CN110146566A - Modified electrode, combination product and its electroluminescent chemiluminescence biosensor and application - Google Patents
Modified electrode, combination product and its electroluminescent chemiluminescence biosensor and application Download PDFInfo
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- CN110146566A CN110146566A CN201910516787.XA CN201910516787A CN110146566A CN 110146566 A CN110146566 A CN 110146566A CN 201910516787 A CN201910516787 A CN 201910516787A CN 110146566 A CN110146566 A CN 110146566A
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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
- G01N27/305—Electrodes, e.g. test electrodes; Half-cells optically transparent or photoresponsive electrodes
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- 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
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- G01N27/308—Electrodes, e.g. test electrodes; Half-cells at least partially made of carbon
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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
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Abstract
The present invention relates to modified electrode, combination product and its electroluminescent chemiluminescence biosensor and applications.The modified electrode includes the illuminator of electrode and modification on the electrodes；The illuminator is mainly poly- [(9,9- dioctyl fluorene -2,7- diyl) -co- (1,4- benzo-{ 2,1 ', 3 }-the thiadiazoles)] point of carboxyl-functional.The electroluminescent chemiluminescence biosensor is without coreaction reagent, the deficiency of system stability present in the existing ECL biosensor assay carried out in the presence of coreaction reagent, measurement can be overcome, and there are lack reproducibility in error, detection, construct the dual signal amplification electrogenerated chemiluminescence strategy of no coreaction reagent type, there is comparatively ideal ECL signal response and excellent ECL luminous efficiency simultaneously, with excellent high sensitivity, specific selectivity and stability, a kind of new method is provided for the detection of nucleic acid.
Description
Technical field
The present invention relates to field of biosensors, send out in particular to modified electrode, combination product and its electroluminescent chemistry
Optical biosensor and application.
Background technique
MicroRNAs (miRNAs) is a kind of endogenous, and nonprotein coding is short (normally about 19-25 base) and single
The RNAs of chain, has been found to be several bioprocess such as hematopoiesis function, the important regulatory factor in cell Proliferation and Apoptosis.
Studies have shown that miRNA can be used as ideal biomarker candidate, and the unconventionality expression of miRNA and various cancers
Occur closely related.Therefore, there is an urgent need to develop highly sensitive, high special and reliable technology detect miRNA.
Electrogenerated chemiluminescence (ECL) not only has the advantages that electrochemical method, such as the simplicity and stability of equipment, and
And also there is dynamic range and higher sensitivity widely than conventional chemical luminous (CL).Thus it is widely used in environment point
Analysis, biochemistry detection and clinical detection.In general, ECL measurement is substantially and carries out in the presence of coreaction reagent.However,
Adding exogenous coreaction reagent in detection solution will affect the environment of test solution, and ECL system is made to lack enough stabilizations
Property, so as to cause measurement error.Although in addition, can also be to avoid addition external source coreaction using dissolved oxygen as coreaction reagent
The defect of reagent, but in the case where dissolved oxygen is as coreaction reagent, the dissolved oxygen of unknown concentration will lead to inevitably
Error and lack reproducibility in the detection.
Therefore, exploitation have high ECL emission effciency and without any coreaction reagent for detect miRNAs it is sensitive,
Simple ECL system has important practical significance.
In view of this, the present invention is specifically proposed.
Summary of the invention
The first object of the present invention is to provide a kind of modified electrode, and the modified electrode is modified with conjugated polymers object point hair
Body of light, the electrode are function of surface polarizing electrode, have the advantages such as high charge carrier mobility, Rapid radiating rate.
The second object of the present invention is to provide the combination product including above-mentioned modified electrode, which is based on nothing
Dual signal amplification electrogenerated chemiluminescence (ECL) the strategy design of coreaction reagent type, it can be used in building and tried without coreaction
Dosage form dual signal amplifies electroluminescent chemiluminescence biosensor.
The third object of the present invention is to provide the electrogenerated chemiluminescence bio-sensing obtained by said combination assembling product
Device, the electroluminescent chemiluminescence biosensor is no coreaction reagent type ECL biosensor, in conjunction with biped DNA walker
(walker) dual amplification strategy can overcome the existing ECL biosensor assay carried out in the presence of coreaction reagent
Present in system stability is insufficient, measurement is there are lacking reproducibility in error, detection, while having comparatively ideal
ECL signal response and excellent ECL luminous efficiency.
The fourth object of the present invention be to provide above-mentioned electroluminescent chemiluminescence biosensor in detection nucleic acid, especially
Application in terms of miRNA.
In order to realize above-mentioned purpose of the invention, the following technical scheme is adopted:
Modified electrode comprising the illuminator of electrode and modification on the electrodes.
The illuminator is mainly poly- [(9,9- dioctyl fluorene -2,7- diyl) -co- (1,4- benzo-of carboxyl-functional
{ 2,1 ', 3 }-thiadiazoles)] point.
Optionally, the electrode is selected from glass-carbon electrode, gold electrode.
Optionally, the electrode is selected from glass-carbon electrode.
As an implementation, PFBT-COOH point is poly- (phenylethylene-maleic anhydride) (PSMA) functionalization in the present invention
PFBT point.
Modified electrode is using conjugated polymer, conjugated polymers object point as illuminator, especially PFBT and carboxyl in the application
The PFBT-COOH point of functionalization, this quasi polymer and polymerization object point have high charge carrier mobility, Rapid radiating rate, easily
In surface-functionalized, a variety of prominent features such as good photostability, the use of this kind of material is building without coreaction reagent type
ECL biosensor provide a good platform, and extend application of the conjugated polymers object point in clinical analysis.
In the present invention, PFBT-COOH point not only shows higher ECL intensity, but also shows superior film forming
Ability and stability.It is not adding any coreaction reagent and is using N2In the case where removing dissolved oxygen, at photomultiplier tube (PMT)
Voltage be set as 800V, when number of stages of amplification is set as 3, the ECL intensity of PFBT-COOH point can reach 18,000a.u., signal
The ECL signal response of intensity ratio PS-COOH-co-PFO point is about 5 times high, 24 times higher than pure PFBTECL signal response.
Therefore, PFBT-COOH point is provided for the building of the ECL sensing platform of no coreaction reagent type and is more preferably selected.
Another object according to the present invention provides a kind of combination product comprising above-mentioned modified electrode, DNA S1, DNA
S2；DNA hair clip H1 and DNA hair clip H2.
The DNA S1 and DNA S2 can complementary pairing, coupling has quencher on the DNA S2.
The DNA hair clip H1 and DNA hair clip H2 assembling can form biped DNA walker under nucleic acid to be detected catalysis；
The biped part of the biped DNA walker can with the DNA S1 complementary pairing, and on the DNA S1 after the pairing
Reserve the restriction enzyme site of DNA excision enzyme.
The combination product does not include coreaction reagent.
Optionally, the combination product further includes the DNA excision enzyme.
Optionally, the DNA excision enzyme is exonucleaseⅲ.
Optionally, the quencher is selected from black hole quencher, ferrocene.
Optionally, the quencher is black hole quencher.
In the present invention, DNA excision enzyme, especially exonucleaseⅲ (Exo III) can carry out ECL signal strength
The amplification of two steps, so that electroluminescent chemiluminescence biosensor obtains the ECL signal significantly increased, dual amplification strategy can assign biography
Sensor more preferably signal amplifying power.
Another object according to the present invention provides the method for detection nucleic acid, and the method includes using said combination to produce
Product detect nucleic acid to be detected.
Optionally, the method for the detection nucleic acid includes the assemble method of electroluminescent chemiluminescence biosensor:
A) using the modified electrode as matrix assembled dna S1, the DNA S2 with the DNA S1 complementary pairing is introduced；
B) under the nucleic acid catalysis to be detected, DNA hair clip H1 and H2 assembling generate biped DNA walker；
C) the biped DNA walker and the DNA excision enzyme are incubated for altogether with the electrode obtained in step a)；
Wherein, step a) and b) without sequencing.
Optionally, DNA S1 described in step a) is in the presence of crosslinking agent in the modified electrode over-assemble；The crosslinking
Agent is EDC and NHS.
Optionally, the preparation method of the modified electrode includes:
The dispersant liquid drop of the illuminator is applied to the surface of the electrode, drying and forming-film obtains the modified electrode.
Optionally, the decentralized medium of the dispersion liquid is selected from secondary distilled water or ultrapure water.
Optionally, the concentration of illuminator described in the dispersion liquid is 0.2mgmL-1~0.4mgmL-1。
It optionally, further include the pretreatment of electrode before the illuminator modifies the electrode.
Optionally, it is described pretreatment include: by the electrode carry out at least 2 times polishing, then in the presence of cleaning agent into
Row ultrasonic cleaning.
Optionally, the polishing is polished using aluminium oxide；The partial size of the aluminium oxide be no more than 0.30 μm, and partial size with
It polishes the increase of number and reduces.
Optionally, the cleaning agent includes ethyl alcohol, water.
Optionally, the power of the ultrasound is 160~200W, and ultrasonic time is 2~5 minutes.
Optionally, the preparation process of the biped DNA walker includes:
DNA hair clip H1 and DNA hair clip H2 is heated to being kept for 5~10 minutes at 90 DEG C~100 DEG C, is preferably heated to 95 DEG C
It is lower to be kept for 5 minutes, it is slowly cooled to room temperature, forms hairpin structure；
Object, DNA hair clip H1 and DNA hair clip H2 are mixed to get mixture, and by mixture at 25 DEG C~65 DEG C
It is incubated for 60~120 minutes, is incubated for 90 minutes preferably at 37 DEG C, obtain biped DNA walker.
Optionally, the incubation time of the biped DNA walker and the DNA excision enzyme on the electrodes is 1~3 small
When, preferably 2 hours.
As an implementation, DNA S1 is assembled using the electrode by illuminator modification as matrix, is not being added
To obtain strong ECL signal under conditions of any coreaction reagent；Then, Black Hole Quencher is introduced by hybridizing with DNA S1
(BHQ) the DNA S2 (BHQ-S2) marked.Since BHQ is to the efficient quenching effect of PFBT-COOH signal, ECL signal reaches " letter
Number close " state, it is thus achieved that function switching signal.
As an implementation, circumscribed by biped DNA walker and DNA that object is catalyzed to hair clip assembling generation
Enzyme is incubated for onto electrode simultaneously.On the one hand, BHQ-S2 is replaced by biped DNA walker to release from electrode surface.It is another
Aspect, DNA excision enzyme can digest blunt or recess 3 ' ends in double-stranded DNA (dsDNA).With the help of DNA excision enzyme,
DNA walker, which is released, replaces another BHQ-S2 again to realize DNA walker cyclic process.As was expected, ECL
Signal is switched to the state of " signal is opened ".Due to the high-luminous-efficiency of conjugated polymers object point illuminator, the high quenching efficiency of BHQ with
And the combination of dual amplification strategy, the biosensor of the ECL proposed " off-on " model realize the hypersensitive of object
Detection.
Optionally, the nucleic acid is RNA, preferably miRNAs.
Optionally, the nucleic acid is miRNAs-155.
Optionally, the sequence of the DNA S1 is as shown in SEQ ID NO:1；
The sequence of the DNA S2 is as shown in SEQ ID NO:2；
The miRNAs-155, sequence is as shown in SEQ ID NO:3；
The sequence of the H1 is as shown in SEQ ID NO:4, and the sequence of the H2 is as shown in SEQ ID NO:5.
Optionally, the range of linearity of above-mentioned detection method is 10amolL-1~5pmolL-1, detection is limited to
3.3amol·L-1。
It is raw to provide the electrogenerated chemiluminescence assembled by above-mentioned assemble method for another free-revving engine according to the present invention
Object sensor.
Electroluminescent chemiluminescence biosensor provided by the invention is constructed using conjugated polymers object point as illuminator
ECL biosensor without coreaction reagent type realizes strong ECL signal under conditions of not adding any coreaction reagent
It obtains, the cyclic process for arriving signal recovery again, i.e. " signal is opened " state-" signal-off " state-of ECL signal is quenched in signal
The cyclic switching of " signal is opened " state completes the sensing of ECL signal.
Compared with prior art, beneficial effects of the present invention include but is not limited to:
(1) modified electrode provided by the invention is function of surface polarizing electrode, has high charge carrier mobility, quick spoke
Penetrate the advantages such as rate.
(2) combination product provided by the invention is the dual signal amplification electrogenerated chemiluminescence based on no coreaction reagent type
(ECL) strategy designs, and can be used in building without coreaction reagent type dual signal and amplifies electroluminescent chemiluminescence biosensor.
(3) co-reactant is not used, in conjunction with biped DNA walking in electroluminescent chemiluminescence biosensor provided by the invention
Device (walker) and enzyme trigger dual amplification strategy, can overcome the existing ECL biology carried out in the presence of coreaction reagent
System stability present in sensor measurement is insufficient, measurement is there are reproducibility is lacked in error, detection, constructs nothing
The dual signal of coreaction reagent type amplifies electrogenerated chemiluminescence strategy, while having comparatively ideal ECL signal response and excellent
Different ECL luminous efficiency.
(4) electroluminescent chemiluminescence biosensor provided by the invention, with excellent highly sensitive when for detecting miRNA
Property and lower detection limit, specific selectivity and stability, the range of linearity for detecting miRNA is 10amolL-1~5pmol
L-1, detect and be limited to 3.3amolL-1, a kind of new method is provided for the detection of miRNA.
Detailed description of the invention
It, below will be to specific in order to illustrate more clearly of the specific embodiment of the invention or technical solution in the prior art
Embodiment or attached drawing needed to be used in the description of the prior art be briefly described, it should be apparent that, it is described below
Attached drawing is some embodiments of the present invention, for those of ordinary skill in the art, before not making the creative labor
It puts, is also possible to obtain other drawings based on these drawings.
Fig. 1 is the building process of electroluminescent chemiluminescence biosensor in one embodiment of the present invention；
Fig. 2 is the native polyacrylamide gel electrophoresis analysis of biped DNA walker in one embodiment of the present invention
As a result；
Fig. 3 is the Cyclic voltamogram characterization result of electroluminescent chemiluminescence biosensor in one embodiment of the present invention
(A) and ECL response results (B)；
Wherein, in Fig. 3 A curve a indicate bare electrode redox curve, curve b indicate PFBT-COOH point modified electrode
Redox curve, curve c indicate incubated dna S1 after redox curve, curve d indicate closed with HT after oxidation also
Virgin curve, curve e indicate that the redox curve after introducing BHQ-S2, curve f indicate that DNA walker and ExoIII are incubated for simultaneously
Redox curve after on to electrode surface；
Curve a indicates that the ECL signal response curve of bare electrode, curve b indicate PFBT-COOH point modified electrode in Fig. 3 B
ECL signal response curve, curve c indicate that incubated dna S1 and the ECL signal response curve after being closed with HT, curve d indicate to introduce
ECL signal response curve after BHQ-S2, after curve e indicates that DNA walker and ExoIII are incubated for onto electrode surface simultaneously
ECL signal response curve；
Fig. 4 be in one embodiment of the present invention electroluminescent chemiluminescence biosensor to the object of various concentration
ECL signal response results；Wherein, Fig. 4 A is ECL response curve, and it is 0,10amol that curve a~i, which respectively corresponds target concentration,
L-1、100amol·L-1、1fmol·L-1、5fmol·L-1、50fmol·L-1、100fmol·L-1、1pmol·L-1And
5pmol·L-1Under response curve；Fig. 4 B is calibration curve；
Fig. 5 is that the electroluminescent chemiluminescence biosensor of excision enzyme is not used in one embodiment of the present invention to difference
The ECL signal response results of the object of concentration and with use triggering enzyme biosensor comparing result；Wherein, Fig. 5 A is
ECL response curve, it is 0.5fmolL that curve a~h, which respectively corresponds target concentration,-1、1fmol·L-1、5fmol·L-1、
10fmol·L-1、50fmol·L-1、100fmol·L-1、0.5pmol·L-1And 5pmolL-1Under response curve；Fig. 5 B
For calibration curve；Fig. 5 C and Fig. 5 D are respectively that target concentration is 5fmolL-1And 1pmolL-1When, using with do not use
The ECL signal of DNA excision enzyme responds comparing result；
Fig. 6 is the selectivity (A) and stability (B) of electroluminescent chemiluminescence biosensor in one embodiment of the present invention
As a result；Wherein, a is blank control in Fig. 6 A, and b~g is respectively that concentration is 100fmolL-1MiRNA-21,100fmolL-1MiRNA-141,100fmolL-1MiRNA-126,100fmolL-1Let-7a, 1fmolL-1MiRNA-
155, the ECL signal response results of the mixture of miRNA-155 and interfering substance；
Fig. 7 is electroluminescent chemiluminescence biosensor in one embodiment of the present invention in different cell pyrolysis liquids
ECL signal response results；Wherein, a is blank control, and b~e is respectively that cell concentration is 10,102、103、104Result.
Specific embodiment
Embodiment of the present invention is described in detail below in conjunction with embodiment, but those skilled in the art will
Understand, the following example is merely to illustrate the present invention, and is not construed as limiting the scope of the invention.It is not specified in embodiment specific
Condition person carries out according to conventional conditions or manufacturer's recommended conditions.Reagents or instruments used without specified manufacturer is
It can be with conventional products that are commercially available.
Wherein: N- (3- (dimethylaminopropyl)-N'- ethyl-carbodiimide hydrochloride (EDC), N- weight ratio amber
Acid imide sodium salt (NHS) and tetrahydrofuran (THF) are purchased from Sigma-Aldrich Co., Ltd (U.S. St. Louis)；
Poly- [(9,9- dioctyl fluorene -2,7- diyl) -co- (1,4- benzo-{ 2,1 ', 3 }-thiadiazoles)] (PFBT) is purchased from beauty
Source of polymer company, state；
Poly- (phenylethylene-maleic anhydride) (PSMA) is purchased from Aladdin Co., Ltd (Chinese Shanghai)；
In the present invention all DNA oligonucleotides and exonuclease III (Exo III) be all by raw work bioengineering (on
Sea) limited liability company synthesizes and purifies；
Phosphate buffer solution (PBS) (pH 7.4,0.10molL-1) by H3PO4It is prepared with NaOH.By slow with PBS
Fliud flushing dissolves the potassium ferricyanide and potassium ferrocyanide, obtains ferricyanide solution (Fe (CN)6 3-/4-, 5mmolL-1, pH 7.4).
Characterization device information used in the present invention is as follows:
ECL measurement is carried out using MPI-A ECL analyzer (Xi'an Rui Mai Electronic Science and Technology Co., Ltd., Chinese Xi'an), is swept
It retouches current potential and is set as 0 to 1.25V, photomultiplier tube is set as 800V；
Electrochemical measurement is carried out using CHI600D electrochemical workstation (Shanghai Chen Hua Co., Ltd instrument, Chinese Shanghai)；
Use the pattern of transmission electron microscope (TEM) characterization material.
The synthesis of 1 PFBT-COOH point of experimental example
Firstly, PFBT and PSMA are dissolved in THF, preparing concentration respectively is 1.0mgmL-1Stock solution.Then,
The 4.0mL PFBT prepared and 800 μ L PSMA are mixed and sonicated to form uniform mixed solution.Then, by 10mL
Secondary distilled water is injected in above-mentioned solution and is stirred overnight.THF is removed finally, evaporating by parital vacuum, obtains PFBT-COOH
Point, is kept in dark place.
The preparation of 1 biosensor of embodiment
The preparation of biped DNA walker
Biped DNA walker is synthesized by object catalysis hair clip assembling process (CHA).
DNA hair clip H1 and DNA hair clip H2 is heated to 95 DEG C, reaction 5min is kept, is then slowly cooled to room temperature and is allowed to
Form hairpin structure；
By 10 μm of olL of the object miRNA-155 of 10 μ L various concentrations and 5 μ L-1DNA hair clip H1 is added to 5 μ L, 10 μ
mol·L-1In DNA hair clip H2；Then, mixture is incubated for 90min at 37 DEG C.Obtain biped DNA walker.
The building of biosensor
Fig. 1 illustrates gradually building and the assembling process of ECL biosensor.
A) after polishing glass-carbon electrode (GCE, Φ=4.0mm) respectively with 0.30 and 0.05 μm of aluminium oxide, ethyl alcohol is successively used
It is cleaned by ultrasonic with secondary distilled water；
B) the secondary distilled water dispersant liquid drop of the PFBT-COOH point of 10 μ L is applied to glassy carbon electrode surface, done in air
After dry film forming, modified electrode is immersed to crosslinking agent (0.02gmL at room temperature-1EDC and 0.01gmL-1NHS 30min in)；
It c) is 10 μm of olL by the concentration of 10 μ L-1DNA S1 be connected to active carboxyl PFBT-COOH point modification
Electrode on, be incubated overnight at 4 DEG C；1.0mmolL is used at room temperature-1After hexyl mercaptan (HT) closes 40min, by BHQ-S2
Compound (10 μ L, 10 μm of olL-1) 37 DEG C in modification to being incubated for 2h on electrode；
D) Exo III of the 10 μ L biped DNA walker prepared and 5 μ L is added drop-wise to the electrode table modified
On face, it is incubated for 2h, obtains electroluminescent chemiluminescence biosensor.
The ECL biosensor of DNA excision enzyme is not used in comparative example 1
The process that ECL biosensor is constructed in this comparative example is substantially the same manner as Example 1, the difference is that only, not
Use DNA excision enzyme.
The characterization of 2 biped DNA walker of experimental example
It is walked with the analysis of polyacrylamide gel electrophoresis (PAGE) method by the biped DNA that object is catalyzed hair clip assembling synthesis
Row device.
The DNA chain prepared is instilled in freshly prepd polyacrylamide gel (16%) groove, it is then slow in 1 × TBE
In fliud flushing, current potential 120V carries out electrophoresis 120min.After ethidium bromide (EB) dyeing, carried out with biogel imaging system
Shooting imaging.As shown in Fig. 2, the loss band in swimming lane 1 corresponds to miRNA-155 (1.0 μm of olL-1), because it has seldom
Nucleotide base and do not allow easy dyeing.Swimming lane 2 and swimming lane 3 are shown respectively corresponds to H2 (1.0 μm of olL-1) and H1 (1.0 μ
mol·L-1) different bands.Occur two independent band (1.0 μm of olL in swimming lane 4-1H2 and 1.0 μm of olL-1
H1), show that H2 and H1 stable in the solution can coexist.As expected, as addition miRNA-155 (1.0 μm of ol
L-1), H1 (1.0 μm of olL-1) and H2 (1.0 μm of olL-1) mixture when, at the top of swimming lane 5, there are apparent DNA is multiple
Close object band, it was demonstrated that hair clip assembling process is catalyzed by object, successfully produces biped DNA walker.
The electrochemical Characterization and ECL characterization of 3 biosensor of experimental example
Electrochemical Characterization
In 5.0mmolL-1[Fe (CN)6]3-/4-Middle measurement cyclic voltammogram (CVs) is prepared in embodiment 1 with characterizing
The gradually building process of biosensor.
As shown in Figure 3A, the CVs of bare electrode shows reversible redox peaks (curve a).PFBT-COOH point modification electricity
(curve b), this is attributed to the modified membrane on electrode surface and hinders electron transmission for the redox peak current decline of pole.Due to
DNA chain hinders the characteristic of electron transmission, detects significantly reduced redox peaks after incubated dna S1 on the electrode of preparation
Electric current (curve c).After being closed with HT, redox peak current slightly rises (curve d).When introducing BHQ-S2, peak current is again
Once slightly (curve e) illustrates that BHQ-S2 is successfully introduced into for decline.Finally, when biped DNA walker and ExoIII are in electrode surface
After going up while being incubated, redox current obviously increases (curve f), this is because one side BHQ-S2 is by biped DNA walking
Device displacement is discharged from electrode surface, meanwhile, DNAS1 is sheared under the auxiliary of ExoIII, discharges DNA walker simultaneously certainly
Another BHQ-S2 is replaced by ground, DNA chain is finally made all to be detached from electrode.
ECL characterization
Fig. 3 B shows the electroluminescent chemiluminescence biosensor being prepared in embodiment 1 in PBS (0.10molL-1,
PH 7.4) in gradually building process ECL signal response curve.As shown in Figure 3B, in PBS, bare electrode is almost without ECL
Signal response (curve a), and (the ECL signal that curve b) shows the last one is corresponding, and response connects for PFBT-COOH point modified electrode
Nearly 18000a.u. shows that PFBT-COOH point has strong ECL luminous efficiency.Electrode is incubated for upper DNA S1 and further uses HT
(response of curve c), ECL signal slightly declines after closing.(the curve d), since BHQ is to PFBT-COOH point when introducing BHQ-S2
Efficient ECL quenching effect, ECL signal response significantly reduce.However, when DNA walker and Exo III are incubated for simultaneously to electricity
(curve e), BHQ-S2 are cemented out by biped DNA walker from electrode surface, so that the ECL signal being quenched is extensive when in pole surface
The multiple state to " on ".
ECL of the 4 ECL biosensor of experimental example for miRNA is detected
Response of the ECL biosensor to object miRNA-155 prepared by embodiment 1
Using miRNA-155 as object, ECL biosensor is to object miRNA-155 prepared by testing example 1
Response.Test results are shown in figure 4.
As can be seen from Figure 4A, anode ECL signal is with miRNA-155 concentration from 10amolL-1To 5pmolL-1Increasing
Add and increases.Signal-to-noise ratio (S/N) be 3 when, calculate detection be limited to 3.3amolL-1, wherein curve a is that no target is deposited
When ECL response.
Fig. 4 B shows the logarithm of measured ECL signal and miRNA-155 concentration in good positive correlation, linear side
Journey is I=45813.8+2573.6lgc, coefficient R2=0.9938.Herein, I represents ECL signal strength, and c represents miRNA-
155 concentration.
Response of the ECL biosensor to object miRNA-155 prepared by comparative example 1
Using miRNA-155 as object, biosensor prepared by test comparison example 1 is to object miRNA-155's
Response.Test results are shown in figure 5.
As shown in Figure 5A, in the absence of ExoIII, ECL intensity is as miRNA-155 concentration is from 0.5fmolL-1Increase
To 5pmolL-1When and be stepped up, signal-to-noise ratio (S/N) be 3 when detection be limited to 0.17fmolL-1.It is deposited with embodiment 1
It carries out two step amplifications (Fig. 4 A) in Exo III to compare, detection limit increases about 50 times.Fig. 5 C and Fig. 5 D are illustrated in same concentrations
In the presence of miRNA-155, exists and there is no the ECL signal strengths of ExoIII to compare.By result it can be found that not having in comparative example
There is the biosensor for carrying out secondary amplification using ExoIII, shows relatively low ECL signal.However, when using
When ExoIII carries out the amplification of two steps, biosensor obtains the ECL signal significantly increased, shows dual amplification plan in the present invention
Summary can assign sensor more preferably signal amplifying power.
The present invention in the prior art it has been reported that method compare
By it is provided by the invention detection object method in the prior art it has been reported that method compare, as a result
As shown in table 1.By result in table 1 it can be found that being shown in the detection of ECL biosensor miRNA provided by the invention
Higher sensitivity and lower detection limit, this is attributed to following reason: (1) PFBT-COOH point has brilliant luminous efficiency；
(2) Resonance energy transfer from PFBT-COOH excitation state to BHQ makes BHQ have the height to the ECL signal of PFBT-COOH point sudden
It goes out efficiency, background signal is low；(3) assembling of combining target object catalysis hair clip and biped DNA walker are put with realizing dual signal
Greatly.
The tactful comparing result of ECL biosensor and other detections miRNA in 1 present invention of table
| Detection method | The range of linearity | Detection limit | Document |
| Fluorescence | 1fmol·L-1~100nmolL-1 | 1fmol·L-1 | 1 |
| Electrochemistry | 5fmol·L-1~5pmolL-1 | 10fmol·L-1 | 2 |
| ECL | 1fmol·L-1~50pmolL-1 | 0.33fmol·L-1 | 3 |
| ECL | 100amol·L-1~1nmolL-1 | 29.5amol·L-1 | 4 |
| ECL | 10amol·L-1~5pmolL-1 | 3.3amol·L-1 | The present invention |
List of documents is as follows:
1.Yin,B.C.；Liu,Y.Q.；Ye,B.C.Sensitive Detection of MicroRNA in Complex
Biological Samples via Enzymatic Signal Amplification Using DNA Polymerase
Coupled with Nicking Endonuclease.Anal.Chem.2013,85,11487-11493.
2.Koo,K.M.；Carrascosa,L.G.；Shiddiky,M.J.A.；Trau,M.Poly(A)Extensions
of MiRNAs for Amplification-Free Electrochemical Detection on Screen-Printed
Gold Electrodes.Anal.Chem.2016,88,2000-2005.
3.Zhang,P.；Lin,Z.F.；Zhuo,Y.；Yuan,R.；Chai,Y.Q.Dual microRNAs-Fueled
DNA Nanogears:A Case of Regenerated Strategy for Multiple
Electrochemiluminescence Detection of MicroRNAs with Single
Luminophore.Anal.Chem.2017,89,1338-1345.
4.Liu,J.L.；Tang,Z.L.；Zhang,J.Q.；Chai,Y.Q.；Zhuo,Y.；Yuan,R.Morphology-
Controlled 9,10-Diphenylanthracene Nanoblocks as Electrochemiluminescence
Emitters for MicroRNA Detection with One-Step DNA Walker
Amplification.Anal.Chem.2018,90,5298-5305.
The selectivity and stability of 5 ECL biosensor of experimental example
The selectivity of ECL biosensor
Select common short single stranded RNA (including miRNA-21, miRNA-141, miRNA-126 and let-7a) as potential
Interfering substance study the selectivity of ECL biosensor provided by the invention.
As shown in Figure 6A, the biosensor proposed is being separately added into miRNA-21 (100fmolL-1)、miRNA-
141(100fmol·L-1)、miRNA-126(100fmol·L-1) and let-7a (100fmolL-1) when ECL signal strength
It is consistent with blank control.However, when in low concentration (1fmolL-1) under when testing miRNA-155, ECL signal strength is aobvious
Write enhancing.In addition, obtained ECL signal strength and concentration is after the mixture of miRNA-155 and interfering substance is added
1fmol·L-1The response of miRNA-155 is compared, without notable difference.The above results show that ECL biology provided by the present invention passes
Sensor being capable of specific detection miRNA-155.
The stability of ECL biosensor
It is 10fmolL in concentration-1MiRNA-155 in the presence of, the steady of biosensor is assessed by continuous scanning
It is qualitative.As shown in Figure 6B, 16 circle of scanning, ECL signal strength do not show apparent fluctuation under continuous cycle potentials, relatively
Standard deviation (RSD) is 0.04%, this shows that ECL biosensor provided by the invention has satisfactory stability.
The application of 6 ECL biosensor of experimental example
By studying miRNA-155 in the cell cracking of human cervix cancer cells (Hela) and breast cancer cell (MCF-7)
Expressing to assess the practicability of biosensor in liquid.
As shown in fig. 7, blank test shows very weak ECL signal transmitting.And as the cell number of Hela increases, ECL
Signal strength increases, and illustrates that miRNA-155 has certain expression in Hela cell.Meanwhile with MCF-7 concentration from 10 increase
To 104A cell, ECL signal strength also obviously increase, and show that miRNA-155 is overexpressed in MCF-7.The above results show
ECL biosensor provided by the invention provides a kind of new method for the detection of miRNA.
Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention., rather than its limitations；To the greatest extent
Present invention has been described in detail with reference to the aforementioned embodiments for pipe, but those skilled in the art should understand that: its
It is still possible to modify the technical solutions described in the foregoing embodiments, or to some or all of the technical features
It is equivalently replaced；And these are modified or replaceed, various embodiments of the present invention skill that it does not separate the essence of the corresponding technical solution
The range of art scheme.
SEQUENCE LISTING
<110>Southwest University, Shapingba District, Chongqing City the People's Hospital, Chongqing City No.9 People's Hospital
<120>modified electrode, combination product and its electroluminescent chemiluminescence biosensor and application
<160> 5
<170> PatentIn version 3.3
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Claims (10)
1. modified electrode, which is characterized in that including the illuminator of electrode and modification on the electrodes；
The illuminator be mainly carboxyl-functional it is poly- [(9,9- dioctyl fluorene -2,7- diyl) -co- (1,4- benzo -2,
1 ', 3 }-thiadiazoles)] point；
The electrode is selected from glass-carbon electrode, gold electrode；
Preferably, the electrode is selected from glass-carbon electrode.
2. combination product characterized by comprising
Modified electrode described in claim 1；DNA S1,DNA S2；DNA hair clip H1 and DNA hair clip H2；
The DNA S1 and DNA S2 can complementary pairing, coupling has quencher on the DNA S2；
The DNA hair clip H1 and DNA hair clip H2 assembling can form biped DNA walker under nucleic acid to be detected catalysis；It is described
The biped part of biped DNA walker can with the DNA S1 complementary pairing, and on the DNA S1 after the pairing reserve
The restriction enzyme site of DNA excision enzyme out；
The combination product does not include coreaction reagent.
3. combination product according to claim 2, which is characterized in that the combination product further includes the DNA excision enzyme；
The DNA excision enzyme is exonucleaseⅲ；
The quencher is selected from black hole quencher, ferrocene；
Preferably, the quencher is black hole quencher.
4. the method for detecting nucleic acid, which is characterized in that including using combination product described in Claims 2 or 33 to core to be detected
Acid is detected.
5. according to the method described in claim 4, it is characterised in that it includes the assembling side of electroluminescent chemiluminescence biosensor
Method:
A) using the modified electrode as matrix assembled dna S1, the DNA S2 with the DNA S1 complementary pairing is introduced；
B) under the nucleic acid catalysis to be detected, DNA hair clip H1 and H2 assembling generate biped DNA walker；
C) the biped DNA walker and the DNA excision enzyme are incubated for altogether with the electrode obtained in step a)；
Wherein, step a) and b) without sequencing；
Preferably, DNA S1 described in step a) is in the presence of crosslinking agent in the modified electrode over-assemble；The crosslinking agent is
EDC and NHS.
6. according to the method described in claim 5, it is characterized in that, the preparation method of the modified electrode includes:
The dispersant liquid drop of the illuminator is applied to the surface of the electrode, drying and forming-film obtains the modified electrode；
Preferably, the decentralized medium of the dispersion liquid is selected from secondary distilled water or ultrapure water；Illuminator described in the dispersion liquid
Concentration be 0.2mgmL-1~0.4mgmL-1。
7. according to the method described in claim 5, it is characterized in that, the preparation process of the biped DNA walker includes:
Object, DNA hair clip H1 and DNA hair clip H2 are mixed to get mixture, and mixture is incubated at 25 DEG C~65 DEG C
It 60~120 minutes, is incubated for 90 minutes preferably at 37 DEG C, obtains biped DNA walker；
Preferably, the incubation time of the biped DNA walker and the DNA excision enzyme on the electrodes is 1~3 hour,
Preferably 2 hours.
8. according to the described in any item methods of claim 4 to 7, which is characterized in that the nucleic acid is RNA, preferably miRNAs.
9. according to the method described in claim 8, it is characterized in that, the nucleic acid is miRNAs-155；
Preferably:
The sequence of the DNA S1 is as shown in SEQ ID NO:1；
The sequence of the DNA S2 is as shown in SEQ ID NO:2；
The miRNAs-155, sequence is as shown in SEQ ID NO:3；
The sequence of the H1 is as shown in SEQ ID NO:4, and the sequence of the H2 is as shown in SEQ ID NO:5.
10. electroluminescent chemiluminescence biosensor is assembled by the assemble method that any one of claim 5,7,8,9 defines
It obtains.
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