CN109270144B - Method for detecting 5-hydroxymethylcytosine based on non-labeled and non-immobilized electrochemical magnetic biosensor - Google Patents
Method for detecting 5-hydroxymethylcytosine based on non-labeled and non-immobilized electrochemical magnetic biosensor Download PDFInfo
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Abstract
The invention belongs to the technical field of biology, and particularly relates to a method for detecting 5-hydroxymethylcytosine by using a non-label non-immobilized electrochemical magnetic biosensor for non-disease diagnosis. Quantitative analysis of 5-hydroxymethylcytosine using an electrochemical magnetic biosensor with screen printed carbon electrodes based on a dual signal amplification strategy of terminal transferase and ru (iii) redox cycling. The method comprises the following specific steps: biotinylation of the 5-hydroxymethylcytosine site in the DNA strand; enrichment of biotinylated 5-hydroxymethylcytosine DNA strands with streptavidin-coated magnetic beads; terminal transferase catalyzes the extension of the polymerized biotinylated 5-hydroxymethylcytosine DNA strand; adding Ru (NH)3)6 3+/Fe(CN)6 3‑The system produces an enhanced electrocatalytic signal; and (3) carrying out quantitative analysis on the electrochemical magnetic biosensor. The lowest detection limit of the method can reach 9.06 fM.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a method for detecting 5-hydroxymethylcytosine by using a non-label non-immobilized electrochemical magnetic biosensor for non-disease diagnosis.
Background
Accurate quantification of 5-hydroxymethylcytosine (5-hmC) remains a great challenge compared to 5-methylcytosine (5-mC) assay, as it has a relatively low abundance and similarity to the more abundant 5-methylcytosine. The bisulfite sequencing method is considered as a standard for 5-methylcytosine determination, but it has the disadvantage that it cannot distinguish between 5-methylcytosine and 5-hydroxymethylcytosine. Currently, various methods for 5-hydroxymethylcytosine determination in genomic DNA have been developed, such as single molecule real-time (SMRT) sequencing, liquid chromatography/tandem mass spectrometry (LC/MS-MS), High Performance Liquid Chromatography (HPLC), Thin Layer Chromatography (TLC), enzymatic radiolabelling and immunoassays, which can effectively distinguish 5-hydroxymethylcytosine from 5-methylcytosine.
Electrochemical biosensors based on enzyme modification have been used to detect low abundance (pM level) 5-hydroxymethylcytosine due to their advantages of low cost, portability and high sensitivity. However, enzyme modification-based methods require expensive DNA sequence-limited enzymes, synthetase cofactor/substrate analogs, and cumbersome base modification steps. To overcome these limitations, an Electrochemiluminescence (ECL) biosensor based on KRuO has been developed45-hydroxymethylcytosine-DNA can be quantified by specific oxidation of 5-hydroxymethylcytosine to 5-formylcytosine (5-fC) and then labeled with the Electrochemiluminescent (ECL) luminescent molecule N- (4-aminobutyl) -N-ethyl isoluminol (ABEI). More recently, the Bayley group has discovered an alternative approach in which 5-hydroxymethylcytosine is chemically modified in situ in one step with biotin, allowing rare 5-hydroxymethylcytosine-containing sequences to be enriched and identified using protein nanopores. However, these methods are only proof of concept and they are not suitable for detecting 5-hydroxymethylcytosine in genomic DNA.
Disclosure of Invention
Aiming at the problems existing in the detection of 5-hydroxymethylcytosine, the invention provides a method for detecting 5-hydroxymethylcytosine based on a non-labeled and non-immobilized electrochemical magnetic biosensor. The invention adopts a double-signal amplification strategy of terminal transferase (TDT) and Ru (III) redox cycle, and utilizes a non-labeling non-immobilized electrochemical magnetic biosensor to accurately quantify 5-hydroxymethyl cytosine in genome DNA.
The invention adopts the following technical scheme:
in a first aspect of the invention, a non-disease diagnostic method for quantitative analysis of 5-hydroxymethylcytosine by an electrochemical magnetic biosensor is provided, which is based on a dual signal amplification strategy of terminal transferase and ru (iii) redox cycling, and the 5-hydroxymethylcytosine is quantitatively analyzed by the electrochemical magnetic biosensor with a Screen Printed Carbon Electrode (SPCE).
Further, theThe non-disease diagnosis application quantitative analysis method for detecting 5-hydroxymethylcytosine based on the electrochemical magnetic biosensor comprises the following specific steps: biotinylation of the 5-hydroxymethylcytosine site in the DNA strand; magnetic Beads (MB) with streptavidin coating enriched biotinylated 5-hydroxymethylcytosine DNA strands; terminal transferase catalyzes the extension of the polymerized biotinylated 5-hydroxymethylcytosine DNA strand; adding Ru (NH)3)6 3+/Fe(CN)6 3-The system produces an enhanced electrocatalytic signal; and (3) carrying out quantitative analysis on the electrochemical magnetic biosensor.
Further, one step of bisulfite-mediated biotinylation of 5-hydroxymethylcytosine sites in the DNA strand was performed with cysteine-biotin.
Further, cysteine-biotin and double-stranded DNA are mixed and added with Na2S2O5And incubating the solution at 42-45 ℃ for 48-50 hours, cooling the solution to room temperature, adjusting the pH value, reacting at room temperature for 5-10 minutes, and removing excessive cysteine-biotin and salt from the sample through a Micro-Bio-Spin P6 column to obtain the chitosan-biotin chitosan/chitosan composite material.
Further, the extension of biotinylated 5-hydroxymethylcytosine DNA strand is catalyzed and polymerized in a terminal transferase reaction system, wherein the terminal transferase reaction system comprises 1 × terminal transferase buffer solution and CoCl2Solution, dNTPs and terminal transferase.
Further, Ru (NH)3)6 3+/Fe(CN)6 3-The system comprises 1-1.5 mM PBS buffer solution, 0.1-0.5M NaCl, 27-30 μ M Ru (NH)3)6 3+And 2 to 5mM Fe (CN)6 3-。
Further, the electrochemical magnetic biosensor records the electrochemical response by using differential pulse voltammetry with 0 to-500 mV, pulse amplitude of 50mV and pulse width of 50ms, and the reference electrode is Ag/AgCl.
In a second aspect of the invention, there is provided the use of the quantitative analysis method described above for the analytical detection of 5-hydroxymethylcytosine in various biological, tissue and nucleic acid sequences not of diagnostic interest for diseases.
Further, 5-hydroxymethylcytosine is 5-hydroxymethylcytosine in genome DNA; the cell can be human cervical cancer cell line (HeLa cell) and human embryonic kidney cell line (HEK 293T) cell.
In a third aspect of the present invention, there is provided a kit for quantitative analysis of 5-hydroxymethylcytosine, said kit comprising cysteine-biotin, Na2S2O5Solution, streptavidin-coated magnetic beads, washing buffer, terminal transferase reaction solution, and Ru (NH)3)6 3+/Fe(CN)6 3-A system reaction solution and a screen printing carbon electrode;
the wash buffer comprises: 0.5M NaCl, 20mM tris-HCl and 1mM EDTA, tris-HCl pH 7.4;
the terminal transferase reaction mixture contained 1 × terminal transferase buffer solution and 0.25mM CoCl2Solution, dNTPs and terminal transferase;
Ru(NH3)6 3+/Fe(CN)6 3-the system reaction liquid comprises: 1 to 1.5mM PBS buffer, 0.1 to 0.5M NaCl, 27 to 30 μ M Ru (NH)3)6 3+And 2 to 5mM Fe (CN)6 3-。
The invention has the following beneficial effects:
(1) the principle is simple, and the cost is reduced: compared with the method based on the enzyme modified electrochemical biosensor, the method of the invention does not need expensive enzyme with limited recognition motif, synthesis of enzyme cofactor/substrate analogue and multiple steps of base modification due to the introduction of terminal transferase, so that the principle is clearer and easier to understand; due to the introduction of the screen printing carbon electrode, compared with single-molecule real-time sequencing, the method disclosed by the invention does not need a complex and expensive instrument, and the cost of an experiment is greatly reduced.
(2) The method can be used for actual sample detection: with an electrochemical magnetic biosensor based on screen printed carbon electrodes, the biosensor can sensitively quantify 5-hydroxymethylcytosine without the need to immobilize DNA on the electrodes. Importantly, the biosensor can be used for accurately quantifying 5-hydroxymethylcytosine in human cervical cancer cell lines (HeLa cells) and human embryonic kidney cell lines (HEK 293T) cells, and can well distinguish cytosine (C), 5-methylcytosine and 5-hydroxymethylcytosine.
(3) The sensitivity is high: the invention uses terminal transferase to catalyze the repeated addition of dNTPs without any DNA template to generate a longer DNA sequence, so that more Ru (NH3) can be generated6 3+Can be electrostatically bound to an extended negatively charged phosphate backbone, and, at the same time, Ru (NH)3)6 3+Acting as a primary electron acceptor, Fe (CN)6 3-And functions as a secondary electron acceptor. Excess Fe (CN)6 3-Ru (II) that can be electrochemically reduced by oxidation, thereby enlarging Ru (NH)3)6 3+The detection limit of the invention is 9.06fM, the sensitivity of the biosensor is improved by 4 orders of magnitude compared with the electrochemical analysis (0.23nM) based on the modification of DNA methyltransferase, 4 orders of magnitude compared with the fluorescence analysis (0.167nM) based on the modification of β -glucosyltransferase, 46 times compared with the optical sensor (0.42pM) based on a specific antibody, and 46 times compared with the optical sensor (0.42pM) based on KRuO4Is 25 times higher than that of the electrochemiluminescence sensor (0.14 pM).
(4) The specificity is good: the invention utilizes the chemical reaction of 5-hydroxymethylcytosine modification which can not occur at any other non-hydroxymethylated nucleic acid base to realize the one-step biotin incorporation at the 5-hydroxymethylcytosine site in DNA, and cytosine (C) and 5-methylcytosine can not react under the same reaction condition, so that the method can detect 5-hydroxymethylcytosine with high specificity.
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The accompanying drawings, which are incorporated in and constitute a part of this specification, are included to provide a further understanding of the invention, and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description, serve to explain the invention and not to limit the invention.
FIG. 1.5-schematic of a non-labeled, non-immobilized electrochemical magnetic biosensor for hydroxymethylcytosine assay. The assay involves two sequential steps: (A) biotinylation of 5-hydroxymethylcytosine sites in DNA mediated by a one-step bisulfite method; (B) biotinylated 5-hydroxymethylcytosine DNA-induced terminal transferase catalyzed polymerization and subsequent Ru (III) redox cycling dual signal amplification strategy.
FIG. 2 (A) Nyquist plot of Electrochemical Impedance Spectroscopy (EIS) for magnetic bead (a)/screen printed carbon electrode, Nyquist plot of Electrochemical Impedance Spectroscopy (EIS) for biotinylated 5-hydroxymethylcytosine DNA/magnetic bead/screen printed carbon electrode before (b) and after (c) addition of terminal transferase assisted base extension, wherein the impedance solution is 5mM Fe (CN) with 0.1M KCl6 3-/4-And (3) solution. (B) Zeta potential of magnetic beads (a), Zeta potential of biotinylated 5-hydroxymethylcytosine DNA/magnetic beads before (b) and after (c) addition of terminal transferase assisted base extension. (C) Magnetic bead/Screen-printed carbon electrode (a), biotinylated 5-hydroxymethylcytosine DNA/MB/SPCE before (b) and after (c) addition of terminal transferase assisted base extension in Ru (NH)3)6 3+And Fe (CN)6 3-The DPV signal response in the mixture of (a). (D) Biotinylated 5-hydroxymethylcytosine DNA/magnetic beads/Screen-printed carbon electrode and Terminal Deoxynucleotidyl Transferase (TDT) assisted base extension in the Presence of (a) Ru (NH) alone3)6 3+，(b)Ru(NH3)6 3+And Fe (CN)6 3-DPV signal in (1).
FIG. 3 (A) DPV response of electrochemical magnetic biosensors to different concentrations of 5-hydroxymethylcytosine DNA (from a to k, 0, 0.01, 0.1, 0.5, 1, 5, 10, 50, 100, 500 and 1000 pM). (B) Linear relationship between DPV peak enhancement current (Δ I) and logarithm of 5-hmC concentration (in the range of 0.01 to 1000 pM). Error bars represent the standard deviation of triplicate measurements.
FIG. 4 (A) conversion of 5-hydroxymethylcytosine to 5-hydroxymethylcytosine-biotin under condition 1 and purification with Micro-Bio-Spin P6 under condition 2; however, cytosine (C) and 5-methylcytosine are not reacted under condition 1. (B) DPV curves of electrochemical magnetic biosensors using control group of reaction buffer only, cytosine (C) of normal DNA, 5-methylcytosine and 5-hydroxymethylcytosine DNA. (C) DPV signal response of electrochemical magnetic biosensors for cytosine (C), 5-methylcytosine and 5-hydroxymethylcytosine DNA of normal DNA. Error bars represent standard deviations of three independent experiments.
FIG. 5-5-hydroxymethylcytosine content in human cervical cancer cell line (HeLa cells) and human embryonic kidney cell line (HEK293T cells).
Detailed Description
It is to be understood that the following detailed description is exemplary and is intended to provide further explanation of the invention as claimed. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of example embodiments in accordance with the invention. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, and it should be understood that when the terms "comprises" and/or "comprising" are used in this specification, they specify the presence of the stated features, steps, operations, and/or combinations thereof, unless the context clearly indicates otherwise.
In order to make the technical solutions of the present invention more clearly understood by those skilled in the art, the technical solutions of the present invention will be described in detail below with reference to specific embodiments.
The experimental principle of the invention is shown in figure 1. Hybridization of single-stranded DNA containing 5-hydroxymethylcytosine with complementary DNA forms double-stranded DNA, and subsequent introduction of a biotin-cysteine derivative enables one-step incorporation of biotin into double-stranded DNA containing 5-hydroxymethylcytosine sites. After removal of excess cysteine-biotin and salts using a Micro-Bio-Spin P6 column, biotinylated double stranded DNA was enriched by specific biotin-streptavidin interaction using magnetic beads with streptavidin coating. Subsequently, the 3' -hydroxyl terminal of the DNA can be extended by terminal transferase-catalyzed repeated addition of dNTPs without any need for any extensionDNA template, resulting in longer DNA sequences. In the electrocatalytic system, Ru (NH)3)6 3+Acting as a primary electron acceptor, Fe (CN)6 3-And functions as a secondary electron acceptor. Ru (NH)3)6 3+Can be electrostatically attracted to the negatively charged phosphate backbone of the DNA on the surface of the magnetic beads and immobilized on the screen printed carbon electrode by means of a magnetic field. Excess Fe (CN)6 3-Ru (II) that can be electrochemically reduced by oxidation, thereby enlarging Ru (NH)3)6 3+Resulting in recycling of ru (iii) and generation of an amplified electrocatalytic current. The electrocatalytic cycle follows the electron transfer kinetics-based mechanism described by equations (1) and (2).
Ru(NH3)6 3++e-→Ru(NH3)6 2+(1)
Ru(NH3)6 2++Fe(CN)6 3-→Ru(NH3)6 3++Fe(CN)6 4-(2)
Biotinylated 5-hydroxymethylcytosine DNA bound to streptavidin-labeled magnetic beads was placed on a screen-printed carbon electrode, the Ru (NH)3)6 3+/Fe(CN)6 3-The system can produce an enhanced electrocatalytic signal. Furthermore, Fe (CN)6 3-Negatively charged, repelled by DNA anions away from the electrode surface, eliminating their direct response to false positive signals. Due to the 5-hydroxymethylcytosine-induced specific one-step bisulfite conversion method and the efficient dual signal amplification of the terminal transferase catalyzed polymerization reaction and the ru (iii) redox cycle mediated amplification, the biosensor can detect 5-hydroxymethylcytosine simply, rapidly, with high sensitivity and selectivity.
The DNA sequences used in the present invention are shown in Table 1 below.
TABLE 1 related DNA sequences
Examples
The quantitative analysis method comprises the following steps:
extraction of total intracellular DNA: the culture medium for human cervical cancer cell line (HeLa cells) and human embryonic kidney cell line (HEK 293T) cells was Dulbecco's Modified Eagle's Medium (DMEM) containing 10% Fetal Bovine Serum (FBS) and 1% penicillin-streptomycin, and was cultured in an incubator containing 5% carbon dioxide at 37 ℃. When the cells grow to the logarithmic growth phase, extracting and purifying the total DNA in the cells by using a cultured cell DNA extraction kit (Epigenek), wherein the extraction and purification operations are strictly carried out according to the instructions attached to the kit, and the concentration of the obtained total DNA is measured by using an ultraviolet-visible spectrophotometer.
Biotinylation of 5-hydroxymethylcytosine sites in DNA mediated by the one-step bisulfite method: one-step bisulfite-mediated biotinylation of 5-hydroxymethylcytosine in DNA was performed with cysteine-biotin. Cysteine-biotin (120. mu.L, 10. mu.M, pH adjusted to 5.0 with 10M sodium hydroxide) and 10. mu.L of double-stranded DNA (5-hydroxymethylcytosine-complementary DNA) with different concentrations were mixed in a 200. mu.L centrifuge tube. Mixing Na2S2O5(5 μ L, 1.0M, pH 5.0) was added to the solution, followed by incubation at 42 ℃ for 48 hours. After the solution was cooled to room temperature, the pH was adjusted to 13 with 10M sodium hydroxide. After 5 minutes of reaction at room temperature, the sample was passed through a Micro-Bio-Spin P6 column (Bio Rad) three times to remove excess cysteine-biotin and salts.
Preparation of 5-hydroxymethylcytosine DNA-Linked magnetic beads 4. mu.L of streptavidin-coated magnetic bead suspension was transferred to the above 200. mu.L centrifuge tube and washed twice with 50. mu.L of washing buffer (0.5M NaCl, 20mM tris-HCl (pH 7.4), 1mM EDTA.) the magnetic beads were separated by removing the supernatant using a magnetic separation rack, 50. mu.L of buffer containing 1 × terminal transferase (10mM Mg (Ac))2,20mM Tris-Ac，50mM KAc，pH＝7.9)，0.25mM CoCl21mM dNTPs, 6U terminal transferase reaction system for enzymatic amplification, and then again with washing buffer to form sensor array.
Electrochemical measurement: will contain 1mM PBS (pH 7.4), 0.1M NaCl, 27. mu.M Ru (NH)3)6 3+And 2mM Fe (CN)6 3-The solution was added to a centrifuge tube containing isolated biotinylated 5-hydroxymethylcytosine DNA/magnetic beads. 100 μ L of the above solution was then transferred to the surface of a screen printed carbon electrode. Biotinylated 5-hydroxymethylcytosine DNA/magnetic bead complexes were immobilized on a screen printed carbon electrode by applying a super magnet below the surface of the working electrode. The electrochemical response was recorded using Differential Pulse Voltammetry (DPV) (reference electrode Ag/AgCl) from 0 to-500 mV, pulse amplitude 50mV, and pulse width 50 ms.
To verify the feasibility of this approach, the preparation of electrochemical magnetic biosensors was characterized by Electrochemical Impedance Spectroscopy (EIS) and zeta potential (fig. 2). Electron transfer resistance (R)et) From [ Fe (CN) ]6]3-/4-Electron transfer kinetics control on the modified screen-printed carbon electrode and can be characterized by the semi-circle diameter of the nyquist plot. As shown in FIG. 2A, the electron transfer resistance (R) of the magnetic beads of the non-stationary screen-printed carbon electrodeet) At 5980 Ω (fig. 2A, curve a). In contrast, due to negatively charged DNA and [ Fe (CN)6]3-/4-The biotinylated 5-hydroxymethyl cytosine DNA/magnetic bead shows larger electron transfer resistance (R) under the action of electrostatic repulsionet) And 7120 Ω (FIG. 2A, curve b), indicating successful modification of 5-hydroxymethylcytosine DNA on magnetic beads. After incubation of biotinylated 5-hydroxymethylcytosine DNA with terminal transferase, the electron transfer resistance (R)et) Increasing to 9980 omega. This can be explained by the fact that: after terminal transferase catalyzed polymerization, the more negatively charged phosphate backbone of biotinylated 5-hydroxymethylcytosine DNA may be hindered [ Fe (CN)6]3-/4-Electron transfer from the solution to the electrode surface (fig. 2A, curve c).
The zeta potential was used to study the attachment of biotinylated 5-hydroxymethylcytosine to the magnetic beads. As shown in FIG. 2B, the addition of biotinylated 5-hydroxymethylcytosine DNA (FIG. 2B, curve B, -4.5. + -. 0.2mV) induced a significant change in zeta potential compared to the control with magnetic beads alone (FIG. 2B, curve a, -2.3. + -. 0.5mV), indicating successful coating of biotinylated 5-hydroxymethylcytosine DNA on the surface of the magnetic beads. When 5-hydroxymethylcytosine DNA was incubated with terminal transferase, a higher negative zeta potential (-10.2. + -. 0.4mV) was observed due to terminal transferase catalyzed polymerization induced by biotinylated 5-hydroxymethylcytosine (FIG. 2B, curve c).
In Ru (NH)3)6 3+And Fe (CN)6 3-Before and after terminal transferase assisted base extension, DPV measurements were recorded using different electrodes including magnetic bead/screen printed carbon electrode, biotinylated 5-hydroxymethylcytosine DNA/magnetic bead/screen printed carbon electrode (fig. 2C). In the control group, which did not have a 5-hydroxymethylcytosine site in the DNA, the current was small (5.59. mu.A) (FIG. 2C, curve a). When the 5-hydroxymethylcytosine site in the DNA was biotinylated, the signal increased to 7.85. mu.A (FIG. 2C, curve b) due to the uptake of large amounts of Ru (NH) on the negatively charged phosphate backbone of the biotinylated 5-hydroxymethylcytosine DNA3)6 3+. After incubation of biotinylated 5-hydroxymethylcytosine DNA/magnetic beads with terminal transferase, the signal increased further to 11.5. mu.A (FIG. 2C, curve C) because of more Ru (NH)3)6 3+Electrostatically bound to an elongated negatively charged phosphate backbone. These results demonstrate the feasibility of the biosensor for the detection of 5-hydroxymethylcytosine.
Under optimal experimental conditions, the present invention evaluated the analytical performance of a magnetoelectric biosensor in response to different concentrations of 5-hydroxymethylcytosine DNA, as shown in FIG. 3A, with increasing 5-hmC DNA concentration from 0.01 to 1000pM, DPV signal was enhanced, the logarithm of current to 5-hydroxymethylcytosine DNA concentration obtained a linear relationship (FIG. 3B) ranging from 0.01 to 1000pM, the detection limit calculated as 9.06 fM. the sensitivity of the biosensor was 4 orders of magnitude higher than that of electrochemical analysis based on modification of DNA methyltransferase (0.23nM), 4 orders of magnitude higher than that of fluorescence analysis based on modification of β -glucosyltransferase (0.167nM), 46 times higher than that of optical sensor based on specific antibody (0.42pM), and 46 times higher than that based on KRuO 45 of the electroluminescent chemiluminescence method (0.14pM)The selective chemical oxidation of hydroxymethylcytosine is 25 times higher, which is sufficient to demonstrate the high sensitivity of the detection of the present invention.
To investigate the specificity of this biosensor, 5-methylcytosine with a single 5-methylcytosine site and normal DNA cytosine (C) were used as negative controls. As shown in FIG. 4C, under the same conditions, a higher peak current change was detected only in the presence of 5-hydroxymethylcytosine. The peak current change (Δ I) for 5-hydroxymethylcytosine was 22 and 39 times higher than for normal DNA cytosine (C) and 5-methylcytosine DNA (FIG. 4C), indicating that the biosensor has high specificity. The high specificity of the present method can be attributed to the fact that the chemical reaction of 5-hydroxymethylcytosine modification cannot take place at any other non-hydroxymethylated nucleobase.
In order to evaluate the feasibility of the biosensor proposed by the present invention in clinical diagnosis, two different cancer cell lines, including, were measured using the biosensor. The concentration of genomic DNA was measured by Q-5000UltramicroUV-Vis spectrophotometer (Quawell, USA). As shown in fig. 5, the 5-hydroxymethylcytosine content in the human cervical cancer cell line (HeLa cells) and the human embryonic kidney cell line (HEK293T cells) was determined to be 0.00793% and 0.00977% of the total nucleotides, respectively, in agreement with the previously reported results determined by dot blot based on β -glucosyltransferase modification (0.007% and 0.009%, respectively, of the total nucleotides).
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
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Claims (9)
1. A quantitative analysis method for detecting 5-hydroxymethylcytosine based on an electrochemical magnetic biosensor for non-disease diagnosis application is characterized in that: quantitative analysis of 5-hydroxymethylcytosine using an electrochemical magnetic biosensor with screen printed carbon electrodes based on a dual signal amplification strategy of terminal transferase and ru (iii) redox cycling; the method comprises the following specific steps: biotinylation of the 5-hydroxymethylcytosine site in the DNA strand; enrichment of biotinylated 5-hydroxymethylcytosine DNA strands with streptavidin-coated magnetic beads; terminal transferase catalyzes the extension of the polymerized biotinylated 5-hydroxymethylcytosine DNA strand; adding intoRu(NH3)6 3+/Fe(CN)6 3-The system produces an enhanced electrocatalytic signal; and (3) carrying out quantitative analysis on the electrochemical magnetic biosensor.
2. The quantitative analysis method as claimed in claim 1, wherein the bisulfite-mediated biotinylation of 5-hydroxymethylcytosine sites in the DNA strand is carried out in one step with cysteine-biotin.
3. The quantitative analysis method according to claim 2, wherein the cysteine-biotin and the double-stranded DNA are mixed and Na is added2S2O5And incubating the solution at 42-45 ℃ for 48-50 hours, cooling the solution to room temperature, adjusting the pH value, reacting at room temperature for 5-10 minutes, and removing excessive cysteine-biotin and salt from the sample through a Micro-Bio-Spin P6 column to obtain the chitosan-biotin chitosan/chitosan composite material.
4. The quantitative analysis method of claim 1, wherein the extension of the biotinylated 5-hydroxymethylcytosine DNA strand is catalyzed in a terminal transferase reaction system comprising 1 × TdT buffer, CoCl2Solution, dNTPs and terminal transferase.
5. The quantitative analysis method according to claim 1, wherein Ru (NH)3)6 3+/Fe(CN)6 3-The system comprises 1-1.5 mM PBS buffer solution, 0.1-0.5M NaCl, 27-30 μ M Ru (NH)3)6 3+And 2 to 5mM Fe (CN)6 3-。
6. A quantitative analysis method according to claim 1, wherein the electrochemical magnetic biosensor records the electrochemical response using differential pulse voltammetry with a pulse amplitude of 50mV and a pulse width of 50ms from 0 to-500 mV, and the reference electrode is Ag/AgCl.
7. Use of the quantitative analysis method according to any one of claims 1 to 6 for the analytical detection of 5-hydroxymethylcytosine in various tissues, cells and various nucleic acid sequences not intended for the diagnosis of diseases.
8. The use of claim 7, wherein the 5-hydroxymethylcytosine is 5-hydroxymethylcytosine in genomic DNA; the cell is human cervical cancer cell line and human embryo kidney cell line.
9. A kit for quantitative analysis of 5-hydroxymethylcytosine is characterized by comprising cysteine-biotin and Na2S2O5Solution, streptavidin-coated magnetic beads, washing buffer, terminal transferase reaction solution, and Ru (NH)3)6 3+/Fe(CN)6 3-A system reaction solution and a screen printing carbon electrode;
the wash buffer comprises: 0.5M NaCl, 20mM tris-HCl and 1mM EDTA, tris-HCl pH 7.4;
the terminal transferase reaction mixture contained 1 × TdT buffer, 0.25mM CoCl2Solution, dNTPs and terminal transferase;
Ru(NH3)6 3+/Fe(CN)6 3-the system reaction liquid comprises: 1 to 1.5mM PBS buffer, 0.1 to 0.5M NaCl, 27 to 30 μ M Ru (NH)3)6 3+And 2 to 5mM Fe (CN)6 3-(ii) a The kit is used according to claim 1.
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| CN103388024A (en) * | 2013-07-04 | 2013-11-13 | 徐州医学院 | Bridge-PCR-based method for detecting DNA hydroxymethylation |
| CN107760762A (en) * | 2017-09-30 | 2018-03-06 | 山东师范大学 | A kind of fluorescence chemical sensor and its detection method of detection DNA adenine methyltransferases |
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| CN103388024A (en) * | 2013-07-04 | 2013-11-13 | 徐州医学院 | Bridge-PCR-based method for detecting DNA hydroxymethylation |
| CN107760762A (en) * | 2017-09-30 | 2018-03-06 | 山东师范大学 | A kind of fluorescence chemical sensor and its detection method of detection DNA adenine methyltransferases |
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| Sensitive and label-free discrimination of 5-hydroxymethylcytosine and 5-methylcytosine in DNA by ligation-mediated rolling circle amplification;Zi-yue Wang et.al;《Chem. Commun.》;20180710;第54卷;第8602-8605页 * |
| Template-Independent, in Situ Grown DNA Nanotail Enabling Label-Free Femtomolar Chronocoulometric Detection of Nucleic Acids;Fan Yang et.al;《Anal. Chem.》;20141104;第86卷;第11905-11907、11910-11911页 * |
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