CN106338539B - DNA capture probe, biosensor and its detection method based on polyadenous purine - Google Patents
DNA capture probe, biosensor and its detection method based on polyadenous purine Download PDFInfo
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- CN106338539B CN106338539B CN201610959082.1A CN201610959082A CN106338539B CN 106338539 B CN106338539 B CN 106338539B CN 201610959082 A CN201610959082 A CN 201610959082A CN 106338539 B CN106338539 B CN 106338539B
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- G—PHYSICS
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- 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
- G01N27/3275—Sensing specific biomolecules, e.g. nucleic acid strands, based on an electrode surface reaction
- G01N27/3277—Sensing specific biomolecules, e.g. nucleic acid strands, based on an electrode surface reaction being a redox reaction, e.g. detection by cyclic voltammetry
Abstract
The invention discloses a kind of DNA capture probe, biosensor and its detection methods based on polyadenous purine.The nucleotide sequence of the DNA capture probe is as shown in SEQ ID No.1 in sequence table, SEQ ID No.2, SEQ ID No.3, SEQ ID No.4 or SEQ ID No.5.The present invention utilizes the interaction of adenine and gold electrode, DNA probe comprising multiple continuous adenines is fixed on gold electrode surfaces, the electrochemica biological sensor based on more adenine dnas is constructed for the first time, it can achieve based on the same effect of sulfydryl modification DNA assembling gold electrode, will start new era of such novel electrochemical Biosensors.The present invention can it is highly sensitive, detect DNA with high specificity, sensitivity may be up to 1pM, easy to operate, have a extensive future.
Description
Technical field
The invention belongs to field of electrochemical detection, more particularly to a kind of DNA capture probe, biology based on polyadenous purine
Sensor and its detection method.
Background technique
The developing history of electrochemistry is long, and the development with advanced science technology and subject is closely related, and electrochemica biological passes
Sensor have specific molecular identification function, disease gene diagnosis, in terms of show wide application
Prospect is the research means when the forward position of neontology, medical domain.
The substantially step of electrochemica biological sensor measurement DNA includes: the fixation of one, DNA capture probe, i.e., by a certain spy
The single stranded DNA (ssDNA) of sequencing column is fixed on the surface of electrode；Two, hybridize, the electrode for being fixed with DNA capture probe is put into
Into the detected solution containing matching DNA to be measured, hybridize DNA to be measured in electrode surface with DNA capture probe
Reaction forms double-stranded DNA (dsDNA)；Three, measurable electrochemical signals are converted by the signal of hybridization reaction；It four, will be electrochemical
It learns signal and amplifies detection.
The fixation of DNA capture probe is the matter of utmost importance in the production of DNA electrochemical sensor, and fixed amount and activity are direct
Influence the sensitivity of sensor.DNA fixing means common at present mainly has covalent bonding method, self-assembly method and Avidin-life
Object element method.Covalent bond and method are so that DNA is fixed to electrode surface by formation covalent bond, such as amido bond, ester bond, ehter bond,
It needs to carry out activating pretreatment to electrode surface, introduces required active group, pass through bifunctional reagent or coupling activator
DNA molecular after derivative is coupled with electrode.But the problem that this method is little due to the chemical reaction efficiency for forming covalent bond,
Cause the density for the DNA capture probe for being assembled in electrode surface not high.Self-assembly method (SAM method) is mainly used for DNA capture probe
In the fixation of gold electrode surfaces, specific method is that the terminal sulfhydryl group of DNA capture probe is formed Iy self-assembled layer by golden sulfide linkage
Achieve the purpose that fixed dna.But this method needs sulfydryl modification, higher cost, and easy to form double in experimental implementation
Sulfide linkage and the accuracy for influencing testing result.Avidin-Biotin method is that Avidin (avidin) is passed through to electrostatic interaction or total
Valence coupled action is connected to electrode surface, will be biological using bio-identification affinity interaction extremely strong between biotin and Avidin
The DNA of element modification is fixed on electrode.Three of the above method is required to modify DNA capture probe, and synthesis cost is higher,
Complex steps.
However, those skilled in the art are difficult to clear how to regulate and control electrochemica biological sensor electrode surface DNA and catch at present
The packing density of probe is obtained, this directly constrains the further development of electrochemica biological sensor, therefore urgently obtains a kind of complete
The electrochemica biological sensor of new DNA capture probe fixing means and excellent effect.
Summary of the invention
The technical problem to be solved by the present invention is to solve regulation electrochemica biological sensor electrode surface DNA capture probe
Packing density problem, to provide a kind of DNA capture probe, biosensor and its detection side based on polyadenous purine
Method.The DNA capture probe and biosensor can high sensitivity, specificity detect DNA to be measured goodly, easy to operate, application
It has a extensive future.
Test of the present inventor by in-depth study and repeatedly, utilizes the phase interaction of adenine and gold electrode
With devising the DNA capture probe comprising multiple continuous adenines (poly A), and such DNA capture probe is fixed
In gold electrode surfaces, by the hybridizing method of " sandwich ", DNA to be measured and signal probe are introduced into gold electrode surfaces, then foundation
Detection hybridization front and back catalytic oxidation-reduction curent change indicates the concentration of DNA to be measured, so as to complete the present invention.
Technical solution of the present invention first is that: a kind of DNA capture probe based on polyadenous purine, nucleotide sequence such as sequence
SEQ ID No.1 in list, SEQ ID No.2, SEQ ID No.3, shown in SEQ ID No.4 or SEQ ID No.5.
In the present invention, the DNA capture probe of the polyadenous purine contain different numbers (5,10,20,30 and
40) adenine, be referred to as poly A5, poly A10, poly A20, poly A30 and poly A40.
In the present invention, preferably, the DNA capture probe is poly A30, SEQ in nucleotide sequence such as sequence table
Shown in ID No.4.
Technical solution of the present invention second is that: a kind of electrochemica biological sensor, by method comprising the following steps system
It is standby to form:
(1) the DNA capture probe based on polyadenous purine, the nucleotide sequence such as sequence table of the DNA capture probe are prepared
Middle SEQ ID No.1, SEQ ID No.2, SEQ ID No.3, shown in SEQ ID No.4 or SEQ ID No.5；
(2) the resulting DNA capture probe based on polyadenous purine of step (1) is added drop-wise on electrode, reacts, electricity must be assembled
Pole；
(3) it is closed with the vacancy of sulfydryls hexanol (MCH) to resulting group of loading electrode of step (2), obtains closed electrode.
In the present invention, step (2) are as follows: the resulting DNA capture probe based on polyadenous purine of step (1) is added drop-wise to electrode
On, reaction obtains a group loading electrode.Wherein, the packing density when DNA capture probe is added drop-wise on electrode is preferably 0.01~
3 μM, be more preferably 0.1 μM.The temperature of the reaction is 20~40 DEG C, preferably 25 DEG C.The time of the assembling is ability
It is the time of domain routine, preferably overnight, that is, it is greater than 12 hours.
In step (2), the electrode is conventional, preferably, the electrode is the clean electrode of processing；More preferably,
The clean electrode of the processing is made by method comprising the following steps: through physics polishing, sulfuric acid electrochemical cleaning, then with ultrapure
Water cleaning down electrode surface, then uses N2Drying.The electrode is preferably gold electrode.
In the present invention, step (3) are as follows: it is closed with the vacancy of sulfydryls hexanol (MCH) to resulting group of loading electrode of step (2),
Obtain closed electrode.Wherein, the concentration of the MCH is preferably 0.01~1mM, is more preferably 0.1mM.The closed time
It is 5~60 minutes, preferably 30 minutes.The closed temperature be this field routine temperature, preferably 25 DEG C.
In the present invention, it has been investigated that confirming, most preferably, above-mentioned electrochemica biological sensor uses the polyA30
As DNA capture probe；The packing density of polyA30 is 0.1 μM；And it is closed 30 minutes in step (3) using 0.1mM MCH.
Technical solution of the present invention third is that: a kind of side for detecting DNA to be measured using above-mentioned electrochemica biological sensor
Method comprising following step:
(I) by DNA to be measured and nucleotide sequence as shown in SEQ ID No.7 in sequence table and 3 ' end connection biotins
Signal probe prehybridization obtains prehybridization object A；
(II) the resulting prehybridization object A of step (I) and such as the described in any item electrochemica biologicals of claim 2~5 are passed
DNA capture probe hybridization on the electrode of sensor, obtains the electrode of hybridization；
(III) hatch after Avidin-horseradish peroxidase being added dropwise on the electrode of step (II) resulting hybridization；Bottom is added
Object TMB, is tested and analyzed.
In the present invention, step (I) are as follows: by DNA to be measured and nucleotide sequence as shown in SEQ ID No.7 in sequence table and
The signal probe prehybridization of 3 ' end connection biotins, obtains prehybridization object A.
Wherein, preferably, the concentration of the DNA to be measured is 0~1000pM, the concentration of the signal probe is 100nM.Compared with
Goodly, the prehybridization is kept 1~10 minute at 60~100 DEG C, is then placed 5~60 minutes for 20~40 DEG C.More preferably, described
The temperature of prehybridization is 80 DEG C.More preferably, the time of the prehybridization is 5 minutes.Preferably, the time of the placement is 20 points
Clock.Preferably, the temperature of the placement is 25 DEG C.Preferably, the prehybridization carries out under buffer.The buffer is this
The buffer of field routine, preferably, the buffer is the 10mM TE buffer solution comprising 1M NaCl, the TE buffer
Including 10mM Tris-HCl and 1mM EDTA, pH7.0.
In the present invention, step (II) are as follows: by the resulting prehybridization object A of step (I) and the electrochemica biological sensor
Electrode on DNA capture probe hybridization, obtain the electrode of hybridization.Wherein, preferably, hybridization described in step (II) is 25~45
DEG C hybridization 30~120 minutes；More preferably, the temperature of the hybridization is 37 DEG C.Preferably, the time of the hybridization is 60 minutes.
Preferably, further including the steps that cleaning electrode after the completion of the hybridization.The cleaning solution of the cleaning is the cleaning of this field routine
Liquid, preferably, being the 10mM TE buffer solution containing 1M NaCl, the TE buffer includes 10mM Tris-HCl and 1mM
EDTA, pH7.0.
In the present invention, step (III) are as follows: Avidin-horseradish peroxidating is added dropwise on the electrode of step (II) resulting hybridization
Enzyme (avidin-HRP enzyme) is hatched afterwards；Substrate TMB is added, is tested and analyzed.Wherein, preferably, the Avidin-horseradish
Peroxidase is 1000 times of diluted Avidin-horseradish peroxidases；Preferably, 1000 times of diluted Avidin-horseradishes
The additive amount of peroxidase is 1~5 μ L, is more preferably 3 μ L.The temperature of the hatching is the temperature of this field routine, preferably
For room temperature, preferably 25 DEG C.The time of the hatching be this field routine time, preferably 15 minutes.Add in step (III)
Enter substrate TMB, the method tested and analyzed is that this field is conventional.In the present invention, electrochemistry inspection is carried out using the method
After survey, according to the size of the absolute value of the electric current measured, curve DNA to be measured accordingly can be fitted according to current curve diagram
Mathematical formulae between quantity and current absolute value, it is however generally that, DNA quantity to be measured is bigger in sample to be tested, correspondingly, measuring
Electric current absolute value it is bigger.
On the basis of common knowledge of the art, above-mentioned each optimum condition, can any combination to get each preferable reality of the present invention
Example.
The reagents and materials used in the present invention are commercially available.
The positive effect of the present invention is that: the DNA probe comprising multiple continuous adenines is fixed on gold by the present invention
Electrode surface constructs the electrochemica biological sensor based on more adenine dnas for the first time, can achieve based on sulfydryl modification DNA
The same effect of gold electrode is assembled, will be started new era of such novel electrochemical Biosensors.The fixing means is not only real
Existing DNA probe is not necessarily to sulfydryl modification step, can be to avoid the formation of cystine linkage in experimental implementation, Er Qieke while reducing cost
The packing density of gold electrode surfaces DNA probe can be regulated and controled by the number for changing continuous adenine in DNA probe.DNA
After capture probe is fixed, on the one hand other bases (G, C, T) also can non-specifically be adsorbed on gold electrode surfaces, influence DNA and catch
It obtains probe and DNA to be measured forms double-strand；On the other hand, it is formed after double-strand and occurs vacancy again in gold electrode surfaces, it can adsorptive enzyme
(such as avidin-HRP enzyme (Avidin-horseradish peroxidase)), and subsequent electrochemical detection is made to generate very high background signal, thus
It is unfavorable for obtaining optimal signal-to-noise ratio.However the present invention adopts further after DNA capture probe is fixed on gold electrode surfaces
The closing for carrying out a period of time to the vacancy point of electrode surface with the sulfydryls hexanol of low concentration, the DNA for being conducive to electrode surface are visited
Needle orderly erects, and is conducive to subsequent hybridization reaction, and signal-to-noise ratio significantly improves.Detection method of the invention can be answered widely
For in DNA detection.The present invention can it is highly sensitive, detect DNA with high specificity, sensitivity may be up to 1pM, easy to operate,
It has a extensive future.
Detailed description of the invention
Fig. 1 is using the schematic diagram for carrying out Electrochemical Detection based on DNA capture probe.
Fig. 2A~2C is the Electrochemical Detection of different assemble methods as a result, wherein A is to be closed using the MCH of 0.1mM；B
To use 0.1 μM of A5 to close；C is without closing step.
Fig. 3 is the result for the signal-to-noise ratio that different assemble methods carry out Electrochemical Detection.
Fig. 4 is the result for the electrode AC impedance that different assemble methods carry out Electrochemical Detection.
Fig. 5 A~5D is the result for the electron transfer rate coefficient that different assemble methods carry out Electrochemical Detection.Wherein, A is
Naked gold electrode；B is to assemble poly A30 but the electrode without closing step；C is assembling poly A30 and with 0.1mM's
The closed electrode of MCH；D is assembling poly A30 and with the closed electrode of 0.1 μM of A5.
Fig. 6 is the result of influence of the number of adenine in the DNA capture probe based on polyadenous purine to signal-to-noise ratio.
Fig. 7 A~7E be containing different number adenines based on the DNA capture probe of polyadenous purine to the shadow of packing density
Loud result.Wherein, A polyA5, B polyA10, C polyA20, D polyA30, E polyA40.
Fig. 8 is the result of influence of the concentration of poly A30 to signal-to-noise ratio.
Fig. 9 is the result of influence of the off-period of the MCH of 0.1mM to signal-to-noise ratio.
Figure 10 is that the DNA to be measured of various concentration carries out the result (i-t curve) of Electrochemical Detection.
Figure 11 is that the DNA to be measured of various concentration carries out the result (histogram) of Electrochemical Detection.
Figure 12 is the result that Electrochemical Detection is carried out using single nucleotide mismatch sequence, completely not complementary not complementary DNA.
Specific embodiment
The present invention is further illustrated below by the mode of embodiment, but does not therefore limit the present invention to the reality
It applies among a range.In the following examples, the experimental methods for specific conditions are not specified, according to conventional methods and conditions, or according to quotient
The selection of product specification.
The single stranded DNA as shown in No.1~12 SEQ ID in sequence table is by life technology biology Co., Ltd
Synthesis.
Room temperature as described in the examples is the room temperature of this field routine, refers generally to 20~40 DEG C.
The building of the DNA capture probe of 1 polyadenous purine of embodiment
Design and synthesize the DNA capture probe of the adenine containing different numbers (5,10,20,30 and 40)
(being referred to as poly A5, poly A10, poly A20, poly A30 and poly A40), nucleotide sequence is respectively such as sequence
In table shown in No.1~5 SEQ ID, artificial synthesized (life technology biology Co., Ltd) obtains polyadenous purine afterwards
DNA capture probe.
Simultaneously synthesizing have and the single stranded DNA of only 5 adenines (i.e. SEQ ID in A5, nucleotide sequence such as sequence table
Shown in No.6), so as to the control as follow-up test.
The biosensor of DNA capture probe of the embodiment 2 containing polyadenous purine
(1) gold electrode (2mm diameter, the limited public affairs of Shanghai Chen Hua instrument by physics polishing and sulfuric acid electrochemical cleaning are taken
Department), with ultrapure water Milli-Q cleaning down electrode surface, then use N2Drying, must handle clean electrode.
(2) the DNA capture probe poly A30 of the resulting polyadenous purine of 5 μ L, 0.1 μM of embodiment 1 is added drop-wise to step (1)
The clean electrode of resulting processing, 25 DEG C of reactions assemble overnight, obtain a group loading electrode.
(3) 30 are closed to the vacancy of resulting group of loading electrode of step (2) using the MCH of 150 μ L0.1mM (being purchased from sigma)
Minute, closed electrode is obtained, as can be used for the biosensor detected.
(4) DNA to be measured of 1nM (its nucleotide sequence is as shown in SEQ ID No.8 in sequence table) and 100nM signal are visited
Needle (its nucleotide sequence connects biotins as shown in SEQ ID No.7 in sequence table, and at 3 ' ends) is containing 1M NaCl's
Prehybridization in 10mM TE buffer solution (including 10mM Tris-HCl and 1mM EDTA, pH7.0), 80 DEG C keep 5 minutes, then
25 DEG C are placed 20 minutes, and prehybridization object A is obtained.
(5) by the DNA capture probe 37 on the resulting prehybridization object A of step (4) and step (3) resulting closed electrode
DEG C hybridization 60 minutes, then with containing 1M NaCl 10mM TE buffer solution (including 10mM Tris-HCl and 1mM EDTA,
PH7.0) cleaning down electrode.
(6) avidin-HRP of 3 μ L energy catalytic oxidation-reduction reactions is added dropwise on the resulting detectable electrode of step (5)
Enzyme (is purchased from eBioscience, 1000 times of dilutions), and incubation at room temperature 15min connect it with the DNA capture probe on the electrode；
Then reaction substrate TMB (purchased from Neogen) the progress Electrochemical Detection analysis that can be catalyzed avidin-HRP enzyme is added.
The detailed schematic of Electrochemical Detection analysis is carried out referring to Fig. 1, specifically, first between adenine and gold electrode
Strong interaction, one section of DNA capture probe rich in polyadenous purine (poly A) is connected to gold electrode surfaces, to be measured
In the presence of DNA (its one section of nucleotide sequence for belonging to genome of E.coli), DNA and DNA capture probe to be measured, life
The signal probe of object element modification is hybridized using " sandwich method ".It is connected to the signal probe of electrode surface and then passes through biology
HRP enzyme is connected to gold electrode surfaces by firm binding force between element-streptomysin, the HRP enzyme captured by electrode surface and bottom
Object reacts, to delicately detect that generated catalytic current is reacted in catalysis.
The Electrochemical Detection of the different assemble methods of embodiment 3
(1) gold electrode (2mm diameter, the limited public affairs of Shanghai Chen Hua instrument by physics polishing and sulfuric acid electrochemical cleaning are taken
Department), with ultrapure water Milli-Q cleaning down electrode surface, then use N2Drying, must handle clean electrode.
(2) the resulting poly A30 of 5 μ L0.1 μM embodiment 1 is added drop-wise to the clean electrode of the resulting processing of step (1),
25 DEG C of reactions, assemble overnight, obtain a group loading electrode.
(3) A5 prepared respectively using the MCH of 0.1mM, 0.1 μM of embodiment 1 is to resulting group of loading electrode of step (2)
Vacancy is closed 30 minutes, and closed electrode A, B are obtained.
In addition, poly A30 assembling must be added dropwise however without any substance by step (4) is directly carried out after step (2) operation
Carry out closed electrode C.
Remaining all step is identical with embodiment 2.
As a result as shown in Fig. 2A~2C, Fig. 3 and table 1.As a result illustrate, closed without using substance and accounted for without any
When position (electrode C), the background signal that Electrochemical Detection goes out is very high, and the signal for detecting 1nM is not also high, signal-to-noise ratio very little, because
This cannot detect the DNA to be measured of low concentration.After carrying out the vacancy of closing gold electrode surfaces using A5 (i.e. electrode B), situation
It cannot improve, background signal is still very high, and the signal for detecting 1nM is declined slightly, and the catalysis peak at the peak CV is also more unobvious.It is former
Because may be to be closed using after 0.1 μM of capture probe poly A30 assembling gold electrode with 0.1 μM of A5, it is equivalent to 0.2 μM
The gold electrode of A30 assembling, more obstruction electron transmission, cause signal to reduce.And the sky of closing gold electrode surfaces is carried out using MCH
Behind position (i.e. electrode A), background signal is substantially reduced, this, which may be because, is closed gold electrode surfaces with sulfydryls hexanol, is hindered pair
The absorption of enzyme；The signal of detection 1nM significantly improves, and reason may be because using after MCH occupy-place, be assembled in gold electrode surfaces
DNA becomes orderly, to be conducive to hybridize；Signal-to-noise ratio also significantly improves, and the peak CV also tends to normally be catalyzed peak.Therefore, selection uses
The gold electrode of the closed polyadenous pyrimidine capture probe assembling of MCH carries out the experiment of electrochemica biological sensor.
1 Electrochemical Detection of table
The electrode crosslinking impedance characterization of the different assemble methods of embodiment 4
(1) gold electrode (2mm diameter, the limited public affairs of Shanghai Chen Hua instrument by physics polishing and sulfuric acid electrochemical cleaning are taken
Department), with ultrapure water Milli-Q cleaning down electrode surface, then use N2Drying, must handle clean electrode.
(2) the resulting poly A30 of 5 μ L, 0.1 μM of embodiment 1 is added drop-wise to the clean electrode of the resulting processing of step (1),
25 DEG C of reactions, assemble overnight, obtain a group loading electrode.
(3) A5 prepared respectively using the MCH of 0.1mM, 0.1 μM of embodiment 1 is to resulting group of loading electrode of step (2)
Vacancy is closed 30 minutes, and closed electrode A, B are obtained.
In addition, poly A30 assembling must be added dropwise however without any substance by step (4) is directly carried out after step (2) operation
Carry out closed electrode C.
In addition, step (1) resulting electrode is known as naked gold electrode.
(4) by the electrode (naked gold electrode, A, B and C) of four kinds of situations obtained above in the 10.0mM containing 0.1M KCl
K3Fe(CN)6/K4Fe(CN)6AC impedance characterization is carried out in solution.
As a result as shown in Fig. 4 and table 2.As a result illustrate, the impedance of naked gold electrode is very small, only 0.479k Ω, assembles
After polyA30 capture probe, impedance is greatly increased as 9.20k Ω, illustrates that polyA30 capture probe has been assembled in gold electrode table
Face causes impedance to increase to hinder the electron transmission of gold electrode surfaces；After using MCH occupy-place 30 minutes of 0.1mM,
Impedance becomes smaller again as 6.48k Ω, this is because the capture probe of electrode surface becomes orderly, to be conducive to after using MCH occupy-place
Electron transmission.MCH processing, polyA30 and polyA5 are assembled altogether after comparison assembling polyA30 capture probe, polyA30 assembling
The impedance of gold electrode, which can be seen that, to be come, and using polyA5 occupy-place, can not achieve the effect that MCH, and impedance is bigger instead, this may
Be because are as follows: due to the presence of polyA5, more adenines are assembled in electrode surface, more hinder the transmitting of electronics.
2 impedance detection of table
The apparent electron transfer rate coefficient of electrode of the different assemble methods of embodiment 5
(1)~(3) step is the same as embodiment 4.
(4) by the electrode (naked gold electrode, A, B and C) of four kinds of situations obtained above in the 10.0mM containing 0.1M KCl
K3Fe(CN)6Cyclic voltammetric detection is carried out in liquid, is swept speed and is respectively set to 0.1V/s, 0.2V/s, 0.3V/s, 0.4V/s, 0.5V/
s、0.6V/s。
It can be with according to Laviron equation [referring to E.Laviron, J.Electroanal.Chem.101 (1979) 19-28]
Estimate the apparent electron transfer rate coefficient (Ks) of each electrode.
As a result as shown in Fig. 5 A~5D and table 3.As a result illustrate, within the scope of (0.1-0.6) mV/s scanning speed, can see
To apparent and almost symmetrical redox peaks.With sweep speed increase, oxidation and reduction peak current simultaneously increase and
It is linearly increased within the scope of (0.1-0.6) mV/s scanning speed, shows that the redox reaction of electrode is the electricity of diffusion control
Chemical process.Naked gold electrode is conducive to electron transmission, Ks=0.65s- due to good conductivity1；When gold electrode surfaces assemble it is more
Gland pyrimidine capture probe, Ks=0.01s-1, this is because gland pyrimidine is fixed on gold electrode surfaces, electron transmission is hindered, so
Apparent electron transfer rate constant can become smaller very much；However after being closed with 0.1mM MCH to gold electrode vacancy, so that non-
Specific adsorption leaves gold electrode surfaces in the DNA of gold electrode surfaces, and makes the capture probe for being fixed on gold electrode surfaces
Become orderly, so the more unused MCH of Apparent electron transfer rate constant is closed as many as 6 times big, i.e., Ks reaches 0.07s-1；When
When being total to the site of assemble method closing gold electrode surfaces using capture probe and polyA5, Apparent electron transfer rate constant is still
0.01s-1, fail the case where changing polyadenous pyrimidine capture probe assembling gold electrode.So selection is phonetic using the closed polyadenous of MCH
The gold electrode of pyridine capture probe assembling.
The detection of 3 electron transfer rate coefficient of table
To sum up, it determines and closed effect is carried out most to the gold electrode that polyadenous pyrimidine capture probe assembles using low concentration MCH
It is good.
Influence of the number of adenine to signal-to-noise ratio in the DNA capture probe of 6 polyadenous purine of embodiment
(1) gold electrode (2mm diameter, the limited public affairs of Shanghai Chen Hua instrument by physics polishing and sulfuric acid electrochemical cleaning are taken
Department), with ultrapure water Milli-Q cleaning down electrode surface, then use N2Drying, must handle clean electrode.
(2) respectively by the resulting poly A5 of 5 μ L, 0.1 μM of embodiment 1, poly A10, poly A20, poly A30 and
Poly A40 is added drop-wise to the clean electrode of the resulting processing of step (1), and 25 DEG C of reactions assemble overnight, obtain a group loading electrode.
Remaining step is same as Example 2.
As a result as shown in Fig. 6 and table 4.As a result illustrate, with the increase of adenine (A) number in capture probe, detect 1nM
The signal of DNA to be measured gradually increases, and reaches saturation when adenine number is 30, and signal is also more and more stable, this is
Because the capture probe density for being assembled in gold electrode surfaces is smaller, is more conducive to hybridize, signal is caused to gradually increase；With capture
The increase of adenine (A) number in probe, the capture probe for being assembled in gold electrode surfaces is also more stable, causes signal more next steady
It is fixed.With the increase of A number in DNA capture probe, background signal is gradually decreased.This may be because the number of A is more, group
Capture probe mounted in gold electrode surfaces is also more stable, and the base A for being adsorbed on electrode surface plays the role of that enzyme is centainly hindered to adsorb.
With the increase of A number, signal-to-noise ratio is gradually increased, and signal-to-noise ratio is maximum when adenine number is 30, so selection poly
A30 carries out subsequent experimental as optimal DNA capture probe.
4 Electrochemical Detection of table
The DNA capture probe and packing density of the different polyadenous purine of embodiment 7
(1) gold electrode (2mm diameter, the limited public affairs of Shanghai Chen Hua instrument by physics polishing and sulfuric acid electrochemical cleaning are taken
Department), with ultrapure water Milli-Q cleaning down electrode surface, then use N2Drying, must handle clean electrode.
(2) respectively by the resulting poly A5 of 5 μ L, 0.1 μM of embodiment 1, poly A10, poly A20, poly A30 and
Poly A40 is added drop-wise to the clean electrode of the resulting processing of step (1), and 25 DEG C of reactions assemble overnight, obtain a group loading electrode.
(3) using the MCH of 150 μ L 0.1mM (being purchased from sigma), the vacancy of resulting group of loading electrode of step (2) is closed
30 minutes, closed electrode is obtained, as can be used for the biosensor detected.
(4) electrolyte that this experiment is used is 10mM TE buffer solution (10mM Tris-HCl, 1mM comprising 1M NaCl
EDTA, pH 7.0), electrolyte need using when Fresh.15 minutes N are passed through before experiment into electrolyte2With thorough
Oxygen is removed, electrode obtained above is first placed in progress timing coulometry detection in the electrolyte after deoxygenation respectively；So
50 μM of RuHex (being purchased from Sigma) is added in the electrolytic solution afterwards, absorption carries out the detection of timing coulometry again after five minutes.
Go out the surface of poly A capture probe by the redox computed quantity of electricity that timing coulometry measures RuHex complex
Density, as a result as shown in Fig. 7 A~7E and table 5.As a result illustrate, as the number of A increases, be assembled in the DNA's of gold electrode surfaces
Superficial density is gradually reduced.This is because the surface area of gold electrode is certain, and theoretically it is assembled in A in gold electrode surfaces
Sum is constant, therefore as the number of A increases, superficial density is gradually reduced.It therefore, can be by changing according to above-mentioned conclusion
Become the number of continuous adenine in DNA probe to regulate and control the packing density of gold electrode surfaces DNA probe.
5 superficial density of table
| DNA capture probe based on polyadenous purine | ΓDNA(pmol/cm2) |
| Poly A5 | 2.1 |
| Poly A10 | 1.1 |
| Poly A20 | 0.9 |
| Poly A30 | 0.6 |
| Poly A40 | 0.4 |
Embodiment 8poly A30 assembles influence of the concentration to signal-to-noise ratio
(1) gold electrode (2mm diameter, the limited public affairs of Shanghai Chen Hua instrument by physics polishing and sulfuric acid electrochemical cleaning are taken
Department), with ultrapure water Milli-Q cleaning down electrode surface, then use N2Drying, must handle clean electrode.
(2) the resulting poly A30 of embodiment 1 is dripped with the volume of 0.02 μM, 0.1 μM and 0.5 μM of 5 μ L of concentration respectively
It is added to the clean electrode of the resulting processing of step (1), 25 DEG C of reactions assemble overnight, obtain a group loading electrode.
Remaining step is same as Example 2.
As a result as shown in Fig. 8 and table 6.As a result illustrate, when assembling 0.02 μM of polyA30 capture probe, detect 100pM
Mesh time-stamped signals are bigger, illustrate the smaller hybridization for being more conducive to DNA of density, but background signal is very big.When 0.5 μM of assembling
When polyA30, detection 100pM mesh time-stamped signals are smaller, it may be possible to because the packing density of electrode surface is big, be unfavorable for DNA
Hybridization.From the experimental results, when assembling 0.1 μM of poly A30, signal-to-noise ratio is maximum.
6 Electrochemical Detection of table
The optimization of 9 off-period of embodiment
(1) gold electrode (2mm diameter, the limited public affairs of Shanghai Chen Hua instrument by physics polishing and sulfuric acid electrochemical cleaning are taken
Department), with ultrapure water Milli-Q cleaning down electrode surface, then use N2Drying, must handle clean electrode.
(2) the resulting poly A30 of embodiment 1 is added drop-wise to step (1) resulting processing with the volume of 0.1 μM of 5 μ L of concentration
Clean electrode, 25 DEG C of reactions, assembles overnight, obtains a group loading electrode.
(3) vacancy of resulting group of loading electrode of step (2) is closed respectively using the 1mM MCH of 150 μ L (being purchased from sigma)
0,5,10,20,30 and 60 minutes, closed electrode is obtained.
Remaining step is same as Example 2.
As a result as shown in Fig. 9 and table 7.As a result illustrate, after sulfydryls hexanol occupy-place, the signal for detecting 1nM target is obvious
Increase.With the extension of off-period, the signal for detecting 1nM target is gradually decreased, and background signal is gradually lowered, but noise
Than being gradually increased, when closed between when being 30 minutes, the signal-to-noise ratio of Electrochemical Detection is maximum.Therefore, selection control closing
Time is 30 minutes.
To sum up, by the optimization of conditions above, the optimal item of Electrochemical Detection is carried out using biosensor of the invention
Part are as follows: use the capture probe of polyA30；And make 0.1 μM of assembling concentration of the capture probe；It is closed with 0.1mM MCH
30 minutes.
7 Electrochemical Detection of table
1 sensitivity technique of effect example
(1) gold electrode (2mm diameter, the limited public affairs of Shanghai Chen Hua instrument by physics polishing and sulfuric acid electrochemical cleaning are taken
Department), with ultrapure water Milli-Q cleaning down electrode surface, then use N2Drying, must handle clean electrode.
(2) the resulting poly A30 of embodiment 1 is added drop-wise to step (1) resulting processing with the volume of 0.1 μM of 5 μ L of concentration
Clean electrode, 25 DEG C of reactions, assembles overnight, obtains a group loading electrode.
(3) vacancy of resulting group of loading electrode of step (2) is closed respectively using the 1mM MCH of 150 μ L (being purchased from sigma)
30 minutes, obtain closed electrode.
(4) respectively by DNA to be measured (its nucleotide sequence such as sequence of 1pM, 10pM, 100pM, 200pM, 500pM and 1nM
In table shown in SEQ ID No.8) and 100nM signal probe (its nucleotide sequence as shown in SEQ ID No.7 in sequence table, and
And in 3 ' end connection biotins) in 10mM TE buffer solution (including 10mM Tris-HCl and 1mM containing 1M NaCl
EDTA, pH7.0) in prehybridization, 80 DEG C keep 5 minutes, then 25 DEG C place 20 minutes, obtain prehybridization object A.
Remaining step is same as Example 2.
As a result as shown in Figure 10~11 and table 8.As a result illustrate, in the presence of no DNA to be measured, can only observe very little
Background current；When DNA to be measured is added, detection electric current is significantly increased, and increases with the increase of DNA concentration to be measured, therefore adopt
Electrochemical Detection is carried out with the DNA capture probe and its self assembly gold electrode of the purine of the present invention containing polyadenous, it is detected
The detection of DNA to be measured is limited to 1pM.
8 Electrochemical Detection of table
| DNA concentration to be measured | 1pM | 10pM | 100pM | 200pM | 500pM | 1nM |
| Electric current (nA) | -44 | -114 | -250 | -401 | -584 | -900.7 |
2 specific detection of effect example
(1) gold electrode (2mm diameter, the limited public affairs of Shanghai Chen Hua instrument by physics polishing and sulfuric acid electrochemical cleaning are taken
Department), with ultrapure water Milli-Q cleaning down electrode surface, then use N2Drying, must handle clean electrode.
(2) the resulting poly A30 of embodiment 1 is added drop-wise to step (1) resulting processing with the volume of 0.1 μM of 5 μ L of concentration
Clean electrode, 25 DEG C of reactions, assembles overnight, obtains a group loading electrode.
(3) vacancy of resulting group of loading electrode of step (2) is closed respectively using the 1mM MCH of 150 μ L (being purchased from sigma)
30 minutes, obtain closed electrode.
(4) respectively by the DNA to be measured of 100pM (its nucleotide sequence is as shown in SEQ ID No.8 in sequence table), monokaryon glycosides
(its nucleotide sequence is successively such as SEQ ID No.10~12 institute in sequence table by sour mismatch SNP (A), SNP (C) and SNP (G)
Show) and by sufficiently excessive (its nucleotide sequence is such as with the complete incomplementarity DNA (NT) of above-mentioned DNA to be measured nucleotide sequence
In sequence table shown in SEQ ID No.9) and 100nM signal probe (its nucleotide sequence such as SEQ ID No.7 institute in sequence table
Show, and in 3 ' end connection biotins) in 10mM TE buffer solution (including 10mM Tris-HCl and 1mM containing 1M NaCl
EDTA, pH7.0) in prehybridization, 80 DEG C keep 5 minutes, then 25 DEG C place 20 minutes, obtain prehybridization object A.
Correspondingly, there is the corresponding not electrode with any DNA hybridization to be measured as blank control.
Remaining step is same as Example 2.
As a result as shown in Figure 12 and table 9.As a result illustrate, the DNA capture probe of the present invention containing polyadenous purine and
The specificity for the Electrochemical Detection that its self assembly gold electrode carries out is very high.
It is well known that the differentiation to single base mismatch has critically important application in single nucleotide polymorphism detection.Pass through
To only there are the single base mismatch DNA chain of mononucleotide difference (SNP (A), SNP (C) and SNP (G)) to probe into spy with DNA to be measured
The opposite sex, obtained signal strength are about 2 times of background signal, and this is only that real DNA to be measured is detected
/ 5th or so of signal strength, it is seen that the self-assembling electrode has the sequence-specific of height.
9 Electrochemical Detection of table
| DNA to be measured | SNP(A) | SNP(C) | SNP(G) | NT | Blank control | |
| Electric current (nA) | -250 | -57.14 | -68.36 | -46.86 | -32.78 | -21.2 |
It should be understood that those skilled in the art can make the present invention various after having read above content of the invention
Change or modification, these equivalent forms also fall within the scope of the appended claims of the present application.
<110>Shanghai Institute of Measurement and Testing Technology
<120>based on DNA capture probe, biosensor and its detection method of polyadenous purine
<130> P1610939C
<160> 12
<170> PatentIn version 3.5
<210> 1
<211> 18
<212> DNA
<213>artificial sequence
<220>
<223>poly A5 capture probe
<400> 1
aaaaacccac caacgctg 18
<210> 2
<211> 23
<212> DNA
<213>artificial sequence
<220>
<223>poly A10 capture probe
<400> 2
aaaaaaaaaa cccaccaacg ctg 23
<210> 3
<211> 33
<212> DNA
<213>artificial sequence
<220>
<223>poly20 capture probe
<400> 3
aaaaaaaaaa aaaaaaaaaa cccaccaacg ctg 33
<210> 4
<211> 43
<212> DNA
<213>artificial sequence
<220>
<223>poly30 capture probe
<400> 4
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa cccaccaacg ctg 43
<210> 5
<211> 53
<212> DNA
<213>artificial sequence
<220>
<223>poly40 capture probe
<400> 5
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa cccaccaacg ctg 53
<210> 6
<211> 5
<212> DNA
<213>artificial sequence
<220>
<223> poly A5
<400> 6
aaaaa 5
<210> 7
<211> 20
<212> DNA
<213>artificial sequence
<220>
<223>it reports sub (reporter)
<400> 7
atcaattcca cagttttcgc 20
<210> 8
<211> 35
<212> DNA
<213>artificial sequence
<220>
<223>DNA to be measured
<400> 8
gcgaaaactg tggaattgat cagcgttggt gggaa 35
<210> 9
<211> 36
<212> DNA
<213>artificial sequence
<220>
<223>incomplementarity DNA(NT)
<400> 9
agactttgat accatactaa atagcaccat ttccaa 36
<210> 10
<211> 35
<212> DNA
<213>artificial sequence
<220>
<223> SNP(A)
<400> 10
gcgaaaactg tggaattgat cagcgtaggt gggaa 35
<210> 11
<211> 35
<212> DNA
<213>artificial sequence
<220>
<223> SNP(C)
<400> 11
gcgaaaactg tggaattgat cagcgtcggt gggaa 35
<210> 12
<211> 35
<212> DNA
<213>artificial sequence
<220>
<223> SNP(G)
<400> 12
gcgaaaactg tggaattgat cagcgtgggt gggaa 35
Claims (11)
1. a kind of electrochemica biological sensor, which is characterized in that it is prepared by method comprising the following steps:
(1) the DNA capture probe based on polyadenous purine, SEQ in the nucleotide sequence of the DNA capture probe such as sequence table are prepared
Shown in ID No.3, SEQ ID No.4 or SEQ ID No.5；
(2) the resulting DNA capture probe based on polyadenous purine of step (1) is added drop-wise on electrode, reacts, obtains a group loading electrode；
Packing density when the DNA capture probe based on polyadenous purine is added drop-wise on electrode is 0.1 μM；The electrode is gold electricity
Pole；
(3) it is closed with vacancy of the sulfydryls hexanol to resulting group of loading electrode of step (2), obtains closed electrode.
2. electrochemica biological sensor as described in claim 1, which is characterized in that
The nucleotide sequence of DNA capture probe as described in step (1) is as shown in SEQ ID No.4；
Alternatively, the temperature of step (2) described reaction is 20 ~ 40 DEG C；
Alternatively, the time of step (2) described assembling is greater than 12 hours；
Alternatively, step (2) described electrode is made by method comprising the following steps: through physics polishing, sulfuric acid electrochemical cleaning, then
With ultrapure water cleaning down electrode surface, N is then used2Drying；
Alternatively, the concentration of step (3) described sulfydryls hexanol is 0.01 ~ 1mM；
Alternatively, step (3) vacancy closed time is 5 ~ 60 minutes；
Alternatively, the closed temperature in step (3) vacancy is 25 DEG C.
3. electrochemica biological sensor as claimed in claim 2, which is characterized in that the temperature of step (2) described reaction is 25
℃。
4. electrochemica biological sensor as claimed in claim 2, which is characterized in that the concentration of step (3) described sulfydryls hexanol
For 0.1mM.
5. electrochemica biological sensor as claimed in claim 2, which is characterized in that step (3) vacancy closed time
It is 30 minutes.
6. a kind of method for detecting DNA to be measured using such as the described in any item electrochemica biological sensors of claim 1 ~ 5, packet
Include following step:
(I) by DNA to be measured and nucleotide sequence as shown in SEQ ID No.7 in sequence table and in the signal of 3 ' end connection biotins
Probe prehybridization obtains prehybridization object A；The DNA to be measured includes nucleotide sequence as shown in SEQ ID NO. 8 in sequence table
Sequence；
(II) by the resulting prehybridization object A of step (I) and such as the described in any item electrochemica biological sensors of claim 2 ~ 5
DNA capture probe hybridization on electrode, obtains the electrode of hybridization；
(III) hatch after Avidin-horseradish peroxidase being added dropwise on the electrode of step (II) resulting hybridization；Substrate is added
TMB is tested and analyzed.
7. detecting the method for DNA to be measured as claimed in claim 6, which is characterized in that
The concentration of step (I) DNA to be measured is 0 ~ 1000pM；
Alternatively, step (I) prehybridization is kept 1 ~ 10 minute at 60 ~ 100 DEG C, then place 5 ~ 60 minutes for 20 ~ 40 DEG C；It is described
Prehybridization carries out under buffer；
Alternatively, hybridization described in step (II) hybridizes 30 ~ 120 minutes at 25 ~ 45 DEG C；
Alternatively, Avidin-horseradish peroxidase described in step (III) is 1000 times of diluted Avidin-horseradish peroxidases；
Alternatively, the additive amount of described 1000 times diluted Avidin-horseradish peroxidases of step (III) is 1 ~ 5 μ L；
Alternatively, the temperature of step (III) described hatching is 25 DEG C；The time of the hatching is 15 minutes.
8. detecting the method for DNA to be measured as claimed in claim 7, which is characterized in that
The temperature of the prehybridization is 80 DEG C；The time of the prehybridization is 5 minutes；The time of the placement is 20 minutes；Institute
The temperature for stating placement is 25 DEG C；
Alternatively, the buffer is the 10mM TE comprising 1M NaCl；
Alternatively, the temperature of the hybridization is 37 DEG C；
Alternatively, the time of the hybridization is 60 minutes；
Alternatively, further including the steps that cleaning electrode after the completion of the hybridization；
Alternatively, the additive amount of 1000 times of diluted Avidin-horseradish peroxidases is 3 μ L.
9. detecting the method for DNA to be measured as claimed in claim 8, which is characterized in that the TE include 10mM Tris-HCl and
1mM EDTA, pH7.0.
10. detecting the method for DNA to be measured as claimed in claim 8, which is characterized in that cleaning solution used in the cleaning is packet
The 10mM TE buffer of the NaCl containing 1M；The TE buffer includes 10mM Tris-HCl and 1mM EDTA, pH7.0.
11. detecting the method for DNA to be measured as claimed in claim 6, which is characterized in that step (I) described signal probe it is dense
Degree is 100nM.
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| CN112162027B (en) * | 2020-09-21 | 2023-08-18 | 上海市计量测试技术研究院 | Electrochemical sensor based on triblock probe and application of electrochemical sensor in detection of transgenic double-stranded RNA |
| CN115165983B (en) * | 2022-06-29 | 2023-10-24 | 上海市计量测试技术研究院 | Reverse stem-loop specific ratio type electrochemical DNA biosensor based on polyadenylation and application thereof |
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