CN109270144A - A method of the electrochemistry magnetic biosensor based on non-marked, on-fixed detects 5-hydroxymethyl cytosine - Google Patents
A method of the electrochemistry magnetic biosensor based on non-marked, on-fixed detects 5-hydroxymethyl cytosine Download PDFInfo
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Abstract
The invention belongs to field of biotechnology, and in particular to a kind of method of the electrochemistry magnetic biosensor detection 5-hydroxymethyl cytosine based on non-marked, on-fixed of non-disease diagnostic uses.Amplify strategy based on the dual signal of terminal enzyme (DNA) and Ru (III) redox cycle, utilizes with the electrochemistry magnetic biosensor quantitative analysis 5-hydroxymethyl cytosine of screen printing carbon electrode.Specific steps are as follows: the biotinylation in 5-hydroxymethyl cytosine site in DNA chain；With the biotinylated 5-hydroxymethyl cytosine DNA chain of the coated enrichment with magnetic bead of Streptavidin；The extension of the biotinylated 5-hydroxymethyl cytosine DNA chain of terminal enzyme (DNA) catalytic polymerization；Ru (NH is added3)6 3+/Fe(CN)6 3‑System generates the electro-catalysis signal of enhancing；Electrochemistry magnetic biosensor quantitative analysis.This method minimum detection limit is up to 9.06fM.
Description
Technical field
The invention belongs to field of biotechnology, and in particular to a kind of non-disease diagnostic uses based on non-marked, on-fixed
The method of the electrochemistry magnetic biosensor detection 5-hydroxymethyl cytosine of change.
Background technique
Compared with 5-methylcytosine (5-mC) measurement, the accurate quantitative analysis of 5-hydroxymethyl cytosine (5-hmC) is still one
A huge challenge, because it has similitude with relatively low abundance and with richer 5-methylcytosine.Sulfurous
Sour hydrogen salt sequencing approach be considered as 5-methylcytosine measurement standard, but it have cannot be distinguished from 5-methylcytosine and 5-
The shortcomings that hydroxymethyl cytosine.Currently, having developed the various sides measured for 5-hydroxymethyl cytosine in genomic DNA
Method, such as unimolecule (SMRT) sequencing, liquid chromatogram/tandem mass spectrum (LC/MS-MS), high performance liquid chromatography (HPLC) in real time,
Thin-layer chromatography (TLC), enzyme radioactivity glycosylated markers and immunoassays, it is phonetic that these methods can effectively distinguish 5- methylol born of the same parents
Pyridine and 5-methylcytosine.
Due to having low cost, portability and highly sensitive advantage, the electrochemica biological sensor based on enzyme modification is
It is used to detect the 5-hydroxymethyl cytosine of low abundance (pM rank).However, method based on enzyme modification need it is expensive by
Enzyme, synthesis enzyme cofactor/substrate analog and the cumbersome base modification step of DNA sequence limitation.In order to overcome this
A little limitations, have developed a kind of electrogenerated chemiluminescence (ECL) biosensor, are based on KRuO4It can be phonetic by 5- methylol born of the same parents
Pyridine specific oxidation quantifies 5-hydroxymethyl cytosine-DNA to 5- formoxyl cytimidine (5-fC), is then sent out with electroluminescent chemistry
Light (ECL) light emitting molecule N- (4- aminobutyl)-N- ethyl different luminol (ABEI) label.Recently, Bayley group has found
A kind of alternative, wherein 5-hydroxymethyl cytosine is can be enriched with rare contain with the in situ step chemical modification of biotin
The sequence of 5-hydroxymethyl cytosine is simultaneously identified with protein nano hole.However, these methods are only proved as concept, it
Be not particularly suited for detection genomic DNA in 5-hydroxymethyl cytosine.
Summary of the invention
5-hydroxymethyl cytosine detection there are aiming at the problem that, the present invention provides a kind of based on non-marked, on-fixed
The method of electrochemistry magnetic biosensor detection 5-hydroxymethyl cytosine.The present invention uses terminal enzyme (DNA) (TDT) and Ru
(III) dual signal of redox cycle amplifies strategy, quasi- using non-marked, on-fixed electrochemistry magnetic biosensor
Determine the electrochemical method of the 5-hydroxymethyl cytosine in amount genomic DNA.
The invention adopts the following technical scheme:
First aspect of the present invention provides a kind of detecting based on electrochemistry magnetic biosensor for non-disease diagnostic uses
The quantitative analysis method of 5-hydroxymethyl cytosine, the dual signal amplification based on terminal enzyme (DNA) and Ru (III) redox cycle
Strategy is utilized with the electrochemistry magnetic biosensor quantitative analysis 5-hydroxymethyl cytosine of screen printing carbon electrode (SPCE).
Further, the non-disease diagnostic uses is phonetic based on electrochemistry magnetic biosensor detection 5- methylol born of the same parents
The quantitative analysis method specific steps of pyridine are as follows: the biotinylation in 5-hydroxymethyl cytosine site in DNA chain；It is affine with strepto-
The 5-hydroxymethyl cytosine DNA chain of coated magnetic bead (MB) the enriched biological element of element；Terminal enzyme (DNA) catalytic polymerization biotinylation
The extension of 5-hydroxymethyl cytosine DNA chain；Ru (NH is added3)6 3+/Fe(CN)6 3-System generates the electro-catalysis signal of enhancing；Electricity
Chemical magnetic biosensor quantitative analysis.
Further, with cysteine-biotin carry out a step bisulfites mediation DNA chain in 5- methylol born of the same parents it is phonetic
The biotinylation in pyridine site.
Further, cysteine-biotin and double-stranded DNA are mixed and Na is added2S2O5Solution is incubated at 42~45 DEG C
Educate 48~50 hours, be cooled to room temperature to solution, adjust pH value, at room temperature react 5~after ten minutes, sample is passed through
Micro-Bio-Spin P6 column remove excessive cysteine-biotin and salt to get.
Further, the biotinylated 5-hydroxymethyl cytosine DNA chain of catalytic polymerization in terminal enzyme (DNA) reaction system
Extension；The terminal enzyme (DNA) reaction system includes 1 × terminal enzyme (DNA) buffer, CoCl2Solution, dNTPs and end turn
Move enzyme.
Further, Ru (NH3)6 3+/Fe(CN)6 3-System include 1~1.5mM PBS buffer solution, 0.1~0.5M NaCl,
27~30 μM of Ru (NH3)6 3+With 2~5mM Fe (CN)6 3-。
Further, electrochemistry magnetic biosensor utilizes 0 to -500mV, impulse amplitude 50mV, and pulse width is
The differential pulse voltammetry of 50ms records electrochemical response, reference electrode Ag/AgCl.
The second aspect of the present invention provides above-described quantitative analysis method in the life of various non-disease diagnostic purposes
Object, tissue, the application in various nucleic acid sequences in 5-hydroxymethyl cytosine analysis detection.
Further, 5-hydroxymethyl cytosine is 5-hydroxymethyl cytosine in genomic DNA；The cell can be people's uterine neck
Cancerous cell line (HeLa cell) and human embryonic kidney cell (HEK 293T) cell.
Third aspect of the present invention provides a kind of kit of quantitative analysis 5-hydroxymethyl cytosine, the kit packet
Include cysteine-biotin, Na2S2O5The coated magnetic bead of solution, Streptavidin, washing buffer, terminal enzyme (DNA) reaction
Liquid, Ru (NH3)6 3+/Fe(CN)6 3-System reaction solution and screen printing carbon electrode；
The washing buffer includes: 0.5M NaCl, 20mM tris-HCl and 1mM EDTA, tris-HCl pH value is
7.4；
Terminal enzyme (DNA) reaction solution includes 1 × terminal enzyme (DNA) buffer, 0.25mM CoCl2Solution, dNTPs and end
Transferase；
Ru(NH3)6 3+/Fe(CN)6 3-System reaction solution includes: 1~1.5mM PBS buffer solution, 0.1~0.5M NaCl, and 27
~30 μM of Ru (NH3)6 3+With 2~5mM Fe (CN)6 3-。
It is that the present invention obtains the utility model has the advantages that
(1) principle is simple, reduces cost: compared to the method for the electrochemica biological sensor based on enzyme modification, the present invention
Method is due to introducing terminal enzyme (DNA), without the expensive enzyme with limited identification motif, enzyme cofactor/substrate analog
Synthesis and base modification multiple steps, keep principle relatively sharp understandable；Due to the introducing of screen printing carbon electrode, phase
Than being sequenced in real time in unimolecule, the present invention greatly reduces the cost of experiment without complicated and expensive instrument.
(2) actual sample detection can be done: utilizing the electrochemistry magnetic biosensor based on screen printing carbon electrode, the life
Object sensor can delicately quantify 5-hydroxymethyl cytosine, it is not necessary that DNA is fixed on the electrode.Importantly, the bio-sensing
It is phonetic that device can be used for 5- methylol born of the same parents in Human cervical cancer cell lines (HeLa cell) and human embryonic kidney cell (HEK 293T) cell
The accurate quantification of pyridine, and can be very good to distinguish cytimidine (C), 5-methylcytosine and 5-hydroxymethyl cytosine.
(3) high sensitivity: the present invention is not catalyzed repetition addition dNTPs using terminal enzyme (DNA) with needing any DNA profiling,
Longer DNA sequence dna is generated, therefore has more Ru (NH3)6 3+Extended negatively charged phosphoric acid can be electrostatically bound to
On skeleton, meanwhile, Ru (NH3)6 3+It plays a role as primary electron receptor, Fe (CN)6 3-It plays and makees as secondary electron receptor
With.Excessive Fe (CN)6 3-It can be by the Ru (II) that oxidizing and electrochemical is reduced, to increase Ru (NH3)6 3+Electrochemistry
Reduction current leads to the recycling of Ru (III) and generates the electro-catalysis electric current of amplification, realizes double amplifications of signal, therefore this hair
The bright high sensitivity detection that may be implemented to 5-hydroxymethyl cytosine.The present invention detects limit 9.06fM, the spirit of the biosensor
Sensitivity improves 4 orders of magnitude than the electrochemical analysis (0.23nM) modified based on dnmt rna, than being based on β-grape
High 4 orders of magnitude of fluorescence analysis (0.167nM) of glycosyl transferase modification, than the optical sensor based on specific antibody
(0.42pM) is 46 times high, than being based on KRuO4Electrochemiluminescsensor sensor (0.14pM) it is 25 times high.
(4) specificity is good: the present invention cannot be in any other non-hydroxyl using the chemical reaction of 5-hydroxymethyl cytosine modification
Occurring at the nucleic acid base of methylation, the step for realizing that 5-hydroxymethyl cytosine site carries out biotin in DNA mixes, and
Cytimidine (C) and 5-methylcytosine under the same reaction conditions, cannot react, therefore the method for the present invention can Gao Te
5- hydroxymethyl cytosine is detected anisotropicly.
Detailed description of the invention
The Figure of description for constituting a part of the invention is used to provide further understanding of the present invention, and of the invention shows
Examples and descriptions thereof are used to explain the present invention for meaning property, does not constitute improper limitations of the present invention.
The signal of the non-marked, the electrochemistry magnetic biosensor of on-fixed of the measurement of Fig. 1 .5- hydroxymethyl cytosine
Figure.The measurement is related to two consecutive steps: 5-hydroxymethyl cytosine site in the DNA that (A) step bisulfite method mediates
Biotinylation；(B) the terminal enzyme (DNA) catalytic polymerization and subsequent Ru of biotinylated 5-hydroxymethyl cytosine DNA induction
(III) redox cycle dual signal amplification strategy.
The nyquist diagram of the electrochemical impedance spectroscopy (EIS) of Fig. 2 (A) magnetic bead (a)/screen printing carbon electrode, biotin
5-hydroxymethyl cytosine DNA/ magnetic bead/screen printing carbon electrode of change is before addition terminal enzyme (DNA) auxiliary base extends (b)
The nyquist diagram of the electrochemical impedance spectroscopy (EIS) of (c) later, middle impedance liquid are the 5mM Fe containing 0.1M KCl
(CN)6 3-/4-Solution.(B) Zeta potential of magnetic bead (a), biotinylated 5-hydroxymethyl cytosine DNA/ magnetic bead is in addition end
Zeta current potential before transferase assists base to extend (b) and later (c).(C) magnetic bead/screen printing carbon electrode (a), biology
The 5-hydroxymethyl cytosine DNA/MB/SPCE of elementization is before addition terminal enzyme (DNA) auxiliary base extends (b) and later (c)
In Ru (NH3)6 3+With Fe (CN)6 3-Mixture in DPV signal response.(D) biotinylated 5-hydroxymethyl cytosine DNA/
The base of magnetic bead/screen printing carbon electrode and terminal deoxynucleotidyl transferase (TDT) auxiliary extends in that there is only (a) Ru
(NH3)6 3+, (b) Ru (NH3)6 3+With Fe (CN)6 3-In DPV signal.
Fig. 3 (A) electrochemistry magnetic biosensor to the 5-hydroxymethyl cytosine DNA of various concentration (from a to k, 0,
0.01,0.1,0.5,1,5,10,50,100,500 and 1000pM) DPV response.(B) DPV peak value strengthening electric current (Δ I) and 5-
Linear relationship between the logarithm of hmC concentration (in the range of 0.01 to 1000pM).Error bar indicates duplicate measurements three times
Standard deviation.
Fig. 4 (A) is under condition 1, and 5-hydroxymethyl cytosine is converted into 5-hydroxymethyl cytosine-biotin, and in condition 2
Lower Micro-Bio-Spin P6 is purified；However, cytimidine (C) and 5-methylcytosine are not react under condition 1
's.(B) only with the control group of reaction buffer, cytimidine (C), 5-methylcytosine and the 5-hydroxymethyl cytosine of normal DNA
The DPV curve of the electrochemistry magnetic biosensor of DNA.(C) cytimidine (C), 5-methylcytosine and the 5- hydroxyl of normal DNA
The DPV signal of the electrochemistry magnetic biosensor of methylcystein DNA responds.Error bar represents the mark of independent experiment three times
Quasi- deviation.
5- methylol born of the same parents in Fig. 5 Human cervical cancer cell lines (HeLa cell) and human embryonic kidney cell (HEK 293T cell)
Pyrimidine content.
Specific embodiment
It is noted that described further below be all exemplary, it is intended to provide further instruction to the present invention.Unless another
It indicates, all technical and scientific terms used herein has logical with general technical staff of the technical field of the invention
The identical meanings understood.
It should be noted that term used herein above is merely to describe specific embodiment, and be not intended to restricted root
According to exemplary embodiments of the present invention.As used herein, unless the context clearly indicates otherwise, otherwise singular shape
Formula be also intended to include plural form, additionally, it should be understood that, when in the present specification use term "comprising" and/or
When " comprising ", existing characteristics, step, operation and/or their combination are indicated.
In order to enable those skilled in the art can clearly understand technical solution of the present invention, below with reference to tool
The embodiment of the body technical solution that the present invention will be described in detail.
Experimental principle of the present invention, as shown in Figure 1.The hybridization shape of single stranded DNA containing 5-hydroxymethyl cytosine and complementary DNA
At double-stranded DNA, biotin-cysteine derivative is subsequently introduced and makes it possible to for one step of biotin being incorporated into containing 5- methylol
The double-stranded DNA in cytimidine site.After removing excessive cysteine-biotin and salt using Micro-Bio-Spin P6 column,
It is interacted using with the coated magnetic bead of Streptavidin by biotin-Streptavidin specificity come enriched biological element
The double-stranded DNA of change.Then, the 3'-hydroxyl terminal of DNA can be catalyzed by terminal enzyme (DNA) repeats addition dNTPs extension, and
Any DNA profiling is not needed, longer DNA sequence dna is generated.In the electro-catalysis system, Ru (NH3)6 3+As primary electron by
Body plays a role, Fe (CN)6 3-It plays a role as secondary electron receptor.Ru(NH3)6 3+Magnetic bead surfaces can be electrostatically drawn to
In the negatively charged phosphoric acid backbone of upper DNA, and it is fixed on screen printing carbon electrode electrode by means of magnetic field.Excessive Fe
(CN)6 3-It can be by the Ru (II) that oxidizing and electrochemical is reduced, to increase Ru (NH3)6 3+Electrochemical reduction electric current, lead
It causes the recycling of Ru (III) and generates the electro-catalysis electric current of amplification.Electro-catalysis circulation is followed by the base of equation (1) and (2) description
In the mechanism of electron transfer kinetics.
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-hydroxymethyl cytosine DNA is placed on silk-screen printing carbon in conjunction with the magnetic bead of marked by streptavidin
On electrode, the Ru (NH3)6 3+/Fe(CN)6 3-System can produce the electro-catalysis signal of enhancing.In addition, Fe (CN)6 3-It is negatively charged,
By DNA anion exclusion far from electrode surface, their direct reactions to false positive signal are eliminated.Due to 5- methylol born of the same parents
A step bisulfite conversion method with specificity for pyrimidine induction and the polymerization reaction and Ru of terminal enzyme (DNA) catalysis
(III) the efficient dual signal amplification for the amplification that redox cycle mediates, this biosensor can be simple, quickly, Gao Ling
It is quick and selectively detect 5-hydroxymethyl cytosine.
Related DNA sequence dna used in the present invention, as shown in table 1 below.
1 associated dna sequence of table
Embodiment
The quantitative analysis method are as follows:
The extraction of intracellular total DNA: Human cervical cancer cell lines (HeLa cell) and human embryonic kidney cell (HEK 293T)
The culture medium of cell is to improve Iger culture containing the Dulbecco of 10% fetal calf serum (FBS) and 1% Pen .- Strep
Base (DMEM) is placed on 37 DEG C, is cultivated in the incubator containing 5% carbon dioxide.Logarithmic growth phase is grown into cell
When, using culture cell DNA extracts kit (Epigentek) total DNA in cell is stripped and is purified, extract with
Purification process is carried out in strict accordance with specification appended by kit, and gained total DNA measures it using ultraviolet-uisible spectrophotometer
Concentration.
The biotinylation in 5-hydroxymethyl cytosine site in the DNA that one step bisulfite method mediates: cysteine-life is used
Object element carries out the 5-hydroxymethyl cytosine biotinylation of step bisulfites mediation in DNA.By cysteine-biotin
(120 μ L, adjust pH to double-stranded DNA (the 5- methylol born of the same parents 5.0) with 10 μ L with various concentration with 10M sodium hydroxide by 10 μM
Pyrimidine-complementary DNA) it is mixed in the centrifuge tube of 200 μ L.By Na2S2O5(5 μ L, 1.0M, pH=5.0) is added in solution, so
It is incubated for 48 hours at 42 DEG C afterwards.It is cooled to room temperature to solution, adjusts pH to 13 with 10M sodium hydroxide.5 are reacted at room temperature
After minute, by sample by Micro-Bio-Spin P6 column (Bio Rad) three times to remove excessive cysteine-biotin
And salt.
It connects the preparation of the magnetic bead of 5-hydroxymethyl cytosine DNA: the coated bead suspension of 4 μ L Streptavidins is shifted
Into the centrifuge tube of above-mentioned 200 μ L, and with 50 μ L washing buffer (0.5M NaCl, 20mM tris-HCl (pH 7.4), 1mM
EDTA it) washes twice.Supernatant is removed using magnetic separation rack to separate magnetic bead.Contain 1 × terminal enzyme (DNA) buffering in 50 μ L
Liquid (10mM Mg (Ac)2, 20mM Tris-Ac, 50mM KAc, pH=7.9), 0.25mM CoCl2, 1 end mM dNTPs, 6U
Enzymatic amplification is carried out in the reaction system of transferase, is then washed again with washing buffer to form sensor array.
Electrochemical measurement: 1mM PBS (pH 7.4) will be contained, 0.1M NaCl, 27 μM of Ru (NH3)6 3+With 2mM Fe
(CN)6 3-Solution be added to containing separation biotinylated 5-hydroxymethyl cytosine DNA/ magnetic bead centrifuge tube in.Then
The 100 above-mentioned solution of μ L are transferred on the surface of screen printing carbon electrode.By applying super magnetic below working electrode surface
Biotinylated 5-hydroxymethyl cytosine DNA/ bead complexes are fixed on screen printing carbon electrode by body.Utilize 0 to -500
MV, impulse amplitude 50mV, pulse width are the differential pulse voltammetry (DPV) (reference electrode Ag/AgCl) of 50ms, are come
Record electrochemical response.
In order to verify the feasibility of this method, the preparation of electrochemistry magnetic biosensor is hindered by the electrochemistry of (Fig. 2)
Anti- spectrum (EIS) and zeta current potential characterize.Electronics transfer resistance (Ret) by [Fe (CN)6]3-/4-In the silk-screen printing carbon electricity of modification
Electron transfer kinetics control on extremely, and can be characterized by half circular diameter of nyquist diagram.As shown in Figure 2 A,
Electronics transfer resistance (the R of the magnetic bead of on-fixed screen printing carbon electrodeet) it is 5980 Ω (Fig. 2A, curve a).Conversely, because negative
Charge DNA and [Fe (CN)6]3-/4-Between electrostatic repulsion, the performance of biotinylated 5-hydroxymethyl cytosine DNA/ magnetic bead
Biggish electronics transfer resistance (R outet), it is that (Fig. 2A, curve b) show successfully to modify 5- methylol born of the same parents on magnetic bead 7120 Ω
Pyrimidine DNA.After biotinylated 5-hydroxymethyl cytosine DNA and terminal enzyme (DNA) are incubated for, electronics transfer resistance (Ret) increase
To 9980 Ω.This can be by the fact that explain: after terminal enzyme (DNA) catalytic polymerization, biotinylated 5- methylol
The more negatively charged phosphate backbone of cytosine dna may hinder [Fe (CN)6]3-/4-Electronics transfer from solution to electrode surface
(Fig. 2A, curve c).
Zeta current potential is for studying connection of the biotinylated 5-hydroxymethyl cytosine on magnetic bead.As shown in Figure 2 B, with
The control group (Fig. 2 B, curve a, -2.3 ± 0.5mV) of only magnetic bead is compared, and biotinylated 5-hydroxymethyl cytosine DNA is added
(Fig. 2 B, curve b, -4.5 ± 0.2mV) induces zeta current potential that significant variation occurs, and shows that biotinylated 5- methylol born of the same parents are phonetic
Pyridine DNA successful application is in magnetic bead surfaces.When 5-hydroxymethyl cytosine DNA and terminal enzyme (DNA) are incubated with, due to biology
The terminal enzyme (DNA) catalytic polymerization of the 5-hydroxymethyl cytosine induction of elementization, then observe higher negative zeta current potential (- 10.2
± 0.4mV) (Fig. 2 B, curve c).
In Ru (NH3)6 3+With Fe (CN)6 3-Mixture in, terminal enzyme (DNA) auxiliary base extend before and after, make
With include magnetic bead/screen printing carbon electrode, biotinylated 5-hydroxymethyl cytosine DNA/ magnetic bead/screen printing carbon electrode
Different electrodes record DPV measured value (Fig. 2 C).In the control group for not having 5-hydroxymethyl cytosine site in DNA, electric current very little
(5.59 μ A) (Fig. 2 C, curve a).After 5-hydroxymethyl cytosine site biotinylation in DNA, signal increases to 7.85 μ A (figure
2C, curve b), because being absorbed in the electronegative phosphoric acid backbone of biotinylated 5-hydroxymethyl cytosine DNA a large amount of
Ru(NH3)6 3+.After biotinylated 5-hydroxymethyl cytosine DNA/ magnetic bead and terminal enzyme (DNA) are incubated for, signal is further increased
To 11.5 μ A (Fig. 2 C, curve c), because of more Ru (NH3)6 3+It is electrostatically bound in extended negatively charged phosphoric acid backbone.
These results demonstrate the feasibility that the biosensor detects 5-hydroxymethyl cytosine.
Under optimum experimental condition, the present invention has evaluated the 5- methylol of magnetoelectricity chemical biosensor response various concentration
The analysis performance of cytosine dna.As shown in Figure 3A, the increase with 5-hmC DNA concentration from 0.01 to 1000pM, DPV letter
Number enhancing.The logarithm of electric current and 5-hydroxymethyl cytosine DNA concentration obtains linear relationship (figure within the scope of 0.01 to 1000pM
3B), detection limit is calculated as 9.06fM.The electrochemistry that the remolding sensitivity of the biosensor is modified based on dnmt rna
Analysis (0.23nM) improves 4 orders of magnitude, higher by 4 than based on β-glucosyltransferase modification fluorescence analysis (0.167nM)
A order of magnitude, it is 46 times higher than the optical sensor (0.42pM) based on specific antibody, than being based on KRuO4It is electroluminescent at chemistry
The 5-hydroxymethyl cytosine selective chemical oxidation of luminescence method (0.14pM) is 25 times high, this is enough to illustrate detection spirit of the invention
The height of sensitivity.
It is phonetic using the 5- methyl born of the same parents with single 5-methylcytosine site in order to study the specificity of this biosensor
Pyridine and normal DNA cytimidine (C) are used as negative control.As shown in Figure 4 C, under the same conditions, only in 5- methylol born of the same parents
Higher peak point current variation can be just detected in the presence of pyrimidine.The peak point current of 5-hydroxymethyl cytosine changes (Δ
I) than normal DNA cytimidine (C) and 5-methylcytosine DNA high 22 and 39 times (Fig. 4 C), show that the biosensor has
High specific.The high specific of this method be attributable to 5-hydroxymethyl cytosine modification chemical reaction cannot any other not
Occur at methylolated nucleic acid base.
In order to assess feasibility of the biosensor proposed by the present invention in clinical diagnosis, surveyed using the biosensor
Two different cancerous cell lines are measured, including.Pass through Q-5000UltramicroUV-Vis spectrophotometer (Quawell, USA)
Measure the concentration of genomic DNA.As shown in figure 5, Human cervical cancer cell lines (HeLa cell) and human embryonic kidney cell (HEK
293T cell) in 5-hydroxymethyl cytosine content be confirmed as the 0.00793% and 0.00977% of total nucleotide respectively, with
Consistent (the respectively total nucleosides of the result by the dot blotting measurement modified based on β-glucosyltransferase being previously reported
Acid 0.007% and 0.009%).
The above embodiment is a preferred embodiment of the present invention, but embodiments of the present invention are not by above-described embodiment
Limitation, other any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present invention,
It should be equivalent substitute mode, be included within the scope of the present invention.
SEQUENCE LISTING
<110>Shandong Normal University
<120>a kind of side of the electrochemistry magnetic biosensor detection 5-hydroxymethyl cytosine based on non-marked, on-fixed
Method
<130>
<160> 4
<170> PatentIn version 3.3
<210> 1
<211> 42
<212> DNA
<213>artificial synthesized
<400> 1
ggtaccatcg atacthmcag atagactaca ctccagaagc tt 42
<210> 2
<211> 41
<212> DNA
<213>artificial synthesized
<400> 2
ggtaccatcg atactmcaga tagactacac tccagaagct t 41
<210> 3
<211> 40
<212> DNA
<213>artificial synthesized
<400> 3
ggtaccatcg atactcagat agactacact ccagaagctt 40
<210> 4
<211> 40
<212> DNA
<213>artificial synthesized
<400> 4
aagcttctgg agtgtagtct atctgagtat cgatggtacc 40
Claims (10)
1. a kind of quantitative point based on electrochemistry magnetic biosensor detection 5-hydroxymethyl cytosine of non-disease diagnostic uses
Analysis method, it is characterised in that: the dual signal based on terminal enzyme (DNA) and Ru (III) redox cycle amplifies strategy, utilize with
The electrochemistry magnetic biosensor quantitative analysis 5-hydroxymethyl cytosine of screen printing carbon electrode.
2. quantitative analysis method according to claim 1, which is characterized in that specific steps are as follows: 5- methylol born of the same parents in DNA chain
The biotinylation in pyrimidine site；With the biotinylated 5-hydroxymethyl cytosine DNA chain of the coated enrichment with magnetic bead of Streptavidin；
The extension of the biotinylated 5-hydroxymethyl cytosine DNA chain of terminal enzyme (DNA) catalytic polymerization；Ru (NH is added3)6 3+/Fe(CN)6 3-
System generates the electro-catalysis signal of enhancing；Electrochemistry magnetic biosensor quantitative analysis.
3. quantitative analysis method according to claim 2, which is characterized in that carry out step Asia with cysteine-biotin
The biotinylation in 5-hydroxymethyl cytosine site in the DNA chain that disulfate mediates.
4. quantitative analysis method according to claim 3, which is characterized in that mix cysteine-biotin and double-stranded DNA
It closes and Na is added2S2O5Solution is incubated for 48~50 hours at 42~45 DEG C, is cooled to room temperature to solution, adjusts pH value, at room temperature
Reaction 5~after ten minutes, sample is removed into excessive cysteine-biotin and salt by Micro-Bio-Spin P6 column, i.e.,
?.
5. quantitative analysis method according to claim 2, which is characterized in that be catalyzed in terminal enzyme (DNA) reaction system poly-
Close the extension of biotinylated 5-hydroxymethyl cytosine DNA chain；The terminal enzyme (DNA) reaction system include 1 × TdT buffer,
CoCl2Solution, dNTPs and terminal enzyme (DNA).
6. quantitative analysis method according to claim 2, which is characterized in that Ru (NH3)6 3+/Fe(CN)6 3-System include 1~
1.5mM PBS buffer solution, 0.1~0.5M NaCl, 27~30 μM of Ru (NH3)6 3+With 2~5mM Fe (CN)6 3-。
7. quantitative analysis method according to claim 2, which is characterized in that electrochemistry magnetic biosensor using 0 to-
500mV, impulse amplitude 50mV, pulse width are the differential pulse voltammetry of 50ms to record electrochemical response, reference electrode
For Ag/AgCl.
8. according to claim 1~7 any quantitative analysis method the biology of various non-disease diagnostic purposes, tissue,
Application in cell and various nucleic acid sequences in 5-hydroxymethyl cytosine analysis detection.
9. application according to claim 8, which is characterized in that 5-hydroxymethyl cytosine is 5- methylol in genomic DNA
Cytimidine；The cell can be Human cervical cancer cell lines, human embryonic kidney cell.
10. a kind of kit of quantitative analysis 5-hydroxymethyl cytosine, which is characterized in that including cysteine-biotin,
Na2S2O5The coated magnetic bead of solution, Streptavidin, washing buffer, terminal enzyme (DNA) reaction solution, Ru (NH3)6 3+/Fe
(CN)6 3-System reaction solution and screen printing carbon electrode；
The washing buffer includes: 0.5M NaCl, 20mM tris-HCl and 1mM EDTA, tris-HCl pH value is 7.4；
Terminal enzyme (DNA) reaction solution includes 1 × TdT buffer, 0.25mM CoCl2Solution, dNTPs and terminal enzyme (DNA)；
Ru(NH3)6 3+/Fe(CN)6 3-System reaction solution includes: 1~1.5mM PBS buffer solution, 0.1~0.5M NaCl, 27~30 μ
M Ru(NH3)6 3+With 2~5mM Fe (CN)6 3-。
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| CN112326637A (en) * | 2020-10-30 | 2021-02-05 | 山东师范大学 | Chemiluminescence biosensor for detecting 5-hydroxymethylcytosine and detection method and application thereof |
| CN113834931A (en) * | 2021-09-02 | 2021-12-24 | 浙江大学 | Method for detecting genome whole DNA methylation by electrochemical magnetic biosensor |
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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 |
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| CN112326637A (en) * | 2020-10-30 | 2021-02-05 | 山东师范大学 | Chemiluminescence biosensor for detecting 5-hydroxymethylcytosine and detection method and application thereof |
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| CN113834931A (en) * | 2021-09-02 | 2021-12-24 | 浙江大学 | Method for detecting genome whole DNA methylation by electrochemical magnetic biosensor |
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