高检测灵敏度等优势。电化学生物传感器在床旁即时诊断、微创检测、肿瘤分型、细胞分化等生物医学领域有广泛的应用前景。Ultrasensitive nucleic acid detection technology is widely used in functional gene detection, biological function evaluation, and disease diagnosis. For example, quantitative nucleic acid detection technologies such as fluorescent quantitative PCR technology and rolling circle amplification technology are based on polymerase chain reaction and fluorescent signal detection methods to achieve quantitative nucleic acid quantification or detection. However, these detection methods have complicated operation steps, expensive optical signal probes, and need Problems with specific testing equipment. Electrochemical sensing technology has always been the most widely used biomedical and chemical analysis and detection platform, with advantages such as simplicity, portability, low cost, and high detection sensitivity. Electrochemical biosensors have broad application prospects in biomedical fields such as immediate bedside diagnosis, minimally invasive detection, tumor typing, and cell differentiation.
选择性多聚腺苷酸化(Alternative cleavage and polyadenylation，APA)是指前体mRNA在成熟的过程中，由于环境的细微变化导致mRNA在不同的剪切位点上进行选择性剪切和多聚腺苷酸化的现象。APA在mRNA 3'非编码区(3'UTR)的形成过程中起着重要的作用，是转录后水平调控的重要组成部分。APA的形成过程主要包括poly(A)位点的选择和多聚腺苷酸化。选择性多聚腺苷酸化信号位点的不同是形成APA的关键。前体mRNA在多聚腺苷酸化信号位点的下游剪切掉10～30nt，接下来，在多聚腺苷酸聚合酶的催化下，在3'端加入多聚腺苷酸poly(A)尾。经典的多聚腺苷酸化位点有AAUAAA，其他非经典的变体包括AUUAAA、AAGAAA和UAUAAA等。研究表明，在不同的癌细胞和癌组织中，基因特别是原癌基因更倾向于使用带有短链3'UTR的mRNA亚型，而且这种亚型与肿瘤的诊断分型和预后密切相关，因此它可以作为一种新型的肿瘤生物标志物，对它们的检测具有良好的临床价值和应用前景。另外，APA还与细胞的分化、细胞定位、RNA的稳定、DNA甲基化、基因的表达与沉默、胚胎组织的发育等诸多的生理功能有密切的关系，因此APA在细胞和生物医学中行使非常重要的功能。目前，检测APA的典型方法主要有印迹杂交法、实时荧光定量PCR、基因芯片、高通量测序技术等方法，但这些方法中存在的一些缺点，如设备昂贵、维护成本高、样品处理繁琐、耗时、检测成本高、操作复杂、检测灵敏度不高、假阳性率高等，限制了它们的应用及普及。因此，发展一种无需PCR扩增、简单、快速、准确、低成本的APA检测方法，克服上述方法的不足之处就具有重要的科学与应用价值。Selective polyadenylation (Alternative cleavage and polyadenylation (APA)) refers to the pre-mRNA maturation process, due to slight changes in the environment, the mRNA undergoes selective splicing and polyadenylation at different splicing sites Phosphorylation. APA plays an important role in the formation of mRNA 3 'non-coding region (3'UTR), and it is an important part of post-transcriptional regulation. The formation of APA mainly includes the selection of poly (A) sites and polyadenylation. The difference in selective polyadenylation signal sites is the key to APA formation. The precursor mRNA is cut off 10-30 nt downstream of the polyadenylation signal site, and then, under the catalysis of polyadenylation polymerase, polyadenylic acid poly (A) is added at the 3 'end. tail. The classical polyadenylation site is AAUAAA, and other non-classical variants include AUUAAA, AAGAAA, and UAUAAA. Studies have shown that in different cancer cells and cancer tissues, genes, especially proto-oncogenes, are more likely to use mRNA subtypes with short-chain 3'UTRs, and this subtype is closely related to the diagnosis and prognosis of tumors. Therefore, it can be used as a new type of tumor biomarker, which has good clinical value and application prospect for their detection. In addition, APA is also closely related to many physiological functions such as cell differentiation, cell localization, RNA stability, DNA methylation, gene expression and silencing, and embryonic tissue development. Therefore, APA is used in cell and biomedicine. Very important function. At present, the typical methods for detecting APA mainly include blotting hybridization, real-time quantitative PCR, gene chip, high-throughput sequencing and other methods, but there are some disadvantages in these methods, such as expensive equipment, high maintenance costs, tedious sample processing, Time-consuming, high detection cost, complicated operation, low detection sensitivity, high false positive rate, etc. have limited their application and popularity. Therefore, the development of a simple, fast, accurate, and low-cost APA detection method that does not require PCR amplification and overcomes the shortcomings of the above methods has important scientific and application value.
发明内容Summary of the Invention
本发明目的在于提供了一种简单、快速、准确、低成本的非编码RNA的电化学传感器的制备方法，其步骤如下：The purpose of the present invention is to provide a simple, fast, accurate and low-cost method for preparing a non-coding RNA electrochemical sensor. The steps are as follows:
(1)四氧化三铁纳米微球的制备(1) Preparation of Fe3O4 nanospheres
把三氯化铁水合物加入到乙二醇中形成澄清溶液，并加入乙酸钠和聚乙二醇，搅拌30～60分钟后，放入水热反应釜中加热反应，冷却到室温后得黑色沉淀，用无水乙醇洗涤沉淀，干燥后得到Fe 3O 4纳米微球； Ferric trichloride hydrate is added to ethylene glycol to form a clear solution, and sodium acetate and polyethylene glycol are added. After stirring for 30 to 60 minutes, it is placed in a hydrothermal reactor to heat the reaction. After cooling to room temperature, a black color is obtained. Precipitate, wash the precipitate with absolute ethanol, and obtain Fe 3 O 4 nanospheres after drying;
其中所述三氯化铁在乙二醇中的质量体积浓度为0.2％～0.5％，水热反应是在120～220℃下反应5～10小时，干燥是在60～100℃下处理6～10小时，乙酸钠与三氯化铁的质量比为3～6:1，聚乙二醇与三氯化铁的质量比为1:2～3:1；The mass-volume concentration of the ferric chloride in ethylene glycol is 0.2% to 0.5%. The hydrothermal reaction is performed at 120 to 220 ° C for 5 to 10 hours, and the drying is performed at 60 to 100 ° C for 6 to For 10 hours, the mass ratio of sodium acetate to ferric chloride is 3 to 6: 1, and the mass ratio of polyethylene glycol to ferric chloride is 1: 2 to 3: 1;
(2)金纳米粒子负载四氧化三铁纳米复合物(Au@Fe 3O 4)的制备 (2) Preparation of Au nanoparticle-supported Fe3O4 nanocomposite (Au @ Fe 3 O 4 )
将步骤(1)Fe 3O 4纳米微球分散于超纯水中，超声分散均匀后，依次加聚乙二醇400、柠檬酸三钠、氯金酸和抗坏血酸，搅拌，用磁铁分离后得到黑色沉淀，用无水乙醇洗涤后得到Au@Fe 3O 4复合物；所述聚乙二醇400、柠檬酸三钠、氯金酸、抗坏血酸的在超纯水中的浓度分别为0.10～0.25mg/mL、1～5mg/mL、2～6mg/mL、1～6mg/mL； Disperse the Fe 3 O 4 nano-microspheres in ultrapure water in step (1). After the ultrasonic dispersion is uniform, add polyethylene glycol 400, trisodium citrate, chloroauric acid, and ascorbic acid in this order. Stir and separate with a magnet to obtain The black precipitate was washed with anhydrous ethanol to obtain Au @ Fe 3 O 4 complex; the concentrations of the polyethylene glycol 400, trisodium citrate, chloroauric acid, and ascorbic acid in ultrapure water were 0.10 to 0.25, respectively. mg / mL, 1 ~ 5mg / mL, 2 ~ 6mg / mL, 1 ~ 6mg / mL;
(3)金纳米粒子/磺酸化杯[8]芳烃/还原氧化石墨烯/电信号物质复合物的制备(3) Preparation of gold nanoparticle / sulfonated calix [8] arene / reduced graphene oxide / electric signal substance complex
将4-磺酸杯[8]芳烃水合物、氧化石墨烯分散到去离子水中，超声后调节pH值为7.0～12.0，回流反应后，离心，弃去上清液，固体用去离子水洗涤3～4次，得到还原的氧化石墨烯-SCX8复合物；将还原的氧化石墨烯-SCX8复合物分散于去离子水中，超声分散均匀后加入HAuCl 4，搅拌、离心分离后弃去上清液，固体用去离子水洗涤，得到Au@RGO-SCX8复合物；把Au@RGO-SCX8复合物超声分散于去离子水中，然后加入电信号物质，搅拌，离心分离得到沉淀，用去离子水洗涤沉淀，最后得到Au@RGO-SCX8-电信号物质复合物； Disperse the 4-sulfonic acid calix [8] arene hydrate and graphene oxide in deionized water, adjust the pH value to 7.0 ～ 12.0 after sonication, and after refluxing, centrifuge, discard the supernatant, and wash the solid with deionized water 3 to 4 times to obtain the reduced graphene oxide-SCX8 composite; disperse the reduced graphene oxide-SCX8 composite in deionized water, and add HAuCl 4 after homogeneous ultrasonic dispersion, stir, centrifuge and discard the supernatant The solid was washed with deionized water to obtain the Au @ RGO-SCX8 complex; the Au @ RGO-SCX8 complex was ultrasonically dispersed in deionized water, then an electric signal substance was added, stirred, and centrifuged to obtain a precipitate, which was washed with deionized water Precipitation, finally the Au @ RGO-SCX8-electric signal substance complex;
所述4-磺酸杯[8]芳烃水合物和氧化石墨在去离子水中的质量浓度均为0.1％～0.5％；HAuCl 4在还原的氧化石墨烯-SCX8复合物分散液中的质量浓度为1％～5％； The mass concentration of the 4-sulfonic calix [8] arene hydrate and graphite oxide in deionized water are both 0.1% to 0.5%; the mass concentration of HAuCl 4 in the reduced graphene oxide-SCX8 composite dispersion is: 1% to 5%;
所述电信号物质为能被4-磺酸杯[8]芳烃水合物识别的电活性物质，每1mL Au@RGO-SCX8复合物分散液中添加0.2mg～1mg电信号物质；电信号物质为甲苯胺蓝、亚甲基蓝或二茂铁。The electric signal substance is an electroactive substance that can be recognized by a 4-sulfonic acid calix [8] arene hydrate, and 0.2 mg to 1 mg of the electric signal substance is added per 1 mL of the Au @ RGO-SCX8 complex dispersion; the electric signal substance is Toluidine blue, methylene blue or ferrocene.
(4)电化学传感器的构建(4) Construction of electrochemical sensors
将Au@Fe 3O 4复合物超声分散在缓冲液Ⅰ中，Au@Fe 3O 4复合物在缓冲液Ⅰ中的浓度为0.5～3mg/mL，并加入捕获探针，捕获探针在缓冲液Ⅰ中浓度为0.5～10μmol/L，在4℃的条件下放置5～20小时，磁铁分离；然后在固体加入缓冲液Ⅰ和己硫醇，固体在缓冲液Ⅰ中的浓度为0.5～3mg/mL，己硫醇在缓冲液Ⅰ中浓度为1～5mmol/L，己硫醇用来封闭非特异性位点，室温放置10～40min后，用磁铁分离，在分离后固体中加入缓冲液Ⅱ和浓度范围在10 -18～10 -9mol/L的目标RNA，分离后固体在缓冲液Ⅱ中的浓度是0.5～5mg/mL；室温下放置1～2小时 后磁铁分离，在分离的固体中依次加入信号探针、辅助探针和Au@RGO-SCX8-电信号物质复合物分散液，室温下放置1～2小时后磁铁分离，将固体分散在磷酸缓冲液中，取分散液滴于丝网印刷电极表面，并在电化学工作站上用示差脉冲伏安法、循环伏安法或交流伏安法确定目标RNA的浓度和峰电流的关系，并获得电流强度与RNA浓度的标准曲线，进而完成电化学传感器的构建； The Au @ Fe 3 O 4 complex was ultrasonically dispersed in the buffer solution I, and the concentration of the Au @ Fe 3 O 4 complex in the buffer solution I was 0.5 to 3 mg / mL, and a capture probe was added, and the capture probe was buffered The concentration in solution Ⅰ is 0.5 ～ 10μmol / L, and it is left for 5-20 hours at 4 ℃, and the magnets are separated. Then, buffer Ⅰ and hexyl mercaptan are added to the solid, and the concentration of solid in buffer Ⅰ is 0.5 ～ 3mg / mL, the concentration of hexyl mercaptan in buffer solution I is 1-5 mmol / L. Hexyl mercaptan is used to block non-specific sites. After standing at room temperature for 10 to 40 minutes, it is separated with a magnet. Buffer solution II and The target RNA at a concentration range of 10 -18 to 10 -9 mol / L. The concentration of the solid in the buffer solution Ⅱ is 0.5 to 5 mg / mL after separation. After standing at room temperature for 1 to 2 hours, the magnet is separated and the solid is separated. Add the signal probe, the auxiliary probe, and the Au @ RGO-SCX8-electric signal substance complex dispersion in order, and leave it at room temperature for 1 to 2 hours. After that, the magnets are separated, the solid is dispersed in the phosphate buffer solution, and the dispersion liquid is dropped on the silk. Screen printing electrode surface, and using differential pulse voltammetry and cyclic voltammetry on the electrochemical workstation AC voltammetry and the peak current to determine the relationship between the concentration of the target RNA, and a standard curve obtained with RNA concentration of current strength, thereby completing build the electrochemical sensor;
其中所述信号探针和辅助探针的初始浓度均为10～20μmol/L，每1mg分离的固体中各添加100～150μL的信号探针和辅助探针；Au@RGO-SCX8-电信号物质复合物分散液(去离子水分散液)的浓度为1～5mg/mL，每1mg分离的固体中添加1～1.5mL的Au@RGO-SCX8-电信号物质复合物分散液，探针用水稀释。The initial concentrations of the signal probes and auxiliary probes are both 10-20 μmol / L, and 100-150 μL of signal probes and auxiliary probes are added to each 1 mg of separated solids; Au @ RGO-SCX8-electrical signal substance The concentration of the complex dispersion (deionized water dispersion) is 1 to 5 mg / mL, and 1 to 1.5 mL of Au @ RGO-SCX8-electric signal substance complex dispersion is added to each 1 mg of separated solids, and the probe is diluted with water. .
所述每0.1mg固体添加2～10μL浓度范围在10 -18～10 -9mol/L的目标RNA。 The target RNA is added in an amount of 2 to 10 μL in a concentration range of 10 -18 to 10 -9 mol / L per 0.1 mg of solid.
所述缓冲液Ⅰ为含有10mmol/L Tris-HCl、1mmol/L EDTA、300mmol/L NaCl、1mmol/L MgCl 2的溶液；缓冲液Ⅱ为含有10mmol/L Tris-HCl、1mmol/LEDTA、300mmol/L NaCl、1mmol/LTCEP的溶液。 The buffer solution I is a solution containing 10mmol / L Tris-HCl, 1mmol / L EDTA, 300mmol / L NaCl, 1mmol / L MgCl 2 ; the buffer solution II is a solution containing 10mmol / L Tris-HCl, 1mmol / LEDTA, 300mmol / L L NaCl, 1 mmol / LTCEP solution.
所述磷酸缓冲液pH为7。The pH of the phosphate buffer was 7.
其中目标基因由于APA作用可产生带有不同长度3’UTR的转录本，从这些3’UTR序列中选取长约40nt序列作为目标序列(target sequences)，并在NCBI数据(https://www.ncbi.nlm.nih.gov/)上检测探针的特异性；然后取5’端20nt序列作为模板，根据碱基配对反向互补原理，获得捕获探针(Capture probe，CP)识别序列；信号探针(Label probe，LP)序列由两部分组成：3’端20nt序列来自于目标序列3’端的20nt序列的反向互补，5’端15nt序列是任意的长约15nt的核酸序列；辅助探针(Auxiliary probe，AP)由两部分组成：5’端的15nt来自于信号探针LP的5’端15nt序列的反向互补，3’端的序列来自于LP的3’端20nt序列的反向互补。捕获探针CP和信号探针LP序列的3’端用巯基修饰，这样可以通过配位结合作用，让两个探针分别连接到Au@Fe3O4复合物和Au@RGO-SCX8复合物上。Among them, the target gene can produce transcripts with 3'UTRs of different lengths due to the action of APA. From these 3'UTR sequences, about 40nt long sequences are selected as target sequences and the NCBI data (https: // www. ncbi.nlm.nih.gov/) to detect the specificity of the probe; then use the 5 'end 20nt sequence as a template to obtain the capture probe (CP) recognition sequence based on the base pairing reverse complementation principle; signal The probe (LP) sequence consists of two parts: the 20 nt sequence at the 3 'end is derived from the reverse complement of the 20 nt sequence at the 3' end of the target sequence, and the 15 nt sequence at the 5 'end is an arbitrary 15 nt nucleic acid sequence; The needle (Auxiliary probe, AP) consists of two parts: the 15 ′ end of the 5 ′ end of the signal probe LP is the reverse complement of the 15 ′ sequence, and the 3 ′ end is the reverse complement of the 20 ′ sequence of the 3 ′ end of the LP. . The 3 ′ ends of the capture probe CP and signal probe LP sequences are modified with thiol groups, so that the two probes can be linked to the Au @ Fe3O4 complex and the Au @ RGO-SCX8 complex respectively by coordination binding.
本发明另一目的是将上述方法制得电化学传感器在选择性多聚腺苷酸化检测中的应用，用电化学法检测由APA现象所产生的不同长度的3’UTR转录本表达量。Another object of the present invention is to use the electrochemical sensor prepared by the above method in selective polyadenylation detection, and use electrochemical method to detect the expression amount of 3'UTR transcripts of different lengths produced by the APA phenomenon.
根据上述步骤(4)中得到的标准曲线计算待测样品中非编码RNA(目标RNA)的浓度或者细胞中非编码RNA的浓度，并根据该结果来评价APA与癌症的关系，并用于床旁即时诊断、微创检测等领域。Calculate the concentration of non-coding RNA (target RNA) in the test sample or the concentration of non-coding RNA in the cell according to the standard curve obtained in step (4), and evaluate the relationship between APA and cancer based on the results, and use it at the bedside Immediate diagnosis, minimally invasive detection and other fields.
本发明通过所设计的捕获探针与信号探针、目标RNA和Au@SCX8-RGO-TB形成超分子纳米复合物，制备成用于检测RNA的生物传感器。The present invention forms a supramolecular nanocomplex through a designed capture probe, a signal probe, a target RNA, and Au @ SCX8-RGO-TB, and is prepared into a biosensor for detecting RNA.
与现有技术相比，本发明具有如下特点：Compared with the prior art, the present invention has the following characteristics:
1、本发明无需核酸的PCR扩增等过程，可以有效的节省样品前处理的时间；1. The present invention does not require processes such as PCR amplification of nucleic acids, which can effectively save time for sample pretreatment;
2、本发明无需复杂的标记和固定DNA探针分子操作过程，其主要利用捕获探针分子末端修饰的巯基与纳米材料表面的金纳米粒子配位形成稳定的探针；2. The present invention does not require complicated labeling and immobilization of DNA probe molecules, and mainly uses a thiol group modified at the end of a capture probe molecule to coordinate with gold nanoparticles on the surface of a nanomaterial to form a stable probe;
3、本发明对DNA探针的碱基序列无特殊要求，因此具有普遍适用性；3. The invention has no special requirements for the base sequence of the DNA probe, and therefore has universal applicability;
4、本发明中电化学传感器中所使用的电信号富集材料为对甲苯胺蓝等识别能力较强的4-磺酸杯[8]芳烃水合物，因此具有较好的电信号放大效果；同时，4-磺酸杯[8]芳烃水合物有较好的化学稳定性，有利于产品的稳定和产业化开发；4. The electric signal enrichment material used in the electrochemical sensor in the present invention is 4-sulfonic acid calix [8] arene hydrate, which has strong recognition ability, such as p-toluidine blue, and therefore has better electric signal amplification effect; At the same time, 4-sulfonic calix [8] arene hydrate has good chemical stability, which is beneficial to the stability of products and industrial development;
5、本发明中电化学传感器所检测的目标RNA是通过特异性较强的DNA捕获探针来识别的，有较强的特异性；5. The target RNA detected by the electrochemical sensor in the present invention is identified by a DNA capture probe with strong specificity, and has strong specificity;
6、本发明中由于电化学传感器具有四氧化三铁的催化效应、4-磺酸杯[8]芳烃水合物的富集效应和金纳米粒子的导电效应，可以有效的放大信号从而有较低的检出限，达到了单细胞检测水平(1.76×10 -19mol/L)； 6. In the present invention, because the electrochemical sensor has the catalytic effect of ferric oxide, the enrichment effect of 4-sulfonic acid calix [8] arene hydrate, and the conductive effect of gold nanoparticles, it can effectively amplify the signal and have a lower The detection limit reached the single cell detection level (1.76 × 10 -19 mol / L);
7、本发明中的方法具有简单可控、检出限低和信号易于采集等优点，可快速检测待测样品的浓度，且灵敏度高；7. The method of the present invention has the advantages of simple controllability, low detection limit, and easy acquisition of signals, and can quickly detect the concentration of the sample to be measured with high sensitivity;
8、本发明中的电化学传感器可以测定RNA的实际浓度，即可以绝对定量；8. The electrochemical sensor in the present invention can measure the actual concentration of RNA, that is, it can be absolutely quantified;
9、本发明中的电化学传感器可直接测定RNA样品，无需将待测RNA样品反转录为cDNA检测。9. The electrochemical sensor in the present invention can directly measure the RNA sample without the need to reverse transcribe the RNA sample to be tested into cDNA detection.
附图说明BRIEF DESCRIPTION OF THE DRAWINGS
图1是Fe 3O 4(A)和Au@Fe 3O 4(B)的透射电子显微镜图； FIG. 1 is a transmission electron microscope image of Fe 3 O 4 (A) and Au @ Fe 3 O 4 (B);
图2是Au@Fe 3O 4纳米复合材料的光电子能谱图； Fig. 2 is a photoelectron spectrum of Au @ Fe 3 O 4 nanocomposite;
图3是Au@Fe 3O 4纳米复合材料和Fe 3O 4的X衍射图； 3 is an X-ray diffraction pattern of Au @ Fe 3 O 4 nanocomposite and Fe 3 O 4 ;
图4是还原氧化石墨烯、还原的氧化石墨烯-SCX8、4-磺酸杯[8]芳烃水合物的红外光谱图；4 is an infrared spectrum chart of reduced graphene oxide, reduced graphene oxide-SCX8, 4-sulfonic acid calix [8] arene hydrate;
图5是还原氧化石墨烯、还原的氧化石墨烯-SCX8复合物的热重图谱；5 is a thermogravimetric spectrum of reduced graphene oxide and reduced graphene oxide-SCX8 composite;
图6是Au@RGO-SCX8复合物的透射电子显微镜图；6 is a transmission electron microscope image of the Au @ RGO-SCX8 composite;
图7是CCND2捕获探针CP和qPCR引物设计原理图；7 is a schematic diagram of the design of the CCND2 capture probe CP and qPCR primers;
图8是电化学传感器的构建和工作原理，图中HT是己硫醇；Figure 8 shows the construction and working principle of an electrochemical sensor, where HT is hexyl mercaptan;
图9是电化学传感器在修饰上不同材料后阻抗的变化；CCND2-L(A图)和CCND2-S(B图)的阻抗图谱；Figure 9 is the impedance change of the electrochemical sensor after modification of different materials; impedance maps of CCND2-L (A picture) and CCND2-S (B picture);
图10是电化学传感器对CCND2-L定量检测结果，A图是DPV结果，B图是根据DPV数据做的标准曲线；Figure 10 shows the results of quantitative detection of CCND2-L by electrochemical sensors. Figure A is the DPV result and Figure B is the standard curve based on DPV data.
图11是电化学传感器对CCND2-S定量检测结果，A图是DPV结果，B图是根据DPV数据 做的标准曲线；Figure 11 shows the results of quantitative detection of CCND2-S by electrochemical sensors. Figure A is the DPV result and Figure B is the standard curve based on the DPV data.
图12是电化学传感器特异性检测结果；A图为不同序列特异性检测结果；B图是人乳腺癌细胞H292和人正常肺细胞Beas-2B中，CCND2基因由于APA调控所产生的CCND2两种转录本(CCND2-L和CCND2-S)表达量的电化学检测结果；Figure 12 shows the specific detection results of electrochemical sensors; Figure A shows the specific sequence detection results of different sequences; Figure B shows the CCND2 gene generated from human breast cancer cells H292 and human normal lung cells Beas-2B. Results of electrochemical detection of transcripts (CCND2-L and CCND2-S);
图13是人乳腺癌细胞H292和人正常肺细胞Beas-2B中APA调控所产生的CCND2两种转录本(CCND2-L和CCND2-S)表达量的qPCR检测结果。FIG. 13 is a qPCR test result of the expression levels of two CCND2 transcripts (CCND2-L and CCND2-S) produced by APA regulation in human breast cancer cells H292 and human normal lung cells Beas-2B.
具体实施方式detailed description
下面通过附图和实施例对本发明作进一步详细说明，但本发明保护范围不局限于所述内容，实施例中方法如无特殊说明均为常规方法，使用的试剂如无特殊说明均为常规市售试剂或按常规方法配制的试剂；The present invention will be further described in detail below with reference to the accompanying drawings and embodiments, but the scope of protection of the present invention is not limited to the content. The methods in the examples are conventional methods unless otherwise specified, and the reagents used are conventional ones unless otherwise specified. Sell reagents or reagents formulated according to conventional methods;
实施例中涉及的还原氧化石墨烯是称取市购氧化石墨烯置于去离子水中超声分散后，用NaOH调pH至10.0～12.0，然后90℃回流反应3～6h，16000rpm离心20～40min，离心水洗2～4次制得。The reduced graphene oxide involved in the examples was weighed commercially available graphene oxide and dispersed in deionized water, then adjusted to pH 10.0 to 12.0 with NaOH, and then refluxed at 90 ° C for 3 to 6 hours, and centrifuged at 16,000 rpm for 20 to 40 minutes. It was made by washing with centrifugal water 2 to 4 times.
实施例1：本非编码RNA的电化学传感器的制备方法步骤如下：Example 1: The method of preparing the non-coding RNA electrochemical sensor is as follows:
1、四氧化三铁(Fe 3O 4)纳米微球的制备 1. Preparation of Fe 3 O 4 nano-microspheres
把1.35g FeCl 3.6H 2O加入到40mL乙二醇中形成澄清溶液，并加入乙酸钠和聚乙二醇，其中乙酸钠与三氯化铁的质量比为3:1，聚乙二醇与三氯化铁的质量比为7:10；搅拌30分钟后，放入水热反应釜中加热至180℃，反应7小时，冷却后到室温得黑色沉淀，用无水乙醇洗涤沉淀，在60℃下处理10小时得到Fe 3O 4纳米微球； Add 1.35g FeCl 3 .6H 2 O to 40mL ethylene glycol to form a clear solution, and add sodium acetate and polyethylene glycol, where the mass ratio of sodium acetate to ferric chloride is 3: 1, polyethylene glycol The mass ratio to ferric chloride is 7:10; after stirring for 30 minutes, it is placed in a hydrothermal reaction kettle and heated to 180 ° C for 7 hours. After cooling to room temperature, a black precipitate is obtained. The precipitate is washed with absolute ethanol. Fe 3 O 4 nanospheres were obtained by processing at 60 ° C for 10 hours;
2、金纳米粒子负载四氧化三铁纳米复合物(Au@Fe 3O 4)的制备 2.Preparation of gold nanoparticle-supported Fe3O4 nanocomposite (Au @ Fe 3 O 4 )
将步骤(1)Fe 3O 4纳米微球10mg分散于10mL的超纯水中，超声分散均匀后，加入聚乙二醇400、柠檬酸三钠、氯金酸和抗坏血酸，搅拌，用磁铁分离后得到黑色沉淀，用无水乙醇洗涤后得到Au@Fe 3O 4复合物；其中聚乙二醇400、柠檬酸三钠、氯金酸、抗坏血酸的在超纯水中的浓度分别为0.10mg/mL、2mg/mL、3mg/mL、2mg/mL； Disperse 10 mg of Fe 3 O 4 nanospheres in step (1) in 10 mL of ultrapure water. After homogeneous ultrasonic dispersion, add polyethylene glycol 400, trisodium citrate, chloroauric acid, and ascorbic acid, stir, and separate with a magnet. A black precipitate was obtained, and the Au @ Fe 3 O 4 complex was obtained after washing with absolute ethanol; the concentrations of polyethylene glycol 400, trisodium citrate, chloroauric acid, and ascorbic acid in ultrapure water were 0.10 mg, respectively. / mL, 2mg / mL, 3mg / mL, 2mg / mL;
采用JEM-2100型透射电子显微镜(日立，日本)对所得的四氧化三铁纳米微球和金纳米粒子负载的四氧化三铁复合物分别进行了表征；由图1A可见，制备成的四氧化三铁呈规整的球状，粒径大约为350nm。负载金纳米粒子之后，四氧化三铁表面有明显的小颗粒(图1B)，证明了金纳米粒子已经负载到四氧化三铁的表面。同时，为了从另外的角度验证金纳米粒子和四氧化三铁已经复合成功，采用光电子能谱(图2)和X衍射(图3)对其进行了表征，结果表明复合物中存在金和铁的特征峰，因此也进一步证明金纳米粒子与四氧化三铁成功复合。The JEM-2100 transmission electron microscope (Hitachi, Japan) was used to characterize the obtained Fe3O4 nanospheres and gold nanoparticle-supported Fe3O4 composites; as shown in Figure 1A, the prepared tetraoxide Triiron has a regular spherical shape with a particle size of about 350 nm. After the gold nanoparticles were loaded, there were obvious small particles on the surface of the iron oxide (Figure 1B), which proved that the gold nanoparticles had been loaded on the surface of the iron oxide. At the same time, in order to verify that the gold nanoparticles and ferric oxide have been successfully compounded from another angle, they were characterized by photoelectron spectroscopy (Figure 2) and X-ray diffraction (Figure 3). The results show that gold and iron are present in the composite. The characteristic peaks of this compound also further prove that gold nanoparticles and triiron tetroxide successfully recombine.
3、金纳米粒子/磺酸化杯[8]芳烃/还原氧化石墨烯/甲苯胺蓝/纳米复合物(Au@SCX8-RGO-TB)复合材料的制备3. Preparation of Au nanoparticle / sulfonated calix [8] arene / reduced graphene oxide / toluidine blue / nanocomposite (Au @ SCX8-RGO-TB) composite
将4-磺酸杯[8]芳烃水合物、氧化石墨烯分散到去离子水中，4-磺酸杯[8]芳烃水合物在去离子水中的质量浓度均为0.2％，氧化石墨烯在去离子水中的质量浓度均为0.3％，超声混合后调节pH值为7，回流反应后，离心，弃去上清液，固体用去离子水洗涤3次，得到还原的氧化石墨烯-SCX8复合物(RGO-SCX8)；将制得的RGO-SCX8分散于去离子水中，超声分散均匀后加入HAuCl 4，HAuCl 4在还原的氧化石墨烯-SCX8复合物分散液中的质量浓度为3％，搅拌、离心分离后弃去上清液，用去离子水洗涤沉淀，得到Au@RGO-SCX8复合物；把Au@RGO-SCX8复合物超声分散于离子水中，然后加入甲苯胺蓝(每1mL Au@RGO-SCX8复合物分散液中添加0.5mg甲苯胺蓝)，搅拌，离心分离得到沉淀，用去离子水洗涤沉淀，最后得到Au@RGO-SCX8-TB复合物； Disperse the 4-sulfonic calix [8] arene hydrate and graphene oxide into deionized water. The mass concentration of 4-sulfonic calix [8] arene hydrate in deionized water is 0.2%, and the graphene oxide is dehydrated. The mass concentration in ionized water was 0.3%. After ultrasonic mixing, the pH was adjusted to 7. After refluxing, the mixture was centrifuged, and the supernatant was discarded. The solid was washed three times with deionized water to obtain a reduced graphene oxide-SCX8 complex. (RGO-SCX8); disperse the prepared RGO-SCX8 in deionized water, and add HAuCl 4 after homogeneous ultrasonic dispersion. The mass concentration of HAuCl 4 in the reduced graphene oxide-SCX8 composite dispersion is 3%. Stir After centrifugation, discard the supernatant, wash the precipitate with deionized water to obtain Au @ RGO-SCX8 complex; ultrasonically disperse the Au @ RGO-SCX8 complex in ionized water, and then add toluidine blue (each 1mL Au @ 0.5mg toluidine blue was added to the RGO-SCX8 complex dispersion, stirred, centrifuged to obtain a precipitate, and the precipitate was washed with deionized water to finally obtain Au @ RGO-SCX8-TB complex;
采用红外光谱、热重分析和透射电镜等技术对制得的SCX8-RGO复合物进行了表征；还原的氧化石墨烯(RGO)的FTIR光谱表明该材料中存在伸展振动峰-OH(3426cm -1)，和含氧官能团C-O/C-C(1040cm -1)，其中也存在共轭C＝C(1625cm -1)。SCX8-RGO中，-OH(3426cm -1)和O-H弯曲峰振动(1400cm -1)明显增强，是由于在SCX8中引入了-OH引起的，同时也存在CH 2(3190cm -1)的伸缩峰。此外，在1167和1040cm -1处出现典型的-SO 3-，这意味着SCX8分子被成功负载到RGO上。因此，红外光谱结果说明复合物中含有SCX8的特征红外峰(图4)，证明材料复合成功。热重分析结果也表明在相同温度下，复合物的失重比RGO要大得多(图5)，表明SCX8作为有机物和RGO成功复合。从透射电镜图中可以看出，RGO-SCX8复合材料为片层结构。同时，经过还原反应，可以看到SCX8-RGO表面存在粒径约为10nm左右的颗粒(图6)，证明金纳米粒子已经成功地修饰到SCX8-RGO表面。在丝网印刷电极上修饰或负载上各种材料之后，其阻抗发生了变化，由此来判断修饰是否成功。 Infrared spectroscopy, thermogravimetric analysis, and transmission electron microscopy were used to characterize the prepared SCX8-RGO composite; the FTIR spectrum of reduced graphene oxide (RGO) indicated that a stretching vibration peak -OH (3426cm -1 ) exists in the material ), And an oxygen-containing functional group CO / CC (1040 cm -1 ), where a conjugate C = C (1625 cm -1 ) is also present. In SCX8-RGO, -OH (3426cm -1 ) and OH bending peak vibration (1400cm -1 ) are significantly enhanced, which is caused by the introduction of -OH in SCX8, and there is also a stretching peak of CH 2 (3190cm -1 ). . In addition, typical -SO 3 -appeared at 1167 and 1040 cm -1 , which means that SCX8 molecules were successfully loaded onto RGO. Therefore, the infrared spectrum results show that the composite contains the characteristic infrared peaks of SCX8 (Figure 4), which proves that the material is successfully compounded. The results of thermogravimetric analysis also show that at the same temperature, the weight loss of the composite is much greater than that of RGO (Figure 5), indicating that SCX8 successfully compounded as an organic compound with RGO. It can be seen from the transmission electron microscope image that the RGO-SCX8 composite material has a sheet structure. At the same time, after the reduction reaction, it can be seen that particles with a diameter of about 10 nm exist on the surface of SCX8-RGO (Figure 6), which proves that the gold nanoparticles have been successfully modified on the surface of SCX8-RGO. After the various materials are modified or loaded on the screen-printed electrode, the impedance changes, thereby judging whether the modification is successful.
4、电化学传感器的构建4. Construction of electrochemical sensors
以CCND2基因的长3’UTR(CCND2-L)和短的3’UTR(CCND2-S)为研究对象，用所构建的传感器对其进行了定量检测；由于CCND2基因中APA信号位点的选取不同，可产生分别带有长和短3’UTR的CCND2转录本CCND2-L和CCND2-S。我们首先从NCBI数据库中下载到人的Dicer1基因序列(GenBank No.NG_016311.1)，然后根据基因3’UTR序列中的APA多聚腺苷酸化位点(AAUAAA)，获得CCND2-L和CCND2-S转录本的序列，然后使用Primer5.0软件设计扩增这些不同转录本的实时定量PCR(qPCR)的引物(图7)，并把引物放入UCSC(http://genome.ucsc.edu)数据库中在人的基因组中blast，当结果显示为特异性扩 增目的片段时选用该引物并开展qPCR实验。同时，截取qPCR反应获得的扩增子amplicon中40nt的序列作为电化学传感器构建用的捕获探针序列(Capture probe，CP)，并在NCBI数据(https://www.ncbi.nlm.nih.gov/)上检测探针的特异性。根据碱基互补配对原理设计，CP与目标RNA的部分序列互补，目标RNA另一部分序列则与信号探针(Label probe，LP)互补配对。信号探针LP与带有电信号物质TB的超分子复合材料相结合，辅助探针(Auxiliary probe，AP)将带有信号探针LP的超分子复合材料进行串联，从而达到信号放大的目的(图8)。本发明用丝网印刷电极进行检测；丝网印刷电可以减小人为误差，降低背景电信号值；所有步骤均在0.5mL离心管中完成。引入磁性材料Fe 3O 4以便分离。Au@Fe 3O 4作为金属纳米粒子不仅有增加导电率，增大表面积的作用，还能与带有-SH的捕获探针CP相连(图7、8)。 The long 3'UTR (CCND2-L) and short 3'UTR (CCND2-S) of the CCND2 gene were used as research objects, and the quantitative detection was performed with the constructed sensor. Due to the selection of APA signal sites in the CCND2 gene Differently, CCND2-L and CCND2-S can be generated with CCND2 transcripts with long and short 3'UTR, respectively. We first downloaded the human Dicer1 gene sequence (GenBank No. NG_016311.1) from the NCBI database, and then obtained CCND2-L and CCND2- based on the APA polyadenylation site (AAUAAA) in the 3'UTR sequence of the gene. S transcript sequence, and then use Primer5.0 software to design primers for real-time quantitative PCR (qPCR) of these different transcripts (Figure 7), and place the primers in UCSC (http://genome.ucsc.edu) The database is blasted in the human genome. When the results show that the target fragment is specifically amplified, the primer is selected and a qPCR experiment is performed. At the same time, the 40 nt sequence of the amplicon amplicon obtained from the qPCR reaction was taken as a capture probe (CP) for the construction of an electrochemical sensor, and the NCBI data (https: //www.ncbi.nlm.nih. gov /). Designed according to the principle of base complementary pairing, CP is complementary to a part of the sequence of the target RNA, and the other part of the target RNA is complementary to the signal probe (Label probe, LP). The signal probe LP is combined with the supramolecular composite material with the electric signal substance TB, and the auxiliary probe (Auxiliary probe (AP)) connects the supramolecular composite material with the signal probe LP in series to achieve the purpose of signal amplification ( Figure 8). The invention uses a screen-printed electrode for detection; screen-printed electricity can reduce human error and reduce the background electrical signal value; all steps are completed in a 0.5 mL centrifuge tube. The magnetic material Fe 3 O 4 is introduced for separation. Au @ Fe 3 O 4 as metal nanoparticles can not only increase the conductivity and surface area, but also be connected to the capture probe CP with -SH (Figures 7 and 8).
(1)构建检测CCND2-L和CCND2-S不同转录本的传感器序列，包括目标RNA(Target RNA)、捕获探针(CP-S/L)、信号标记探针(LP-S/L)、辅助探针(AP-S/L)，探针用灭菌水稀释；(1) Construct sensor sequences for detecting different transcripts of CCND2-L and CCND2-S, including target RNA (Target RNA), capture probe (CP-S / L), signal-labeled probe (LP-S / L), Auxiliary probe (AP-S / L), the probe is diluted with sterilized water;
其中捕获探针序列为CP-S：GACGCGTCTCTCTCTTTCGG-(CH 2) 6-SH；CP-L：AAGGCAGCT GACTATATCAT-(CH 2) 6-SH； The capture probe sequence is CP-S: GACGCGTCTCTCTCTTTCGG- (CH 2 ) 6 -SH; CP-L: AAGGCAGCT GACTATATCAT- (CH 2 ) 6 -SH;
目标RNA为CCND2基因的长3’UTR(CCND2-L)和短的3’UTR(CCND2-S)，CCND2-L序列为：ATGATATAGTCAGCTGCCTTTTAAGAGGTCTTATCTGTTC；CCND2-S序列为CCGAAAGAGAGAGACGCGTCCATAATCTGGTCTCTTCTTC；The target RNA is the long 3’UTR (CCND2-L) and the short 3’UTR (CCND2-S) of the CCND2 gene. The sequence of CCND2-L is: ATGATATAGTCAGCTGCCTCTTATAGAGGTCTTATCTGTTC; the sequence of CCND2-S is CCGAAAGAGAGAGAGACGCGTCTCCATAATCTGGTCTCTTCTTC;
信号探针(LP-S)序列为：TACTCCCCCAGGTGCGAAGAAGAGACCAGATTATG-(CH 2) 6-SH；LP-L：TACTCCCCCAGGTGCGAACAGATAAGACCTCTTAA-(CH 2) 6-SH； The signal probe (LP-S) sequence is: TACTCCCCCAGGTGCGAAGAAGAGACCAGATTATG- (CH 2 ) 6 -SH; LP-L: TACTCCCCCAGGTGCGAACAGATAAGACCTCTTAA- (CH 2 ) 6 -SH;
辅助探针(AP-S)序列为：GCACCTGGGGGAGTACATAATCTGGTCTCTTCTTC；AP-L：GCACCTGGGGGAGTATTAAGAGGTCTTATCTGTTC。Auxiliary probe (AP-S) sequence is: GCACCTGGGGGAGTACATAATCTGGTCTCTTCTTC; AP-L: GCACCTGGGGGAGTATTAAGAGGTCTTATCTGTTC.
(2)将10mg Au@Fe 3O 4复合物超声分散在10mL缓冲液Ⅰ(含有10mmol/L Tris-HCl、1mmol/L EDTA、300mmol/L NaCl、1mmol/L MgCl 2的溶液)中，并加入捕获探针(CP)，捕获探针在缓冲液Ⅰ中浓度为10μmol/L，在4℃的条件下放置12小时，磁铁分离；然后在固体加入缓冲液Ⅰ和己硫醇，固体在缓冲液Ⅰ中的浓度为1mg/mL，己硫醇在缓冲液Ⅰ中浓度为5mmol/L，己硫醇用来封闭非特异性位点，室温放置30min后，用磁铁分离，在分离后固体中加入缓冲液Ⅱ(含有10mmol/L Tris-HCl、1mmol/LEDTA、300mmol/L NaCl、1mmol/L TCEP的溶液)和浓度范围在10 -18～10 -9mol/L的不同浓度目标RNA，分离后固体在缓冲液Ⅱ中的浓度是2mg/mL；室温下放置2小时后磁铁分离，在分离的固体中依次加入信号探针(LP)、辅助探针(AP)和Au@RGO-SCX8-TB复合物分散液，室温下放置2小时后 磁铁分离，将固体分散在磷酸缓冲液(pH＝7)中，取磷酸缓冲液分散液滴于并覆盖在丝网印刷电极表面，并在电化学工作站上用示差脉冲伏安法确定目标RNA的浓度和峰电流的关系(电信号也可以用循环伏安法或交流伏安法实现)，并获得电流强度与RNA浓度的标准曲线，进而完成电化学传感器的构建；其中所述信号探针和辅助探针的初始浓度均为10μmol/L，每1mg分离的固体中各添加100μL的信号探针和辅助探针；Au@RGO-SCX8-TB复合物分散液的浓度为1mg/mL，每1mg固体中添加1mL的Au@RGO-SCX8-TB复合物分散液； (2) Ultrasonic disperse 10 mg of Au @ Fe 3 O 4 complex in 10 mL of buffer solution I (a solution containing 10 mmol / L Tris-HCl, 1 mmol / L EDTA, 300 mmol / L NaCl, 1 mmol / L MgCl 2 ), and Add capture probe (CP), the concentration of capture probe in buffer solution I is 10μmol / L, leave it at 4 ℃ for 12 hours, and separate the magnet; then add buffer solution I and hexyl mercaptan to the solid, and the solid is in buffer The concentration in Ⅰ is 1 mg / mL, and the concentration of hexyl mercaptan in buffer solution I is 5 mmol / L. Hexyl mercaptan is used to block non-specific sites. After standing at room temperature for 30 minutes, it is separated by a magnet. Buffer is added to the solid after separation. Solution Ⅱ (a solution containing 10mmol / L Tris-HCl, 1mmol / LEDTA, 300mmol / L NaCl, 1mmol / L TCEP) and target RNA at different concentrations ranging from 10 -18 to 10 -9 mol / L. The concentration in Buffer II is 2mg / mL; after 2 hours at room temperature, the magnets are separated, and the signal probe (LP), auxiliary probe (AP), and Au @ RGO-SCX8-TB compound are added to the separated solids in sequence. The dispersion solution was left at room temperature for 2 hours, and the magnet was separated. The solid was dispersed in a phosphate buffer solution (pH = 7), and the phosphate buffer solution was dispersed. Drop on and cover the surface of the screen-printed electrode, and determine the relationship between the concentration of the target RNA and the peak current by differential pulse voltammetry on the electrochemical workstation (electric signals can also be realized by cyclic voltammetry or alternating current voltammetry) And obtain a standard curve of current intensity and RNA concentration to complete the construction of the electrochemical sensor; the initial concentrations of the signal probe and auxiliary probe are both 10 μmol / L, and 100 μL of signal is added to each 1 mg of separated solid Probes and auxiliary probes; the concentration of Au @ RGO-SCX8-TB complex dispersion is 1mg / mL, and 1mL of Au @ RGO-SCX8-TB complex dispersion is added per 1mg of solid;
图9为不同修饰电极分别在0.1M KCl:1mM K 4[Fe(CN) 6]/K 3[Fe(CN) 6]混合溶液中的阻抗图谱；图9A、B分别为CCND2-L和CCND2-S的阻抗图；a:SPCE；b:SPCE/Au@Fe 3O 4；c:SPCE/Au@Fe 3O 4/CP；d:SPCE/Au@Fe 3O 4/CP/HT；e:SPCE/Au@Fe 3O 4/CP/HT/Target；f:SPCE/Au@Fe 3O 4/CP/HT/Target/Au@SCX8-RGO-TB-LP。与a相比，b的阻抗明显降低，是因为Au@Fe 3O 4中Au具有良好的导电性可以促进电子的转移，也进一步说明Au@Fe 3O 4是复合成功的。随着材料的逐步修饰，c、d、e的阻抗是逐渐增大的。但f的阻抗大幅度下降，这是因为Au@SCX8-RGO-TB-LP中含有大量的金纳米粒子增加了电子的转移率。说明生物传感器已被成功构建；本实施例中丝网印刷电极上修饰上的物质的导电性与得到的阻抗值是一致的，由此判断电极的修饰是成功的。 Figure 9 shows the impedance spectra of different modified electrodes in 0.1M KCl: 1mM K 4 [Fe (CN) 6 ] / K 3 [Fe (CN) 6 ] mixed solutions; Figures 9A and B are CCND2-L and CCND2, respectively. -S impedance chart; a: SPCE; b: SPCE / Au @ Fe 3 O 4 ; c: SPCE / Au @ Fe 3 O 4 / CP; d: SPCE / Au @ Fe 3 O 4 / CP / HT; e : SPCE / Au @ Fe 3 O 4 / CP / HT / Target; f: SPCE / Au @ Fe 3 O 4 / CP / HT / Target / Au @ SCX8-RGO-TB-LP. Compared with a, the impedance of b is significantly lower, because Au has a good conductivity in Au @ Fe 3 O 4 and can promote the transfer of electrons, which further proves that Au @ Fe 3 O 4 was successfully compounded. With the gradual modification of the material, the impedance of c, d, and e gradually increases. However, the impedance of f drops significantly because Au @ SCX8-RGO-TB-LP contains a large amount of gold nanoparticles to increase the electron transfer rate. It shows that the biosensor has been successfully constructed; in this embodiment, the conductivity of the modified substance on the screen-printed electrode is consistent with the obtained impedance value, and thus it is judged that the modification of the electrode is successful.
图10是CCND2-L电化学传感器的检出曲线；其中图10A为CCND2-L的DPV检测曲线，DPV扫描范围：0～-0.5V；图10B为CCND2-L的检出曲线；在10 -17-10 -11M浓度范围内，电流强度与样品浓度的线性回归方程为：I(μA)＝-0.047log C-1.64，相关系数R 2为0.993，检出限度为9.5×10 -18mol/L。同理，图11A为CCND2-S的DPV检出曲线，图11B为CCND2-S的检出曲线；在10 -18-10 -11mol/L浓度范围内，其电流强度与样品浓度的线性方程为：I(μA)＝-0.086log C-2.20，相关系数R 2为0.992；计算其检出限度为1.76×10 -19mol/L。 FIG 10 is a graph of detection CCND2-L electrochemical sensor; wherein FIG. 10A is a DPV CCND2-L of the detection curve, DPV scanning range: 0 ~ -0.5V; FIG. 10B is a graph detection CCND2-L; at 10 - In the range of 17 -10 -11 M concentration, the linear regression equation of the current intensity and sample concentration is: I (μA) = -0.047log C-1.64, the correlation coefficient R 2 is 0.993, and the detection limit is 9.5 × 10 -18 mol / L. Similarly, Figure 11A is the DPV detection curve of CCND2-S, and Figure 11B is the detection curve of CCND2-S; in the concentration range of 10 -18 -10 -11 mol / L, the linear equation of its current intensity and sample concentration It is: I (μA) =-0.086log C-2.20, and the correlation coefficient R 2 is 0.992; the detection limit is calculated to be 1.76 × 10 -19 mol / L.
实施例2：电化学传感器特异性检测Example 2: Specific detection of electrochemical sensors
本实施例采用实施例1中的电化学传感器检测1MT-S、2-MT-S和1MT-L、2MT-L四种错配序列；错配序列浓度稀释至10 -10M进行电化学检测；参照实施例步骤4(2)的方法制备复合物磷酸缓冲液分散液，不同在于将目标RNA替换为本实施例中的错配序列，将制得的磷酸缓冲液分散液滴于并覆盖在丝网印刷电极表面，测DPV，DPV扫描范围：0～-0.5V； This embodiment uses the electrochemical sensor in Example 1 to detect four types of mismatch sequences: 1MT-S, 2-MT-S, 1MT-L, and 2MT-L; the concentration of the mismatch sequence is diluted to 10 -10 M for electrochemical detection. ; Refer to the method of step 4 (2) of the example to prepare a complex phosphate buffer dispersion, the difference is that the target RNA is replaced with the mismatch sequence in this example, and the prepared phosphate buffer dispersion is dropped on and covered Screen printing electrode surface, measuring DPV, DPV scanning range: 0 ～ -0.5V;
其中错配序列分别为：The mismatch sequences are:
1MT-S：CCG AAA GAG CGA GAC GCG TC CAT AAT CTG GTC TCT TCT TC；1MT-S: CCG, AAA, GAG, CGA, GAC, GCG, CAT, AAT, CTG, GTC, TCT, TCT;
2MT-S：CCG AAA GAG CAA GAC GCG TC CAT AAT CTG GTC TCT TCT TC；2MT-S: CCG, AAA, GAG, CAA, GAC, GCG, TC, CAT, AAT, CTG, GTC, TCT, TCT;
1MT-L：ATG ATA TAG CCA GCT GCC TT TTA AGA GGT CTT ATC TGT TC；1MT-L: ATG, ATA, TAG, CCA, GCT, TT, TTA, AGA, GGT, CTT, ATC, TGT;
2MT-L：ATG ATA TAG CAA GCT GCC TT TTA AGA GGT CTT ATC TGT TC；2MT-L: ATG, TAG, CAA, GCT, GCC, TT, TGA, AGA, GGT, CTT, ATC, TGT;
结果见图12A，图中结果显示对目标DNA进行一个或两个碱基的突变，检测到四种错配序列所测得的电流强度均很小，基本在0.15μA左右，而CCND2-L(target-L)、CCND2-S(target-S)、以及肺癌细胞H292及人正常肺细胞Beas-2B中的CCND2-S和CCND2-L序列的电流强度明显远高于4个错配序列，因此结果显示本实施例制得的电化学生物传感器具有较高的特异性。The results are shown in Figure 12A. The results show that the target DNA was mutated by one or two bases, and the current intensity measured when the four mismatch sequences were detected was very small, which was basically about 0.15 μA, and CCND2-L ( (target-L), CCND2-S (target-S), and the CCND2-S and CCND2-L sequences in lung cancer cells H292 and human normal lung cells Beas-2B have significantly higher current intensities than the four mismatched sequences, so The results show that the electrochemical biosensor prepared in this embodiment has high specificity.
实施例3：电化学传感器在人肺癌细胞H292及人正常肺细胞Beas-2B中CCND2-S和CCND2-L的检测中的应用Example 3: Application of electrochemical sensors in the detection of CCND2-S and CCND2-L in human lung cancer cells H292 and human normal lung cells Beas-2B
(1)选用人正常肺细胞及人肺癌细胞进行RNA的提取，提取方法如下：(1) Select human normal lung cells and human lung cancer cells for RNA extraction. The extraction method is as follows:
1)除去细胞培养瓶中的培养基，加入1mL PBS清洗细胞，并吸去丢掉；1) Remove the medium from the cell culture flask, wash the cells by adding 1mL PBS, and aspirate and discard;
2)添加0.5mL Trizol，用手震动5min，使细胞裂解，将其收集到1.5mL的离心管中；2) Add 0.5mL Trizol, shake by hand for 5min to lyse the cells, and collect them into a 1.5mL centrifuge tube;
3)加入0.1mL氯仿，剧烈震荡15s，室温放置2-3min；3) Add 0.1mL of chloroform, shake vigorously for 15s, and leave at room temperature for 2-3min;
4)4℃12000rpm离心15min；(开始准备离心所需新管；准备RNA收集柱；准备酶-RDD混合体系：10μL DNase I stock solution和70μL Buffer RDD)；4) Centrifuge at 12,000 rpm for 15 min at 4 ° C; (start to prepare new tubes for centrifugation; prepare RNA collection column; prepare enzyme-RDD mixed system: 10 μL DNase Stock Solution and 70 μL Buffer RDD);
5)转移上层清液至新的离心管中，加入等体积的70％乙醇，上下颠倒使其混合均匀(不能离心)，使用移液枪吸打9次，并马上开始下一步操作；5) Transfer the supernatant to a new centrifuge tube, add an equal volume of 70% ethanol, and mix it upside down (cannot centrifuge), pipette 9 times with a pipette, and immediately start the next operation;
6)转移样品至RNeasy Mini收集柱内，小心盖上，室温下8000g离心15s，弃液相，重复使用收集柱进行下一步操作；6) Transfer the sample to the RNeasy Mini collection column, carefully cover it, centrifuge at 8000g for 15s at room temperature, discard the liquid phase, and reuse the collection column for the next operation;
7)添加350μL Buffer RW1，8000rpm离心15s，弃掉液相；7) Add 350μL Buffer RW1, centrifuge at 8000rpm for 15s, and discard the liquid phase;
8)添加10μL DNase I stock solution和70μL Buffer RDD混合液，室温(20-30℃)放置孵育15min(准备新的收集管，制胶)；8) Add 10 μL DNase Stock Solution and 70 μL Buffer RDD, and incubate at room temperature (20-30 ° C) for 15 min (preparation of a new collection tube, making gel);
9)添加350μL Buffer RW1，8000rpm离心15s，弃液相；9) Add 350μL Buffer RW1, centrifuge at 8000rpm for 15s, and discard the liquid phase;
10)添加500μL Buffer RPE，8000rpm离心15s，弃液相；10) Add 500μL Buffer RPE, centrifuge at 8000rpm for 15s, and discard the liquid phase;
11)添加500μL Buffer RPE，8000rpm离心2min，弃液相；11) Add 500μL Buffer RPE, centrifuge at 8000rpm for 2min, discard the liquid phase;
12)使用新的收集管，最大转速离心1min；12) Use a new collection tube and centrifuge at the maximum speed for 1min;
13)添加30-50μL RNase-free-water至收集柱内薄膜(正中)，12000rpm离心2min；13) Add 30-50 μL RNase-free-water to the membrane (center) in the collection column, and centrifuge at 12000 rpm for 2 min;
14)使用Nanodrop 2000对RNA浓度进行测定及琼脂糖凝胶电泳初步检测RNA质量情况。14) Nanodrop 2000 was used to measure the RNA concentration and agarose gel electrophoresis was used to detect the RNA quality.
(2)采用实施例1步骤4(2)方法制备复合物的磷酸缓冲液分散液，不同在于将目标RNA替换为本实施例步骤(1)中提取的RNA序列，将制得的磷酸缓冲液分散液滴于并覆盖在丝网印刷电极表面，采用示差脉冲伏安法DPV进行检测，扫描范围0～-0.5V；检测结果为H292L电流强度为-1.0232μA，H292-S电流强度为-1.2681μA，Beas-2B-L电流强度为-1.0029 μA，Beas-2B-S电流强度为-1.2193μA(图12A)；(2) The complex phosphate buffer solution was prepared by using the method in step 4 (2) of Example 1, except that the target RNA was replaced with the RNA sequence extracted in step (1) of this example, and the prepared phosphate buffer solution was used. The dispersed liquid droplets are covered on the surface of the screen-printed electrode, and the differential pulse voltammetry DPV is used for detection. The scanning range is 0 to -0.5V. The test result is that the H292L current intensity is -1.0232μA and the H292-S current intensity is -1.2681. μA, the current intensity of Beas-2B-L is -1.0029 μA, and the current intensity of Beas-2B-S is -1.2193μA (Figure 12A);
然后将上述电流强度值带入实施例1步骤4(2)中获得的检出曲线(图10、11)中计算目标RNA的浓度，图12B为人乳腺癌细胞H292和人正常肺细胞Beas-2B中CCND2两种转录本(CCND2-L和CCND2-S)的表达量的电化学检测结果，结果为Beas-2B CCND2-L的浓度为2.15×10 -14M；Beas-2B CCND2-S的浓度为4.52×10 -12M；H292 CCND2-L的浓度为5.84×10 -14M；H292 CCND2-S的浓度为1.66×10 -11M；在人正常细胞及肺癌细胞中，CCND2-S的电流强度明显高于CCND2-L。 Then the above current intensity value is brought into the detection curve (Figures 10 and 11) obtained in step 4 (2) of Example 1 to calculate the target RNA concentration. Figure 12B is human breast cancer cell H292 and human normal lung cell Beas-2B The results of electrochemical detection of the expression levels of the two CCND2 transcripts (CCND2-L and CCND2-S) in the medium, the result is that the concentration of Beas-2B CCND2-L is 2.15 × 10 -14 M; the concentration of Beas-2B CCND2-S 4.52 × 10 -12 M; H292 CCND2-L concentration is 5.84 × 10 -14 M; H292 CCND2-S concentration is 1.66 × 10 -11 M; CCND2-S current in human normal cells and lung cancer cells The strength is significantly higher than CCND2-L.
采用qPCR法对电化学检测结果进行验证，使用PrimerScript RT reagent Kit试剂盒完成qPCR实验，具体操作如下：The qPCR method was used to verify the electrochemical detection results, and the PrimerScript RT reagent kit was used to complete the qPCR experiment. The specific operations are as follows:
(1)将实施例3步骤(1)制得的RNA反转录合成cDNA，使用PrimerScript RT reagent Kit试剂盒完成cDNA的制备；(1) cDNA was synthesized by reverse transcription of the RNA prepared in step (1) of Example 3, and the cDNA was prepared using the PrimerScript RT reagent kit.
(2)实时荧光定量PCR(2) Real-time PCR
1)按以下组分配制qPCR反应体系，总体积20μl(反应液在冰上进行配制，配制过程注意避光)1) Prepare the qPCR reaction system according to the following components, with a total volume of 20 μl (the reaction solution is prepared on ice, and the preparation process should be protected from light)
Figure PCTCN2018092124-appb-000001
Figure PCTCN2018092124-appb-000001
引物序列如下：The primer sequences are as follows:
Figure PCTCN2018092124-appb-000002
Figure PCTCN2018092124-appb-000002
2)配制完成后混合均匀，再平均分装到96孔板中，盖膜，1500rpm离心2min；2) After the preparation is completed, mix evenly, and then distribute it evenly into a 96-well plate, cover the membrane, and centrifuge at 1500 rpm for 2 min;
3)采用两步法进行qPCR反应；按照最适反应条件在荧光定量PCR仪上设置下列参数：3) Two-step qPCR reaction is performed; set the following parameters on the real-time PCR instrument according to the optimal reaction conditions:
Figure PCTCN2018092124-appb-000003
Figure PCTCN2018092124-appb-000003
图13为人乳腺癌细胞H292和人正常肺细胞Beas-2B中CCND2两种转录本(CCND2-L和CCND2-S)表达量的qPCR相对表达量分析结果；可以看出不管是在人正常细胞还是在人肺癌细胞中，CCND2-S的表达量也是明显高于CCND2-L的表达量的；此结果与电化学的检测结果类似。Figure 13 is the result of qPCR relative expression analysis of CCND2 transcripts (CCND2-L and CCND2-S) in human breast cancer cell H292 and human normal lung cell Beas-2B; it can be seen that whether in human normal cells or in human normal cells In human lung cancer cells, the expression of CCND2-S is also significantly higher than that of CCND2-L; this result is similar to the electrochemical detection result.
用本发明构建的电化学传感器测定RNA和APA的结果与传统的qPCR的测定结果一致。但是qPCR一般只用于相对定量，很少用于绝对定量，而本发明中的电化学传感器可以测定RNA的实际浓度，即可以绝对定量。The results of using the electrochemical sensor constructed by the present invention to measure RNA and APA are consistent with the results of traditional qPCR. However, qPCR is generally only used for relative quantification, and rarely used for absolute quantification. The electrochemical sensor in the present invention can measure the actual concentration of RNA, that is, absolute quantification.
本发明实施例提供的非编码RNA的电化学检测方法具有更高的灵敏度，可以获得更准确的结果。The electrochemical detection method for non-coding RNA provided by the embodiment of the present invention has higher sensitivity and can obtain more accurate results.
Figure PCTCN2018092124-appb-000004
Figure PCTCN2018092124-appb-000004
Figure PCTCN2018092124-appb-000005
Figure PCTCN2018092124-appb-000005
Figure PCTCN2018092124-appb-000006
Figure PCTCN2018092124-appb-000006
Figure PCTCN2018092124-appb-000007
Figure PCTCN2018092124-appb-000007
Figure PCTCN2018092124-appb-000008
Figure PCTCN2018092124-appb-000008

Claims (10)

  1. 一种非编码RNA的电化学传感器的制备方法，其特征在于，步骤如下：A method for preparing a non-coding RNA electrochemical sensor, characterized in that the steps are as follows:
    (1)四氧化三铁纳米微球的制备(1) Preparation of Fe3O4 nanospheres
    把三氯化铁水合物加入到乙二醇中形成澄清溶液，并加入乙酸钠和聚乙二醇，搅拌30～60分钟后，放入水热反应釜中加热反应，冷却到室温后得黑色沉淀，用无水乙醇洗涤沉淀，干燥后得到Fe 3O 4纳米微球； Ferric trichloride hydrate is added to ethylene glycol to form a clear solution, and sodium acetate and polyethylene glycol are added. After stirring for 30 to 60 minutes, it is placed in a hydrothermal reactor to heat the reaction. After cooling to room temperature, a black color is obtained. Precipitate, wash the precipitate with absolute ethanol, and obtain Fe 3 O 4 nanospheres after drying;
    (2)金纳米粒子负载四氧化三铁纳米复合物的制备(2) Preparation of gold nanoparticle-supported ferric oxide nanocomposite
    将步骤(1)Fe 3O 4纳米微球分散于超纯水中，超声分散均匀后，依次加聚乙二醇400、柠檬酸三钠、氯金酸和抗坏血酸，搅拌，用磁铁分离后得到黑色沉淀，用无水乙醇洗涤后得到Au@Fe 3O 4复合物； Disperse the Fe 3 O 4 nano-microspheres in ultrapure water in step (1). After the ultrasonic dispersion is uniform, add polyethylene glycol 400, trisodium citrate, chloroauric acid, and ascorbic acid in this order. Stir and separate with a magnet to obtain Black precipitate, washed with anhydrous ethanol to obtain Au @ Fe 3 O 4 complex;
    (3)金纳米粒子/磺酸化杯[8]芳烃/还原氧化石墨烯/电信号物质复合物的制备(3) Preparation of gold nanoparticle / sulfonated calix [8] arene / reduced graphene oxide / electric signal substance complex
    将4-磺酸杯[8]芳烃水合物、氧化石墨烯分散到去离子水中，超声后调节pH值为7.0～12.0，回流反应后，离心，弃去上清液，固体用去离子水洗涤3～4次，得到还原的氧化石墨烯-SCX8复合物；将还原的氧化石墨烯-SCX8复合物分散于去离子水中，超声分散均匀后加入HAuCl 4，搅拌、离心分离后弃去上清液，固体用去离子水洗涤，得到Au@RGO-SCX8复合物；把Au@RGO-SCX8复合物超声分散于去离子水中，然后加入电信号物质，搅拌，离心分离得到沉淀，用去离子水洗涤沉淀，最后得到Au@RGO-SCX8-电信号物质复合物； Disperse the 4-sulfonic acid calix [8] arene hydrate and graphene oxide in deionized water, adjust the pH value to 7.0 ～ 12.0 after sonication, and after refluxing, centrifuge, discard the supernatant, and wash the solid with deionized water 3 to 4 times to obtain the reduced graphene oxide-SCX8 composite; disperse the reduced graphene oxide-SCX8 composite in deionized water, and add HAuCl 4 after homogeneous ultrasonic dispersion, stir, centrifuge and discard the supernatant The solid was washed with deionized water to obtain the Au @ RGO-SCX8 complex; the Au @ RGO-SCX8 complex was ultrasonically dispersed in deionized water, then an electric signal substance was added, stirred, and centrifuged to obtain a precipitate, which was washed with deionized water Precipitation, finally the Au @ RGO-SCX8-electric signal substance complex;
    (4)电化学传感器的构建(4) Construction of electrochemical sensors
    将Au@Fe 3O 4复合物超声分散在缓冲液Ⅰ中，并加入捕获探针，在4℃的条件下放置5～20小时，磁铁分离，然后在固体加入缓冲液Ⅰ和己硫醇，固体在缓冲液Ⅰ中的浓度为0.5～3mg/mL，己硫醇用来封闭非特异性位点，室温放置10～40min后，用磁铁分离，在分离后固体中加入缓冲液Ⅱ和浓度范围在10 -18～10 -9mol/L的目标RNA，分离后固体在缓冲液Ⅱ中的浓度是0.5～5mg/mL；室温下放置1～2小时后磁铁分离，在分离的固体中依次加入信号探针、辅助探针和Au@RGO-SCX8-电信号物质复合物分散液，室温下放置1～2小时后磁铁分离，将固体分散在磷酸缓冲液中，取分散液滴于丝网印刷电极表面，并在电化学工作站上用示差脉冲伏安法、循环伏安法或交流伏安法确定目标RNA的浓度和峰电流的关系，并获得电流强度与RNA浓度的标准曲线，进而完成电化学传感器的构建。 Disperse the Au @ Fe 3 O 4 complex in buffer solution I and add capture probes. Place it at 4 ° C for 5-20 hours. The magnets are separated. Then add buffer solution I and hexyl mercaptan to the solid. The concentration in the buffer solution I is 0.5 to 3 mg / mL. Hexyl mercaptan is used to block non-specific sites. After standing at room temperature for 10 to 40 minutes, it is separated with a magnet. After the separation, the buffer solution II is added and the concentration range is 10 -18 ～ 10 -9 mol / L target RNA, the concentration of solids in buffer solution Ⅱ is 0.5 ～ 5mg / mL after separation; after 1 to 2 hours at room temperature, the magnets are separated, and signal detection is sequentially added to the separated solids. Needle, auxiliary probe, and Au @ RGO-SCX8-electric signal substance composite dispersion. After standing at room temperature for 1 to 2 hours, the magnets are separated, the solid is dispersed in a phosphate buffer solution, and the dispersion liquid is dropped on the surface of the screen printing electrode. And using differential pulse voltammetry, cyclic voltammetry, or alternating current voltammetry to determine the relationship between the target RNA concentration and the peak current on the electrochemical workstation, and obtain a standard curve of the current intensity and RNA concentration to complete the electrochemical sensor Build.
  2. 根据权利要求1所述的非编码RNA的电化学传感器的制备方法，其特征在于：步骤(1)中三氯化铁在乙二醇中的质量体积浓度为0.2％～0.5％，水热反应是在120～220℃下反应5～10小时，干燥是在60～100℃下处理6～10小时，乙酸钠与三氯化铁的质量比为3～6:1，聚乙二醇与三氯化铁的质量比为1:2～3:1。The method for preparing a non-coding RNA electrochemical sensor according to claim 1, characterized in that the mass-volume concentration of ferric chloride in ethylene glycol in step (1) is 0.2% to 0.5%, and the hydrothermal reaction It is a reaction at 120 to 220 ° C for 5 to 10 hours, and drying is performed at 60 to 100 ° C for 6 to 10 hours. The mass ratio of sodium acetate to ferric chloride is 3 to 6: 1, and polyethylene glycol and The mass ratio of ferric chloride is 1: 2 to 3: 1.
  3. 根据权利要求1所述的非编码RNA的电化学传感器的制备方法，其特征在于：步骤(2)中聚乙二醇400、柠檬酸三钠、氯金酸、抗坏血酸的在超纯水中的浓度分别为0.10～0.25mg/mL、1～5mg/mL、2～6mg/mL、1～6mg/mL。The method for preparing a non-coding RNA electrochemical sensor according to claim 1, wherein in step (2), the polyethylene glycol 400, trisodium citrate, chloroauric acid, and ascorbic acid in ultrapure water The concentrations were 0.10 to 0.25 mg / mL, 1 to 5 mg / mL, 2 to 6 mg / mL, and 1 to 6 mg / mL.
  4. 根据权利要求1所述的非编码RNA的电化学传感器的制备方法，其特征在于：步骤(3)中4-磺酸杯[8]芳烃水合物和氧化石墨在去离子水中的质量浓度均为0.1％～0.5％；HAuCl 4的在还原的氧化石墨烯-SCX8复合物分散液中的质量浓度为1％～5％。 The method for preparing a non-coding RNA electrochemical sensor according to claim 1, wherein in step (3), the mass concentration of the 4-sulfonic acid calix [8] arene hydrate and graphite oxide in deionized water are both 0.1% to 0.5%; the mass concentration of HAuCl 4 in the reduced graphene oxide-SCX8 composite dispersion is 1% to 5%.
  5. 根据权利要求1所述的非编码RNA的电化学传感器的制备方法，其特征在于：步骤(3)***号物质为能被4-磺酸杯[8]芳烃水合物识别的电活性物质，每1mL Au@RGO-SCX8复合物分散液中添加0.2mg～1mg电信号物质。The method for preparing a non-coding RNA electrochemical sensor according to claim 1, characterized in that the electric signal substance in step (3) is an electroactive substance that can be recognized by the 4-sulfonic acid calix [8] arene hydrate, 0.2mg ～ 1mg of electric signal substance is added to 1mL of Au @ RGO-SCX8 complex dispersion.
  6. 根据权利要求5所述的非编码RNA的电化学传感器的制备方法，其特征在于：电信号物质为甲苯胺蓝、亚甲基蓝或二茂铁。The method for preparing a non-coding RNA electrochemical sensor according to claim 5, wherein the electrical signal substance is toluidine blue, methylene blue or ferrocene.
  7. 根据权利要求1所述的非编码RNA的电化学传感器的制备方法，其特征在于：步骤(4)中Au@Fe 3O 4复合物在缓冲液Ⅰ中的浓度为0.5～3mg/mL，捕获探针在缓冲液Ⅰ中浓度为0.5～10μmol/L，己硫醇在缓冲液Ⅰ中浓度为1～5mmol/L； The method for preparing a non-coding RNA electrochemical sensor according to claim 1, characterized in that the concentration of the Au @ Fe 3 O 4 complex in the buffer solution I in step (4) is 0.5 to 3 mg / mL, and capture The concentration of the probe in the buffer solution I is 0.5 to 10 μmol / L, and the concentration of the hexyl mercaptan in the buffer solution I is 1 to 5 mmol / L;
    信号探针和辅助探针的初始浓度均为10～20μmol/L，每1mg分离的固体中各添加100～150μL的信号探针和辅助探针；Au@RGO-SCX8-电信号物质复合物分散液的浓度为1～5mg/mL，每1mg分离的固体中添加1～1.5mL的Au@RGO-SCX8-电信号物质复合物分散液。The initial concentrations of the signal probe and auxiliary probe are both 10 to 20 μmol / L, and 100 to 150 μL of signal probe and auxiliary probe are added to each 1 mg of separated solids; Au @ RGO-SCX8-electric signal substance complex is dispersed The concentration of the solution is 1 to 5 mg / mL, and 1 to 1.5 mL of the Au @ RGO-SCX8-electric signal substance complex dispersion is added to each 1 mg of the separated solid.
  8. 根据权利要求7所述的非编码RNA的电化学传感器的制备方法，其特征在于：缓冲液Ⅰ为含有10mmol/L Tris-HCl、1mmol/L EDTA、300mmol/L NaCl、1mmol/L MgCl 2的溶液；缓冲液Ⅱ为含有10mmol/L Tris-HCl、1mmol/L EDTA、300mmol/L NaCl、1mmol/LTCEP的溶液。 The method for preparing a non-coding RNA electrochemical sensor according to claim 7, characterized in that: the buffer solution I comprises 10 mmol / L Tris-HCl, 1 mmol / L EDTA, 300 mmol / L NaCl, 1 mmol / L MgCl 2 Solution; Buffer II is a solution containing 10mmol / L Tris-HCl, 1mmol / L EDTA, 300mmol / L NaCl, 1mmol / LTCEP.
  9. 根据权利要求1所述的非编码RNA的电化学传感器的制备方法，其特征在于：每0.1mg分离后固体添加2～10μL浓度范围在10 -18～10 -9mol/L的目标RNA。 The method for preparing a non-coding RNA electrochemical sensor according to claim 1, characterized in that 2 to 10 μL of the target RNA having a concentration in the range of 10 -18 to 10 -9 mol / L is added per 0.1 mg of the solid after separation.
  10. 权利要求1-9任一项所述的非编码RNA的电化学传感器的制备方法制得的电化学传感器在选择性多聚腺苷酸化检测中的应用，其特征在于：用电化学法检测由APA现象所产生的不同长度的3’UTR转录本表达量。The application of the electrochemical sensor prepared by the method for preparing a non-coding RNA electrochemical sensor according to any one of claims 1 to 9 in selective polyadenylation detection, characterized in that: The expression of 3'UTR transcripts of different lengths produced by the APA phenomenon.
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