CN109706225A - Electrochemical detection method of the palladium nano-particles based on rolling circle amplification mediation to microRNA - Google Patents
Electrochemical detection method of the palladium nano-particles based on rolling circle amplification mediation to microRNA Download PDFInfo
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Abstract
Present disclose provides the palladium nano-particles mediated based on rolling circle amplification to the electrochemical detection method of microRNA, by miRNA to be measured and after carrying out DNA probe specific binding, the primer sequence A2 and circular template DNA of DNA probe exposure carry out rolling circle amplification, it is single-stranded to obtain the DNA rich in guanine, it is single-stranded for template with the DNA rich in guanine, palladium salt is added and reducing agent carries out reduction reaction, then carries out Electrochemical Detection.Disclosed method is used for the detection of miRNA-21, and the range of linearity is 50aM~100fM, and the ultralow detection with 8.6aM limits, while having excellent selectivity.
Description
Technical field
The disclosure belongs to technical field of electrochemical detection, is related to the palladium nano-particles pair mediated based on rolling circle amplification
The electrochemical detection method of microRNA.
Background technique
Here statement only provides background information related with the disclosure, without necessarily constituting the prior art.
MicroRNAs (miRNAs) is as a kind of promising Non-Invasive disease early diagnosis and prognosis (especially cancer
Disease) biomarker, provide important information to understand the generation, transfer and further biomedical applications of cancer.
The analysis of miRNA is of great significance.However, according to known to the disclosed invention people, due to miRNA have it is small in size, in biological sample
The accurate detection of the intrinsic characteristics such as expression is low in product, family member's sequence is similar, miRNA is still challenging.In order to
Meet the needs of miRNA analysis, the highly sensitive and selective detection method of exploitation is imperative.
In order to realize high detection sensitivity, rolling circle amplification (rolling circle amplification, RCA) is because of its speed
Degree is fast, reaction condition is mild, amplification efficiency is high, biology point is introduced into the ability that target identification is combined with amplification of signal
Analysis field.In linear RCA reaction, a short primer is extended to the long single stranded DNA with a large amount of tandem repetitive sequences
(ssDNA), these repetitive sequences are complementary with circular shuttering.Due to being difficult to directly acquire the electrochemical signals of DNA base, by these
The effective signal reading method that duplicate DNA sequence dna is converted into detectable electric signal is most important to biosensor.It reads
Taking the most common method of RCA product is that RCA product and the short chain DNA of marking signal indicator (nano particle, ferrocene, enzyme) are miscellaneous
Interaction is mended.However, the process redundancy of this marking signal indicator is time-consuming, inconvenient for operation according to known to the disclosed invention people.
Summary of the invention
In order to solve the deficiencies in the prior art, purpose of this disclosure is to provide the palladium nano-particles mediated based on rolling circle amplification
To the electrochemical detection method of microRNA, this method target miRNA induces RCA reaction to generate the long ssDNA for being largely rich in G, and
Carry out fabricated in situ palladium nano-particles as amplified signal indicator, for detecting miRNA spirit with higher as effective template
Sensitivity and selectivity.
To achieve the goals above, the technical solution of the present invention is as follows:
On the one hand, primer of a kind of palladium nano-particles mediated based on rolling circle amplification to the Electrochemical Detection of microRNA
Group, including DNA probe and template DNA,
The DNA probe be stem-loop structure DNA, the DNA probe contain the sequence A1 that can hybridize with miRNA and
The primer sequence A2 of rolling circle amplification reaction can be caused, primer sequence A2 to form stem with a part of complementary be coordinated of sequence A1；
The template DNA is the single stranded DNA that 5 ' ends and 3 ' ends can be connected into cyclic structure by DNA ligase, mould
Hold a part of sequence after connecting into cyclic structure can be complementary with the primer sequence A2 of DNA probe in the 5 ' ends of plate DNA and 3 '；
After the primer sequence A2 of DNA probe coordination complementary with cricoid template DNA is formed, it can cause and polymerize richness
DNA containing guanine is single-stranded.
DNA probe can be specifically bound with target miRNA in the disclosure, and the stem-loop knot of DNA probe can be made in conjunction with after
Structure is opened, to expose the primer sequence A2 that can be realized rolling circle amplification, by being rich in guanine, energy in the product of rolling circle amplification
The synthesis of the palladium nano-particles of specific modality is effectively facilitated, enough so as to using palladium nano-particles as the amplification of Electrochemical Detection
Signal designation agent.
On the other hand, electrification of a kind of palladium nano-particles mediated based on rolling circle amplification to the Electrochemical Detection of microRNA
Biosensors, including above-mentioned primer sets and gold electrode, DNA probe are connect by Au-S key with gold electrode, Au-S key mapping in
One end of DNA probe far from primer sequence A2.
The disclosure is enough by DNA probe and the more firm connection of gold electrode by Au-S bond energy, and improves the biography of sensor
Perceptual energy.Meanwhile Au-S key being located remotely to one end of the DNA probe of primer sequence A2, so that miRNA is special with DNA probe
Property combine after, the primer sequence A2 in DNA probe can be discharged, to realize the purpose of rolling circle amplification.
A kind of third aspect, preparation method of above-mentioned electrochemica biological sensor, is put into DNA probe solution for gold electrode
In, it is incubated for, the sulfydryl of DNA probe one end far from primer sequence A2 and gold electrode is made to carry out complexation reaction.
Fourth aspect, a method of palladium nano-particles are prepared based on rolling circle amplification mediation, utilize above-mentioned primer sets or electricity
Chemical biosensor, by miRNA to be measured and after carrying out DNA probe specific binding, the primer sequence A2 of DNA probe exposure
Rolling circle amplification is carried out with circular template DNA, DNA of the acquisition rich in guanine is single-stranded, is so that the DNA rich in guanine is single-stranded
Template, is added palladium salt and reducing agent carries out reduction reaction, obtains palladium nano-particles.
The form of the palladium nano-particles of disclosure preparation is related with miRNA to be measured, can be as Electrochemical Detection miRNA's
Amplified signal indicator.
5th aspect, the palladium nano-particles based on rolling circle amplification mediation utilize the electrochemical detection method of microRNA
Above-mentioned electrochemica biological sensor, by miRNA to be measured and after carrying out DNA probe specific binding, the primer of DNA probe exposure
Sequence A2 and circular template DNA carries out rolling circle amplification, and DNA of the acquisition rich in guanine is single-stranded, with the DNA rich in guanine
Single-stranded is template, and palladium salt is added and reducing agent carries out reduction reaction, then carries out Electrochemical Detection.
The method of the Electrochemical Detection microRNA of the disclosure is not necessarily to marking signal indicator, and has good selectivity
With higher sensitivity, detection is limited down to 8.6aM.
The disclosure has the beneficial effect that
Present disclose provides a kind of label-free electrochemistry of hypersensitive of the palladium nano-particles of fabricated in situ mediated based on RCA
Detect the sensor and method of miRNA.It is had good selectivity using the method that the sensor carries out Electrochemical Detection miRNA
With higher sensitivity, detection is limited down to 8.6aM.
Detailed description of the invention
The Figure of description for constituting a part of this disclosure is used to provide further understanding of the disclosure, and the disclosure is shown
Meaning property embodiment and its explanation do not constitute the improper restriction to the disclosure for explaining the disclosure.
Fig. 1 is schematic illustration of the palladium nano-particles based on RCA mediation in embodiment to miRNA-21 Electrochemical Detection；
Fig. 2 is the gel electrophoresis phenogram in embodiment, and A is polyacrylamide gel electrophoresis (PAGE), and 1 is HP, and 2 are
MiRNA-21,3 is that HP+miRNA-21,4 is CT, and 5 be HP+miRNA-21+CT+T4 ligase, and 6 be HP+CT+T4 ligase, M
It is agarose electrophoresis for marker, B, 1 RCA induced for object, 2 be the control experiment without miRNA-21, M DNA
marker；
Fig. 3 is the electron micrograph for the palladium nano-particles that the RCA product prepared in embodiment and RCA are mediated, and A is
The high resolution transmission electronmicroscopy photo of RCA product, B are that the high-resolution for the palladium nano-particles that RCA is mediated transmits electron microscopic
Mirror photo, C are the selection area for the palladium nano-particles energy disperse spectroscopy that RCA is mediated, and D is the energy spectrogram of O, and E is the energy spectrogram of P, F
For the energy spectrogram of Pd；
Fig. 4 is the biosensor prepared in embodiment in 0.1M HClO4The characterization curve of middle electrochemical behavior, A are
Electrochemical impedance spectrogram (EIS), a are naked AuE, and b HP/AuE, c miRNA-21/HP/AuE, d are RCA product/
MiRNA-21/HP/AuE contains 5.0mM [Fe (CN) in 0.1M KCl6]3-/4-, illustration is the equivalent circuit of Nyquist plots,
B is cyclic voltammogram, and a is naked AuE, and b HP/AuE, c are RCA product/miRNA-21/HP/AuE, d PdNPs/RCA
Product/miRNA-21/HP/AuE, for scanning range from -0.2 to 1.6V, sweep speed 50mV/s, C are various concentration
The DPV of miRNA-21 schemes, a 0fM, b 1fM, c 100fM；
Fig. 5 is in embodiment under 100pM miRNA-21, different parameters to the characterization curve of biosensor analysis performance,
A is HP concentration, and B is CT concentration, and C is the RCA reaction time；
Fig. 6 is the characterization curve of biosensor prepared in embodiment, A be various concentration miRNA-21 (0,0.01,
0.05,0.1,0.5,1,5,10,50,100 and 500fM) under DPV signal, B be corresponding calibration curve；
Fig. 7 is that the selectivity of the biosensor prepared in embodiment characterizes histogram, and a sRNA, b tRNA, c are
MiRNA-141, d let-7d, e miRNA-21.
Specific embodiment
It is noted that described further below be all exemplary, it is intended to provide further instruction to the disclosure.Unless another
It indicates, all technical and scientific terms used herein has usual with disclosure person of an ordinary skill in the technical field
The identical meanings of understanding.
It should be noted that term used herein above is merely to describe specific embodiment, and be not intended to restricted root
According to the illustrative embodiments of the disclosure.As used herein, unless the context clearly indicates otherwise, otherwise singular
Also it is intended to include plural form, additionally, it should be understood that, when in the present specification using term "comprising" and/or " packet
Include " when, indicate existing characteristics, step, operation, device, component and/or their combination.
Palladium salt described in the disclosure is compound containing palladium.
Refer to the sequence containing several duplicate GGGTTGGGCGGGATGGG rich in guanine described in the disclosure.
Highly sensitive, selectivity and marking signal indicator bring mistake can not be met simultaneously in view of existing detection miRNA
The deficiency of journey redundancy consumption, in order to solve technical problem as above, the present disclosure proposes the palladium nanometers mediated based on rolling circle amplification
Electrochemical detection method of the grain to microRNA.
A kind of exemplary embodiment of the disclosure provides a kind of palladium nano-particles pair mediated based on rolling circle amplification
The primer sets of the Electrochemical Detection of microRNA, including DNA probe and template DNA,
The DNA probe be stem-loop structure DNA, the DNA probe contain the sequence A1 that can hybridize with miRNA and
The primer sequence A2 of rolling circle amplification reaction can be caused, primer sequence A2 to form stem with a part of complementary be coordinated of sequence A1；
The template DNA is the single stranded DNA that 5 ' ends and 3 ' ends can be connected into cyclic structure by DNA ligase, mould
Hold a part of sequence after connecting into cyclic structure can be complementary with the primer sequence A2 of DNA probe in the 5 ' ends of plate DNA and 3 '；
After the primer sequence A2 of DNA probe coordination complementary with cricoid template DNA is formed, it can cause and polymerize richness
DNA containing guanine is single-stranded.
DNA probe can be specifically bound with target miRNA in the disclosure, and the stem-loop knot of DNA probe can be made in conjunction with after
Structure is opened, to expose the primer sequence A2 that can be realized rolling circle amplification, by being rich in guanine, energy in the product of rolling circle amplification
The synthesis of the palladium nano-particles of specific modality is effectively facilitated, enough so as to using palladium nano-particles as the amplification of Electrochemical Detection
Signal designation agent.
In one or more embodiments of the embodiment, one end of the DNA probe far from primer sequence A2 is modified with mercapto
Base.It is convenient to be connect with noble metal electrode.
In one or more embodiments of the embodiment, one end of template DNA is modified with phosphate group.It is easier to make mould
Plate DNA forms ring-type.
In order to be specifically bound with miRNA-21, in one or more embodiments of the embodiment, DNA is visited
Needle is TTT TTT TTT CAA CAT CAG TCT GAT AAG CTA ACT GAT GTT GAT by the sequence at 5 ' ends and 3 ' ends
ATT；
Template DNA is AAC ATC AAA AAC CCA ACC CGC CCT ACC CAA by the sequence at 5 ' ends and 3 ' ends
AAA ATG TAA TAT C。
The another embodiment of the disclosure provides a kind of palladium nano-particles pair mediated based on rolling circle amplification
The electrochemica biological sensor of the Electrochemical Detection of microRNA, including above-mentioned primer sets and gold electrode, DNA probe pass through Au-S
Key is connect with gold electrode, and Au-S key is located remotely from one end of the DNA probe of primer sequence A2.
The disclosure is enough by DNA probe and the more firm connection of gold electrode by Au-S bond energy, and improves the biography of sensor
Perceptual energy.Meanwhile Au-S key being located remotely to one end of the DNA probe of primer sequence A2, so that miRNA is special with DNA probe
Property combine after, the primer sequence A2 in DNA probe can be discharged, to realize the purpose of rolling circle amplification.
In one or more embodiments of the embodiment, the Au-S key mapping is in 5 ' ends of DNA probe.
The third of the disclosure, will be golden embodiment there is provided a kind of preparation method of above-mentioned electrochemica biological sensor
Electrode is put into DNA probe solution, is incubated for, make far from primer sequence A2 DNA probe one end sulfydryl and gold electrode into
Row complexation reaction.
In one or more embodiments of the embodiment, incubation time is 10~14h.
In order to optimize the orientation of DNA probe and reduce non-specific adsorption, one or more embodiments of the embodiment
In, after incubation, closed using 6- sulfydryl -1- hexanol.Off-period is 0.5~1.5h.
In order to modify DNA probe preferably in the surface of gold electrode, one or more embodiments of the embodiment,
Preprocess method to gold electrode is first to be polished using polishing agent to gold electrode, then rush to the gold electrode after polishing
It washes, electrochemical cleaning is then carried out in sulfuric acid solution, finally cleaned using ultrapure water.The ultrapure water, also known as UP water,
Reach the water of 18M Ω * cm (25 DEG C) for resistivity.
In the series embodiment, in electrochemical cleaning, electric potential scanning range is steady until obtaining between -0.2V~1.6V
Fixed cyclic voltammogram.
Embodiment there is provided a kind of sides that palladium nano-particles are prepared based on rolling circle amplification mediation for the 4th kind of the disclosure
Method, using above-mentioned primer sets or electrochemica biological sensor, by miRNA to be measured and after carrying out DNA probe specific binding,
The primer sequence A2 and circular template DNA of DNA probe exposure carry out rolling circle amplification, and DNA of the acquisition rich in guanine is single-stranded, with institute
It is single-stranded for template, addition palladium salt and reducing agent progress reduction reaction, acquisition palladium nano-particles to state the DNA rich in guanine.
The form of the palladium nano-particles of disclosure preparation is related with miRNA to be measured, can be as Electrochemical Detection miRNA's
Amplified signal indicator.
In one or more embodiments of the embodiment, miRNA solution to be measured is added dropwise to DNA probe or connection DNA
It on the gold electrode of probe, is incubated for, then is added dropwise anti-containing DNA profiling, DNA ligase and DNA ligase under human body temperature
The solution of liquid is answered, continuation is incubated under human body temperature, is then added dropwise and is contained archaeal dna polymerase and dNTPs, is maintained in human body
At a temperature of be incubated for, obtain rich in guanine DNA it is single-stranded, will be enriched in guanine DNA it is single-stranded be placed in palladium salt solution,
It heats reducing agent and carries out reduction reaction.Human body temperature refers to the temperature of human normal, generally 36.2~37.2 DEG C in the disclosure.
In the series embodiment, the palladium salt is time chlorine palladium acid sodium, and the reducing agent is sodium borohydride.
Embodiment there is provided the palladium nano-particles mediated based on rolling circle amplification to microRNA's for the 5th kind of the disclosure
Electrochemical detection method by miRNA to be measured and carries out DNA probe specific binding using above-mentioned electrochemica biological sensor
Afterwards, the primer sequence A2 and circular template DNA of DNA probe exposure carry out rolling circle amplification, and DNA of the acquisition rich in guanine is single-stranded,
It is single-stranded for template with the DNA rich in guanine, palladium salt is added and reducing agent carries out reduction reaction, then carries out electrochemistry inspection
It surveys.
The method of the Electrochemical Detection microRNA of the disclosure is not necessarily to marking signal indicator, and has good selectivity
With higher sensitivity, detection is limited down to 8.6aM.
In one or more embodiments of the embodiment, miRNA solution to be measured is added dropwise to DNA probe or connection DNA
It on the gold electrode of probe, is incubated for, then is added dropwise anti-containing DNA profiling, DNA ligase and DNA ligase under human body temperature
The solution of liquid is answered, continuation is incubated under human body temperature, is then added dropwise and is contained archaeal dna polymerase and dNTPs, is maintained in human body
At a temperature of be incubated for, obtain rich in guanine DNA it is single-stranded, will be enriched in guanine DNA it is single-stranded be placed in palladium salt solution,
It heats reducing agent and carries out reduction reaction, finally carry out Electrochemical Detection.
In the series embodiment, the palladium salt is time chlorine palladium acid sodium, and the reducing agent is sodium borohydride.
In one or more embodiments of the embodiment, the Electrochemical Detection is differential pulse voltammetry.
In the series embodiment, the solution that differential pulse voltammetry uses is HClO4Solution.
In the series embodiment, it is 0.8V~1.3V that the condition of differential pulse voltammetry, which is scanning range, amplitude is 50 ±
5mV, pulse width are 50 ± 5ms, and the pulse period is 0.5 ± 0.01s.
In order to enable those skilled in the art can clearly understand the technical solution of the disclosure, below with reference to tool
The technical solution of the disclosure is described in detail in the embodiment of body.
Embodiment
Reagent and material
The DNA and miRNA (shown in table 1) of high-efficient liquid phase chromatogram purification are purchased from the raw limited public affairs of work biotechnology of Chinese Shanghai
Department.Phi29DNA polymerase, T4DNA ligase, pyrocarbonic acid diethyl ester (DEPC), trishydroxymethylaminomethane-hydrochloric acid (Tris-
HCl), ethylenediamine tetra-acetic acid (EDTA), 4- (2- ethoxy) piperazine -1- ethanesulfonic acid sodium (HEPES) and deoxyribonucleotide three
The solution of phosphoric acid (dNTPs) mixture is purchased from Chinese Shanghai Sheng Gong Biotechnology Co., Ltd.Sodium borohydride (NaBH4), iron cyanogen
Change potassium (K3Fe(CN)6) and potassium ferrocyanide (K4Fe(CN)6) buy from traditional Chinese medicines and control interest chemical reagent Co., Ltd.Secondary chlorine palladium acid sodium
(Na2PdCl4), 6- sulfydryl -1- hexanol (MCH) and trishydroxymethylaminomethane (2- carboxyethyl) liquid hydrogen phosphide hydrochloric acid (TCEP) purchase
From Chinese Shanghai Aladdin.TE buffer (10mM Tris-HCl, 100mM NaCl, 1.0mM EDTA, pH 8.0) is for diluting
DNA.Tris buffer (40mM Tris-HCl, 10mM MgCl2, pH 7.8) and for diluting circular template.Other reagents are point
Grade is analysed, without being further purified.Consumptive material is all made of 0.1%DEPC processing, high pressure steam sterilization.It is ultrapure used in all solution
Water is obtained from excellent general water purification system (resistivity=18.25M Ω cm).
Nucleic acid sequence information used in 1 the present embodiment of table
Note: HS indicates that sulfydryl, p indicate phosphate group, and inclination indicates that sequence A1, underscore indicate primer sequence A2, thick word
Body is base mismatch.
Instrument
Electrochemical measurement is carried out in CHI760E electrochemical workstation, using traditional three-electrode system, including metal working
Make electrode (AuE, Φ=3mm), silver/silver chlorate (saturation potassium chloride) reference electrode and platinum filament are to electrode.High-resolution transmission electricity
The formation of mirror (HRTEM, JEM-2200FS) observation palladium nano-particles.Native polyacrylamide is carried out using DYCZ-24F electrophoresis apparatus
Gel electrophoresis (PAGE), PAGE glue are imaged on Bio-Rad Gel Doc XR+System (Bio-Rad).Ago-Gel electricity
Swimming carries out in DYCP-31DN electrophoresis apparatus, and Ago-Gel is imaged using Tanon-2500R gel imaging system.
The modification of metal working electrode
Before being modified, gold electrode is polished with aluminium oxide (50nm), then uses ultrapure water, ethyl alcohol and ultrapure water
It successively rinses, removes the impurity such as alumina powder.By polished electrode 0.5M H2SO4Middle electrochemical cleaning, electric potential scanning model
It is trapped among between -0.2V~1.6V, until obtaining stable cyclic voltammogram.Then gold electrode is cleaned with ultrapure water, and uses nitrogen
Drying.The gold electrode pre-processed uses 0.1 μM of hairpin probe (HP) to be incubated for 12h immediately.In order to optimize the orientation and reduction of HP
Non-specific adsorption closes 1h with 1mM MCH, completes the production of HP/AuE sensor.
The identification of miRNA-21 and RCA reaction
The miRNA-21 of 5 μ L various concentrations is dripped on the HP/AuE sensor made, and 37 DEG C of incubation 1h are then washed
Free miRNA-21.RCA is reacted, 10 μ L include 0.25 μM of circular template (CT), and 0.2U T4 ligase and 1 × T4 connect
The solution for connecing enzyme reaction solution is added drop-wise on the sensor for having hybridized miRNA-21,37 DEG C of incubation 1h.Then, RCA reaction is by adding
10 μ L RCA buffers (1mM dNTPs and 0.2U phi29DNA polymerase) is added to cause, 37 DEG C of incubation 1h.Obtained RCA product
Template as fabricated in situ palladium nano-particles.
The synthesis and Electrochemical Detection of palladium nano-particles
After the identification of miRNA-21 and RCA reaction, the synthesis of palladium nano-particles is that gold electrode is immersed 10mM
HEPES (150mM KCl, pH 7.5) includes 150 μM of Na2PdCl4Solution in 30min, then with 300 μM of NaBH4Reduction
30min.Reaction is carried out in room temperature and in the dark.In order to measure electrochemical signals, in 0.1M HClO4In carried out difference arteries and veins
It rushes volt-ampere (DPV), scanning range is 0.8V~1.3V, amplitude 50mV, pulse width 50ms, pulse period 0.5s.
Gel electrophoresis
PAGE is used to verify HP and the CT hybridization of target induction.PAGE 1 × tbe buffer liquid (90mm Tris, 90mm boric acid,
2mm EDTA, pH 8.3) in carry out, constant current 30mA runs glue 1.5h at 10 DEG C.Obtained gel is dyed with SYBR gold
40min, and be imaged in Bio-Rad Gel Doc XR+ system.
Agarose gel electrophoresis demonstrates the RCA reaction of target induction.Sample is placed on 0.7% Ago-Gel, 1 ×
Electrophoresis is carried out in TAE buffer (40mm Tris, 20mm acetic acid, 2mm EDTA, pH 8.3), constant pressure 120V runs glue at room temperature
1h.After ethidium bromide (EB) dyes 15min, it is imaged using Tanon-2500R gel imaging system.
Results and discussion
The testing principle of the present embodiment
As shown in Figure 1, HP mainly includes two regions: (i) A1 hybridizes with miRNA-21；(ii) A2 is drawing for RCA reaction
Object.Firstly, HPs self-assembles to gold electrode surfaces by Au-S key.In the presence of miRNA-21, the stem-loop structure of HP due to
The hybridization of miRNA-21 is opened, thus the exposure part A2.When T4 ligase and CT are introduced into, the exposed region A2 and CT are miscellaneous
It hands over, and causes RCA reaction in the presence of phi29DNA polymerase and dNTPs.Therefore, RCA reaction generate it is several containing
(GGGTTGGGCGGGATGGG) the long ssDNAs of tandem sequence is repeated, these richness G long ssDNAs carry out fabricated in situ palladium as template
Nano particle exports electrochemical signals.On the contrary, in the absence of miRNA-21, HP keeps stem-loop structure, the region A2 it is general hidden
Ensconce the stem of HP.Since the solution temperature between the 3 ' tail portions of CT and HP is less than 15 DEG C, RCA reaction and subsequent series reaction
It will not occur.Therefore, strategy is expanded based on the palladium nano-particles that this RCA is mediated, the sensitivity to miRNA-21 may be implemented
Property and selective enumeration method.
Gel electrophoresis characterization
Above-mentioned principle is verified using gel electrophoresis.As shown in Figure 2 A, swimming lane 1,2 and 4 respectively represented HP,
MiRNA-21 and CT；Swimming lane 3 shows that a bp value is similar to the band of the sum of band in swimming lane 1 and swimming lane 2, shows that HP successfully knows
Not miRNA-21.When CT and T4 ligase is added, swimming lane 5 occur mobility be lower than swimming lane 3 band, show CT and
The successful cross in the region A2 that HP is completely exposed.As control, HP is directly and CT and T4 ligase is not when having miRNA-21
Mixing.It can be observed that corresponding band in swimming lane 1 and 4 in swimming lane 6, it was demonstrated that HP in no miRNA-21 cannot and CT
Hybridization.
Agarose gel electrophoresis confirms the feasibility of target induction RCA reaction, as shown in Figure 2 B.The present embodiment design
RCA reaction is carried out in the presence of miRNA-21, obtained product injection swimming lane 1.As a result occur one in swimming lane 1
The extremely low bright band of a mobility.According to the instruction of DNA marker (lane M), base contained by product is much larger than in swimming lane 1
14kbp shows the successful progress of the RCA reaction of targeted induction.On the contrary, swimming lane 2 does not have in the case where no miRNA-21
Band illustrates that no miRNA-21 cannot trigger RCA reaction.These results are consistent with the result that PAGE is obtained, and show that RCA is anti-
It should can only be carried out in the presence of target miRNA-21.
The characterization for the palladium nano-particles that RCA is mediated
Using the formation of the palladium nano-particles on high resolution transmission electronmicroscopy characterization RCA product.As shown in figure 3, right
In RCA product, it can be observed that about 2.5 μm of coils of a diameter, as shown in Figure 3A, this coil may be in non-stretched and disk
Around DNA chain around the buffer salt assembled, picture contrast of the RCA product under high electron irradiation can be considerably increased.By RCA
Product and Na2PdCl4And NaBH4After incubation, the nano particle that diameter is about 100nm is observed, as shown in Figure 3B, they are along figure
Similar coil shape flocks together in 3A.In order to further characterize these particles, in the selection area energy disperse spectroscopy of Fig. 3 C
(EDS) elemental analysis has been carried out.First vegetarian noodles, which is swept, to be demonstrated O, P and Pd element and coexists, respectively as shown in Fig. 3 D, 3E, 3F.O and P member
The phosphate backbone of RCA product is usually derived from derived from RCA product, especially P element, and the presence of Pd element confirms these
The property of nano particle.Importantly, the distribution of O, P and Pd are almost the same, illustrate palladium nano-particles only along RCA product shape
At.These features, which are demonstrated, successfully synthesizes palladium nano-particles using RCA product as effective template.
The electrochemical Characterization of biosensor
In order to prove the assembling process of biosensor, biosensor is being included into 5.0mM [Fe (CN)6]3-/4-'s
Electrochemical AC impedance (EIS) is carried out in 0.1M KCl, to characterize the different modifying process of AuE, as shown in Figure 4 A.Illustration is shown
Equivalent circuit is applied based on Zview software, by solution resistance (Rs), constant phase element (CPE), Warburg (diffusion)
Impedance (Zw) and electron transmission resistance (Ret) composition.There are a small semicircular area, (curve a) shows naked AuE to naked AuE
Surface charge transfer is very fast.After HP is incubated for and MCH is closed, the Ret significantly increased (537.5 Ω) is obtained, this is because
The upper negatively charged phosphoric acid backbone of HP and [Fe (CN)6]3-/4-The electrostatic repulsion of anion shows the successful fixation of HP on AuE
(curve b).After assembled HP/AuE sensor and miRNA-21 are incubated for, Ret significantly increases to 1398 Ω (curve c), table
HP on bright sensor successfully identifies miRNA-21.Finally, RCA of the sensor of miRNA-21 hybridization for target induction is anti-
It answers, obtains Ret (the curve d), this is because a large amount of long ssDNAs is greatly hindered that increased dramatically to 2197 Ω
[Fe(CN)6]3-/4-Diffusion to the surface AuE, it was demonstrated that the successful generation of RCA reaction.
Biosensor is in 0.1M HClO4The characterization of middle electrochemical behavior.As shown in Figure 4 B, naked AuE has yin in 0.87V
There are multiple anode peaks, these peaks (curve a) corresponding with the peak of gold in pole peak between 1.1V and 1.4V.When in AuE modification HP and
MCH, cathode peak are obviously reduced, and anode peak becomes the broad peak for being located at 1.4V, this is because HP and MCH cover electrode surface
Cover (curve b).In identification miRNA-21 and after RCA reaction occurs, covering due to RCA product to electrode surface, peak value subtracts
Small (curve c).Further, fabricated in situ palladium nano-particles on RCA product.There is a sun in 1.04V or so in curve d
Pole peak, and in reverse scan, a wider cathode peak occur from 0.8V, it was demonstrated that the formation of palladium nano-particles.In addition, by
Palladium nano-particles are covered in the surface AuE, the cathode peak of gold further decreases at 0.87V.Especially observed in 1.45V or so
One apparent anode peak, this is because RCA product G base is oxidized, this oxidation is due to the palladium nano-particles that are newly formed
Electrocatalysis and become obvious.These results further demonstrate the successful generation of RCA reaction and the formation of palladium nano-particles.
In addition, choosing palladium in the oxidation peak of 1.04V or so, miRNA-21 is analyzed with DPV.As shown in Figure 4 C, curve a is in nothing
There is one small background (curve a is at the peak of 1.04V) when miRNA-21, this background is PdIIWith nucleic acid base on the HP of self assembly
The synergistic effect of N atom cause.When 1fM miRNA-21, generated since a large amount of palladium nano-particles synthesize on RCA product
Peak point current (the curve b) significantly increased.Moreover, peak point current further increases when the concentration of miRNA-21 increases to 100fM
(curve c), because more palladium nano-particles synthesize on the RCA product that more objects induce.These results are clearly
Demonstrate feasibility of the biosensor for miRNA-21 analysis.
Optimal conditions
In order to obtain optimal biosensor analysis performance, it is real to have studied HP concentration, CT concentration, RCA incubation time etc.
Test the influence of parameter.Fig. 5 A illustrates HP concentration to whether there is or not the influences of the DPV of miRNA-21 response (Δ I).Obviously, Δ I is with HP
The increase of fixed concentration and be increased up 0.1 μM of HP and reach maximum value.At low concentration HP, the self assembly of electrode surface HP is close
Degree is higher, HP and miRNA-21 combination is more, and signal output is higher.However, when the self assembly density of HP is excessively high, due to steric hindrance
Effect will limit hybridizing for HP and miRNA-21.Therefore, the most suitable concentration of HP is 0.1 μM.CT concentration influences such as Fig. 5 B, Δ I
Increase with the increase of CT concentration, and tend to be steady in 0.25 μM of CT, shows HP and CT maximum combined efficiency.
In order to obtain maximum signal amplification effect, influence of the RCA reaction time to sensor performance is had studied, such as Fig. 5 C
It is shown.In the initial stage of RCA reaction, length dna product is synthesized, and Δ I is increased considerably.With the lengthening in reaction time, Δ I
It is lasting to increase, reach maximum value in 60min.
Under most suitable experiment condition, examined using miRNA-21 of the biosensor developed to various concentration
It surveys.As shown in Figure 6A, Δ I is gradually increased from 0fM to 500fM with the concentration of miRNA-21.Moreover, curent change and miRNA-
The logarithm of 21 concentration is in a linear relationship between 50aM-100fM, as shown in Figure 6B.Obtained linear gauging equation is Δ I (μ
A)=1.7164log (CmiRNA-21(fM))+4.2791, related coefficient 0.993.Detection can be calculated and be limited to 8.6aM
MiRNA-21 (according to 3 σ criterion), this detection limit the detection limit of the electrochemical detection method of existing miRNA.
Selectivity, reproducibility and stability
Same concentrations (10fM) different types of miRNAs, including miRNA-21, single base are detected using biosensor
The miRNA-21 (sRNA) of mispairing, miRNA-21 (tRNA), miRNA-141 and the let-7d of three base mispairings select to verify
Property.As shown in fig. 7, sensor is smaller to the response of sRNA compared with miRNA-21, and to tRNA, miRNA-141 and let-7d
Response almost can be ignored.These are the result shows that the biosensor developed has very well the detection of miRNA-21
Selectivity.
In order to assess the reproducibility of biosensor, detected using 4 HP/AuE sensors prepared under the same conditions
10fM miRNA-21.The result shows that relative standard deviation (RSD) is 5.2%, show that biosensor has acceptable electricity
Interpolar reproducibility.
In order to study the stability of biosensor, the HP/AuE sensor of preparation is placed 4 in 4 DEG C of refrigerator respectively
It, 1 day, 3 days, 5 days, 7 days and 15 days, for detecting miRNA-21.Compared with freshly prepd sensor, response is stablized.I.e.
After Shi Baocun 15 days, sensor is still able to maintain 88.1% original response, illustrates that biosensor is with good stability.
Actual sample detection
In order to research institute exploitation biosensor actual sample analysis in practical applicability, by various concentration
MiRNA-21, which is added in the Healthy Human Serum for dilute 10 times, has carried out standard sample-adding recovery experiment.As shown in table 2, RSDs <
7.33%, the rate of recovery 92%~110% illustrates that the biosensor of the present embodiment can be used as super sensitivity detection in clinical analysis
The tool of miRNA-21.
The measurement (n=3) of miRNA-21 in the Healthy Human Serum of 10 times of the dilution of table 2..
To sum up, present disclose provides a kind of hypersensitive of the palladium nano-particles of fabricated in situ mediated based on RCA is label-free
The sensor of electrochemistry miRNA detection.The biosensor to miRNA-21 have good selectivity with higher sensitivity,
It detects limit down to 8.6aM.It can obtain these satisfactory results and be attributed to following two reason.One is target inductions
RCA, specific target identification and signal are amplified phase by generating carrier of the ssDNA of a large amount of richness G as signal designation agent by it
In conjunction with.In addition, the formation that these ssDNA rich in G power on active palladium nano-particles realizes cascade signal amplification.The sensing
Device sensitivity with higher and selectivity have stronger actual sample analysis ability, have in disease early diagnosis wide
Wealthy application prospect.
The foregoing is merely preferred embodiment of the present disclosure, are not limited to the disclosure, for the skill of this field
For art personnel, the disclosure can have various modifications and variations.It is all within the spirit and principle of the disclosure, it is made any to repair
Change, equivalent replacement, improvement etc., should be included within the protection scope of the disclosure.
SEQUENCE LISTING
<110>Shandong University
<120>palladium nano-particles mediated based on rolling circle amplification are to the electrochemical detection method of microRNA
<130>
<160> 7
<170> PatentIn version 3.3
<210> 1
<211> 50
<212> DNA
<213>primer
<400> 1
tttttttttc aacatcagtc tgataagcta actgatgttg atattacatt 50
<210> 2
<211> 22
<212> RNA
<213> miRNA-21
<400> 2
uagcuuauca gacugauguu ga 22
<210> 3
<211> 43
<212> DNA
<213>primer
<400> 3
aacatcaaaa acccaacccg ccctacccaa aaaatgtaat atc 43
<210> 4
<211> 22
<212> RNA
<213>primer
<400> 4
uagcuuaucg gacugauguu ga 22
<210> 5
<211> 22
<212> RNA
<213>primer
<400> 5
uugcuuaucg gacugaucuu ga 22
<210> 6
<211> 22
<212> RNA
<213> miRNA-141
<400> 6
uaacacuguc ugguaaagau gg 22
<210> 7
<211> 22
<212> RNA
<213> let-7d
<400> 7
agagguagua gguugcauag uu 22
Claims (10)
1. a kind of primer sets of palladium nano-particles mediated based on rolling circle amplification to the Electrochemical Detection of microRNA, feature
It is, including DNA probe and template DNA,
The DNA probe is the DNA of stem-loop structure, and the DNA probe contains the sequence A1 that can hybridize with miRNA and can
Cause the primer sequence A2 of rolling circle amplification reaction, primer sequence A2 to form stem with a part of complementary be coordinated of sequence A1；
The template DNA is the single stranded DNA that 5 ' ends and 3 ' ends can be connected into cyclic structure by DNA ligase, template DNA
5 ' ends and 3 ' hold connect into a part of sequence after cyclic structure can be complementary with the primer sequence A2 of DNA probe；
After the primer sequence A2 of DNA probe coordination complementary with cricoid template DNA is formed, it can cause and polymerize rich in bird
The DNA of purine is single-stranded.
2. primer sets as described in claim 1, characterized in that one end of the DNA probe far from primer sequence A2 is modified with mercapto
Base；
Or, one end of template DNA is modified with phosphate group；
Or, DNA probe is TTT TTT TTT CAA CAT CAG TCT GAT AAG CTA ACT by the sequence at 5 ' ends and 3 ' ends
GAT GTT GAT ATT；
Template DNA is AAC ATC AAA AAC CCA ACC CGC CCT ACC CAA AAA by the sequence at 5 ' ends and 3 ' ends
ATG TAA TAT C。
3. a kind of palladium nano-particles mediated based on rolling circle amplification are to the electrochemical biosensor of the Electrochemical Detection of microRNA
Device, characterized in that including primer sets of any of claims 1 or 2 and gold electrode, DNA probe is connected by Au-S key and gold electrode
It connects, Au-S key is located remotely from one end of the DNA probe of primer sequence A2；
Preferably, the Au-S key mapping is in 5 ' ends of DNA probe.
4. a kind of preparation method of electrochemica biological sensor as claimed in claim 3, characterized in that gold electrode is put into DNA
It in probe solution, is incubated for, the sulfydryl of DNA probe one end far from primer sequence A2 and gold electrode is made to carry out complexation reaction.
5. preparation method as claimed in claim 4, characterized in that incubation time is 10~14h；
Or, being closed after being incubated for using 6- sulfydryl -1- hexanol.
6. preparation method as claimed in claim 4, characterized in that the preprocess method to gold electrode is first to use polishing agent
Gold electrode is polished, then the gold electrode after polishing is rinsed, electrochemical cleaning is then carried out in sulfuric acid solution, most
It is cleaned afterwards using ultrapure water；
Preferably, in electrochemical cleaning, electric potential scanning range is between -0.2V~1.6V, until obtaining stable cyclic voltammetric
Figure.
7. a kind of method for preparing palladium nano-particles based on rolling circle amplification mediation, characterized in that using described in as claimed in claim 1 or 22
Primer sets or electrochemica biological sensor as claimed in claim 3, pass through miRNA to be measured and carry out DNA probe specificity knot
After conjunction, the primer sequence A2 and circular template DNA of DNA probe exposure carry out rolling circle amplification, and it is mono- to obtain the DNA rich in guanine
Chain, it is single-stranded for template with the DNA rich in guanine, palladium salt is added and reducing agent carries out reduction reaction, obtains palladium nanometer
Grain.
8. the method for claim 7, characterized in that miRNA solution to be measured is added dropwise to DNA probe or connection DNA is visited
It on the gold electrode of needle, is incubated under human body temperature, then is added dropwise and is reacted containing DNA profiling, DNA ligase and DNA ligase
The solution of liquid, continuation are incubated under human body temperature, are then added dropwise and are contained archaeal dna polymerase and dNTPs, are maintained in human temperature
Be incubated under degree, obtain rich in guanine DNA it is single-stranded, will be enriched in guanine DNA it is single-stranded be placed in palladium salt solution, add
It heat-treats agent and carries out reduction reaction.
9. the palladium nano-particles based on rolling circle amplification mediation are to the electrochemical detection method of microRNA, characterized in that exploitation right
Benefit require 3 described in electrochemica biological sensor, by miRNA to be measured and carry out DNA probe specific binding after, DNA probe
Exposed primer sequence A2 and circular template DNA carries out rolling circle amplification, and DNA of the acquisition rich in guanine is single-stranded, is rich in described
The DNA of guanine single-stranded is template, and palladium salt is added and reducing agent carries out reduction reaction, then carries out Electrochemical Detection.
10. electrochemical detection method as claimed in claim 9, characterized in that miRNA solution to be measured is added dropwise to DNA probe
Or it on the gold electrode of connection DNA probe, is incubated under human body temperature, then be added dropwise containing DNA profiling, DNA ligase and DNA
The solution of enzyme reaction solution is connected, continuation is incubated under human body temperature, is then added dropwise and is contained archaeal dna polymerase and dNTPs, is kept
It is incubated under human body temperature, obtains that the DNA rich in guanine is single-stranded, the DNA that will be enriched in guanine single-stranded is placed on palladium
In salting liquid, heating reducing agent carries out reduction reaction, finally carries out Electrochemical Detection；
Preferably, the palladium salt is time chlorine palladium acid sodium, and the reducing agent is sodium borohydride；
Or, the Electrochemical Detection is differential pulse voltammetry；
Preferably, the solution that differential pulse voltammetry uses is HClO4Solution；
Preferably, it is 0.8V~1.3V that the condition of differential pulse voltammetry, which is scanning range, and amplitude is 50 ± 5mV, pulse width
For 50 ± 5ms, the pulse period is 0.5 ± 0.01s.
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| CN110736777A (en) * | 2019-09-19 | 2020-01-31 | 江南大学 | electrochemical-ELISA immunosensor based on rolling circle amplification DNA enzyme and covalent organic framework |
| CN110736777B (en) * | 2019-09-19 | 2020-12-01 | 江南大学 | electrochemical-ELISA immunosensor based on rolling circle amplification DNA enzyme and covalent organic framework |
| CN111187806A (en) * | 2020-01-09 | 2020-05-22 | 中国人民解放军陆军军医大学第一附属医院 | MicroRNA detection method based on 3D DNA nano-net structure dual-signal amplification technology |
| CN111187806B (en) * | 2020-01-09 | 2021-01-08 | 中国人民解放军陆军军医大学第一附属医院 | MicroRNA detection method based on 3D DNA nano-net structure dual-signal amplification technology |
| CN111172246A (en) * | 2020-01-13 | 2020-05-19 | 天津国科医工科技发展有限公司 | Electrochemical nucleic acid detection method based on DNA walking and rolling circle amplification signal amplification |
| CN111172246B (en) * | 2020-01-13 | 2022-10-28 | 天津国科医工科技发展有限公司 | Electrochemical nucleic acid detection method based on DNA walking and rolling circle amplification signal amplification |
| CN112014453A (en) * | 2020-07-24 | 2020-12-01 | 南京师范大学 | Method for detecting microcystin based on annular allosteric DNA electrochemical reaction |
| CN112014453B (en) * | 2020-07-24 | 2023-01-31 | 南京师范大学 | Method for detecting microcystin based on annular allosteric DNA electrochemical reaction |
| CN113061649A (en) * | 2021-04-02 | 2021-07-02 | 福州大学 | Surface enhanced Raman spectrum sensor for detecting microRNA and preparation method thereof |
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