CN107589163A - A kind of electrochemical sensor preparation method for the detection of MECP2 mutators - Google Patents
A kind of electrochemical sensor preparation method for the detection of MECP2 mutators Download PDFInfo
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Abstract
The present invention relates to RTT diagnostic genes --- the preparation method and application of the electrochemical sensor of Methylated CpG binding proteins 2 (MECP2) detection in Gene Mutation, belong to technical field of electrochemical detection.It is characterized in that：Synthesis obtains PdPt nanodendrites nano materials first, and melamine and PdPt nanodendrites then are combined to form into nano composite material, then single stranded DNA signal probe is mixed with the composite, and bio signal probe is made；Then the modification of Jenner's popped rice is used for the fixation of amidized DNA capture probes by deposited Au technology with sensor interface, so as to be prepared for the electrochemical sensor of MECP2 detection in Gene Mutation, the sensor is successfully used in the detection that single base mutation occurs for MECP2 genes.The advantage of the invention is that it is simple, quickly, high sensitivity, high specificity, conveniently.The present invention provides new detection method for Precise Diagnosis RTT.
Description
Technical field：
The present invention relates to a kind of preparation method of electrochemical sensor of clinically Precise Diagnosis MECP2 mutators and
Using the interlayer type biology sensor that palladium platinum nanodendrites-melamine is prepared as signal probe being based especially on, for examining
MECP2 gene mutations are surveyed, belong to field of electrochemical detection.
Background technology：
RTT syndromes also known as rett's syndrome (Rett syndrome, RTT), disintegrate with A Si Burgers syndrome, childhood
Sexual dysfunction, autism, the autism that is not true to type, non-specific wide hair sexual development obstacle are collectively known as pervasive developmental disorders, are
A kind of disease for having a strong impact on children's spirit motor development, mainly involves women, and the incidence of disease is 1/10000~1/15000.With
The continuous development of medical science, it is determined that RTT Disease-causing gene is the Methylated CpG binding proteins -2 positioned at chromosome x q28 regions
(MECP2) gene, at present in world wide it has been reported that more than 780 various mutations types, but 8 kinds of focuses is primarily present and are dashed forward
Change site (p.Arg106Trp, p.Arg133Cys, p.Thr158Met, p.Arg168X, p.Arg255X, p.Arg270X,
P.Arg294X and p.Arg306Cys), therefore this diagnosis of 8 kinds of hot spot mutations for RTT is precisely detected with prevention with weight
The medical science and social value wanted.
Detection of the tradition for MECP2 mutators depends on DNA sequence analysis and denaturing high-performance liquid chromatography.
But processing of these methods to sample is complex and needs the instrument and operating technology of expensive specialty, therefore in the last few years
Gene diagnosis technology is more and more applied to the Precise Diagnosis of hereditary disease.Currently used gene diagnosis mainly has PCR- single-stranded
Conformational polymorphism analysis, gene sequencing and biochip technology.But PCR method is easily by complicated ingredient in biological sample
Influence, detection efficiency it is low and easily there is false positive and make its application be restricted.And gene sequencing not only need it is special
Instrument and equipment, well-trained staff and detection process is cumbersome time-consuming, therefore it is not suitable for routine clinical detection.Therefore
Prepare simple, high sensitivity, the detection of the detection method of high specificity for hereditary disease mutator seems most important.In recent years
Come, based on nano material electrochemical DNA biosensor due to simplicity, quickly, cost is low, the advantage such as sensitivity height and it is extensive
Detection applied to mutator.
In electrochemical DNA biosensor application, sensitivity right and wrong of the signal amplification technique for raising DNA sensor
It is often important.In the last few years, the amplification that signal is used for using material with catalytic property is more taken seriously.Palladium platinum nanodendrites
It is of great interest in Material Field in the last few years due to its remarkable bimetal properties and superior catalytic property, and
And gradually increasing application is obtained in electrochemical sensor field.But in order to further lifted it is selected draw materials urge
Change performance, melamine mirrors our eyes.Because melamine has symmetrical three amino, by it and palladium platinum nanodendrites
Enriched palladium platinum nanodendrites can be played a part of by being combined together, so as to realize further enhancing for catalytic performance.Therefore by this
Both composite PdPt nanodendrites-melamine for combining to form have an optimal catalytic performance, and because
There are PdNPs and PtNPs presence, can also be combined with signal probe, pass through the specific binding of signal probe and target sequence
It can be used for building sandwich type biology sensor.
The present invention is built based on PdPt nanodendrites-melamine nano composite materials structure bio signal probe
Preparation method and the application of a kind of electrochemical DNA biosensor of MECP2 mutators detection have been stood, has been Precise Diagnosis RTT
Provide new thinking.
The content of the invention：
It is an object of the invention to provide the electrochemical DNA biology that the MECP2 mutators of Precise Diagnosis RTT a kind of detect to pass
The preparation method of sensor and application, its feature comprise the following steps：
(1) palladium platinum nanodendrites (PdPt nanodendrites)-melamine (Melamine)-single-stranded deoxyribose core
The preparation of sour (ssDNA) detection probe；
(2) electrochemical DNA biosensor is established, determines MECP2 mutators, draws standard curve.
The preparation process of PdPt nanodendrites-melamine-ssDNA compounds of the present invention specifically include with
Lower step, it is characterised in that comprise the following steps：
(1) preparation of PdPt nanodendrites nano materials：
To 1mL Na2PdCl4(5mM) and K2PtCl40.01g Pluronic F127 are added in (15mM) mixed solution,
And this three is set fully to mix under conditions of ultrasound.After resulting solution adds 1mL ascorbic acid solutions (0.4M), it is placed at once
On magnetic stirring apparatus, 3h is stirred at room temperature under conditions of 600r/min.Reaction terminate after, by resulting solution collect be put at a high speed from
12000r/min in scheming, 20min is centrifuged, cleaned be cleaned by ultrasonic altogether three times with ultra-pure water after centrifugation every time.Finally collect gained
Precipitation, is placed in drying at room temperature, 4 DEG C standby.
(2) preparation of PdPt nanodendrites-melamine nano composite materials：
1mL melamine (0.1mmol L-1) solution is taken to be added to the 1mL PdPt nanodendrites prepared
In (1mg mL-1) solution, it is placed on magnetic stirring apparatus, reaction 16h is stirred at room temperature under the conditions of 800e/min, after reaction completely
Resulting solution centrifuges 30min, is cleaned be cleaned by ultrasonic altogether three times with ultra-pure water after centrifugation every time through 12000r/min.Gained precipitates
It is dissolved in 2mL fixers, 4 DEG C standby.
(3) preparation of PdPt nanodendrites-melamine-ssDNA compounds：
The detection probe of biotech firm's synthesis is added into 4 DEG C of stirrings in PdPt nanodendrites-melamine solution
Centrifuge after overnight, and cleaned with PBS (0.1M, pH=7.4).By the PdPt nanodendrites-melamine- of synthesis
SsDNA is dispersed in 1mL hybridization solutions again, and 4 DEG C save backup.
The heretofore described DNA fragmentation established electrochemical DNA biosensor, determine MECP2 mutators, draws
Standard curve, it is characterised in that comprise the following steps：
(1) respectively with 0.3 μm and 50nm Al2O3Powder by polishing electrode into minute surface, then respectively by ultra-pure water, anhydrous
Order each 5min of ultrasound electrode of ethanol, ultra-pure water, drying at room temperature are standby；
(2) dried electrode is immersed in 1% chlorauric acid solution, constant-voltage method -0.2V depositions 30s；
(3) electrode washing is totally added dropwise to 10 μ L afterwards with ultra-pure water, the DNA capture probe solution of 1 μM of amino labeled, 4 DEG C
It is incubated overnight.
(4) electrode washing after incubation is totally added dropwise to 6 μ L, 100 μM 6- sulfydryls hexanol (MCH) solution afterwards with ultra-pure water
It is incubated at room temperature 30min.
(5) electrode cleaning buffer solution (the 10mM Na after above-mentioned MCH is closed2HPO4, 2mM KH2PO4, 37mM
NaCl, 2.7mM KCl, pH 7.4) rinse well and dried in nitrogen.
(6) the target dna solution of various concentrations is added dropwise and 37 DEG C of hybridization 2h is placed on electrode.
(7) 10 μ L detection probe mixed liquors are added dropwise on electrode after the drying and are placed in 37 DEG C of incubation 2h.
(8) it is placed in nitrogen and dries after the electrode after incubation is rinsed well with cleaning buffer solution.
(9) electrode is placed in 5mL, 0.1M PBS (0.1M Na2HPO4, 0.1M KH2PO4, 0.1M KCl) in carry out table
Sign, 20 μ L, 1.5M H are added every 100s2O2, measure its chrono-amperometric variable-current value.
(10) it is linear according to gained current variation value and MECP mutated gene segment concentration, drawing curve.
Compared with prior art, the electrochemical DNA biology of the MECP2 mutators detection of a kind of Precise Diagnosis of the invention
The preparation method of sensor and application, its protrude the characteristics of be：
(1) new PdPt nanodendrites-melamine nano composite materials are incorporated into as signal probe
In the preparation of electrochemical DNA biosensor, the catalytic performance of material is not only effectively raised, and improves biomolecule
Supported quantity, and then improve sensitivity and the biocompatibility of electrochemical DNA biosensor；
(2) form of gold and the shadow of chemical property are deposited to the sensing interface of sensor by studying different sedimentation times
Ring, construct one and conduct electricity very well, specific surface area is big, the sensing interface of good biocompatibility, not only contributes to capture dna spy
The fixation of pin, and further improve the sensitivity of sensor.
(3) electrochemical DNA biosensor prepared by this method can provide new method for Precise Diagnosis RTT, and then expand
Application of the simple and quick gene diagnosis in routine clinical；
(4) identical nano material and method of modifying are used, using capture probe, signal probe and target dna
Specific recognition, only a variety of monogenic inheritance disease Precise Diagnosis, Gao Ling need to can be achieved by changing the nucleotide sequence of probe
Quick detection, in addition, the method is easy, quickly, commercialization is easy to implement, so as to promote the development of translational medicine.
Brief description of the drawings：
Fig. 1 is the structure schematic diagram of the electrochemical DNA biosensor of the present invention.
Fig. 2 is the transmission electron microscope picture of the signal probe of the present invention, and EDS schemes.
Fig. 3 is that the chrono-amperometric that the electrochemical DNA biosensor of the present invention obtains when detecting MECP2 mutators becomes
The linear relationship of galvanic current and concentration, and the specificity and reappearance of sensor.
Embodiment：
The present invention is further elaborated with reference to specific embodiment, it should be appreciated that these embodiments are merely to illustrate
The present invention rather than limitation the scope of the present invention.
Embodiment 1
Na of the step 1. to 1mL2PdCl4(5mM) and K2PtCl40.01g Pluronic are added in (15mM) mixed solution
F127, and this three is fully mixed under conditions of ultrasound.After resulting solution adds 1mL ascorbic acid solutions (0.4M), stand
Quarter is placed on magnetic stirring apparatus, and 3h is stirred at room temperature under conditions of 600r/min, and solution colour trip brown color is changed into aterrimus.Instead
After should terminating, resulting solution is collected and is put in 12000r/min in supercentrifuge, centrifuge 20min, every time with ultrapure after centrifugation
Water cleaning is cleaned by ultrasonic three times altogether.Finally collect gained to precipitate, be placed in drying at room temperature, 4 DEG C standby；
Step 2. takes 1mL melamine (0.1mmol L-1) solution to be added to the 1mL PdPt prepared
In nanodendrites (1mg mL-1) solution, it is placed on magnetic stirring apparatus, reaction is stirred at room temperature under the conditions of 800e/min
16h, resulting solution centrifuges 30min, cleans common ultrasonic cleaning with ultra-pure water after centrifugation every time through 12000r/min after reacting completely
Three times.Gained precipitation is dissolved in 2mL fixers, and 4 DEG C standby；
The detection probe that step 3. synthesizes biotech firm is added 4 in PdPt nanodendrites-melamine solution
Centrifugation (noticing that the speed of stirring is unsuitable too fast, need gentle stirring) after DEG C being stirred overnight, and with PBS (0.1M, pH=7.4) clearly
Wash.The PdPt nanodendrites-melamine-ssDNA of synthesis are dispersed in 1mL hybridization solutions again, 4 DEG C of preservations are standby
With；
Step 4. is respectively with 0.3 μm and 50nm Al2O3Polishing electrode into minute surface, is then pressed ultra-pure water, nothing by powder respectively
Order each 5min of ultrasound electrode of water-ethanol, ultra-pure water, drying at room temperature are standby；
Step 5. immerses dried electrode in 1% chlorauric acid solution, constant-voltage method -0.2V depositions 30s；
Step 6. electrode washing is totally added dropwise afterwards with ultra-pure water 10 μ L, the DNA capture probe solution of 1 μM of amino labeled, and 4
DEG C be incubated overnight；
Step 7. electrode washing after incubation is totally added dropwise afterwards with ultra-pure water 6 μ L, 100 μM of 6- sulfydryls hexanol (MCH)
Solution is incubated at room temperature 30min；
Step 8. by above-mentioned MCH close after electrode cleaning buffer solution (10mM Na2HPO4, 2mM KH2PO4, 37mM
NaCl, 2.7mM KCl, pH 7.4) rinse well and dried in nitrogen；
The target dna solution of various concentrations is added dropwise step 9. is placed in 37 DEG C of hybridization 2h on electrode；
10 μ L detection probe mixed liquors are added dropwise on the electrode of step 10. after the drying and are placed in 37 DEG C of incubation 2h；
Step 11. is placed in nitrogen after the electrode after incubation is rinsed well with cleaning buffer solution and dried；
Electrode is placed in 5mL, 0.1M PBS (0.1M Na by step 12.2HPO4, 0.1M KH2PO4, 0.1M KCl) in carry out
Characterize, 20 μ L, 1.5M H are added every 100s2O2, measure its chrono-amperometric variable-current value；
Step 13. is linear according to gained current variation value and MECP2 mutator DNA fragmentation concentration, draws work
Make curve；Measurement result shows that MECP2 mutated gene segment concentration is linear in the range of 1fM-1nM, linear correlation system
Number is 0.9965, and detection is limited to 0.33fM；
Step 14. is by the sensor of the present invention in 4 DEG C of preservations, discontinuity detection sensor current response, after storing 28 days
Current-responsive is still the 90.2% of initial current, shows the sensor of structure and has good stability.
Step 15. present invention takes DNA biosensor 5 prepared by same batch, under the same conditions to 100pM's
MECP2 mutator DNA fragmentations are measured respectively, each determination of electrode 3 times, as a result the relative standard deviation of response current
Less than 2.19%, illustrate that difference is small in sensor batch, sensor reappearance is good；
The sensor of the present invention is used to detect target nucleic acid sequence by step 16., and base mismatch, as a result base mismatch is electric
Stream response seems insignificant relative to target nucleic acid sequence, illustrates the specific good of sensor, can distinguish target sequence very well
Row.Corresponding capture probe and detection probe are changed, by other 7 kind mutation position of the sensor of this structure for MECP2
The detection of point, as a result shows very strong current-responsive value, illustrates that the sensor that the present invention is built can enter to MECP2 mutators
The accurate detection of row.
Described above is only the preferred embodiment of the present invention, it is noted that for the common skill of the art
For art personnel, under the precondition for not departing from the principle of the invention, some improvements and modifications can also be made, these improve and
Retouching also should be regarded as protection scope of the present invention.
Claims (3)
1. a kind of electrochemical sensor preparation method for the detection of MECP2 mutators, it is characterised in that comprise the following steps：
(1) palladium platinum nanodendrites (PdPt nanodendrites)-melamine (Melamine)-single stranded deoxyribonucleic acid
(ssDNA) preparation of detection probe；
(2) electrochemical DNA biosensor is established, determines MECP2 mutators, draws standard curve.
2. the preparation process of PdPt nanodendrites-melamine-ssDNA compounds is specific according to claim 1
Comprise the following steps, it is characterised in that comprise the following steps：
(1) PdPt nanodendrites preparation：
To 1mL Na2PdCl4(5mM) and K2PtCl40.01g Pluronic F127 are added in (15mM) mixed solution, ultrasound makes
It dissolves.After resulting solution adds 1mL ascorbic acid solutions (0.4M), room temperature is stirred in 600r/min as on magnetic stirring apparatus
3h is reacted, reaction terminates rear resulting solution through 12000r/min, centrifuges 20min, respectively cleaned with ultra-pure water 3 times.Gained is precipitated
Be placed in drying at room temperature it is complete after, 4 DEG C save backup.
(2) preparation of PdPt nanodendrites-melamine nano composite materials：
Take 1mL melamine (0.1mmol L-1) solution is added to the 1mL PdPt nanodendrites (1mg prepared
mL-1) in solution, reaction 16h is stirred at room temperature, resulting solution has been reacted through 12000r/min, centrifuges 30min, 3 are cleaned with ultra-pure water
It is secondary.
(3) preparation of PdPt nanodendrites-melamine-ssDNA compounds
Detection probe is added in PdPt nanodendrites-melamine solution after being stirred overnight at room temperature and centrifuged, and use PBS
(0.1M, pH=7.4) is cleaned.The PdPt nanodendrites-melamine-ssDNA of synthesis are dispersed in 1mL hybridization again
In liquid, 4 DEG C save backup.
3. according to claim 1 establish electrochemical DNA biosensor, MECP2 mutators are determined, it is bent to draw standard
Line, it is characterised in that comprise the following steps：
(1) respectively with 0.3 μm and 50nm Al2O3Powder by polishing electrode into minute surface, then respectively by ultra-pure water, absolute ethyl alcohol,
Order each 5min of ultrasound electrode of ultra-pure water, drying at room temperature are standby；
(2) dried electrode is immersed in 1% chlorauric acid solution, constant-voltage method -0.2V depositions 30s.
(3) electrode washing is totally added dropwise to 10 μ L, the DNA capture probe solution of 1 μM of amino labeled, 4 DEG C of incubations afterwards with ultra-pure water
Overnight.
(4) electrode washing after incubation is totally added dropwise to 6 μ L, 100 μM 6- sulfydryls hexanol (MCH) solution room temperature afterwards with ultra-pure water
It is incubated 30min.
(5) electrode cleaning buffer solution (the 10mM Na after above-mentioned MCH is closed2HPO4, 2mM KH2PO4, 37mM NaCl,
2.7mM KCl, pH7.4) rinse well and dried in nitrogen.
(6) the target dna solution of various concentrations is added dropwise and 37 DEG C of hybridization 2h is placed on electrode.
(7) 10 μ L detection probe mixed liquors are added dropwise on electrode after the drying and are placed in 37 DEG C of incubation 2h.
(8) it is placed in nitrogen and dries after the electrode after incubation is rinsed well with cleaning buffer solution.
(9) electrode is placed in 5mL, 0.1M PBS (0.1M Na2HPO4, 0.1M KH2PO4, 0.1M KCl) in characterized, every
100s adds 20 μ L, 1.5M H2O2, measure its chrono-amperometric variable-current value.
(10) it is linear according to gained current variation value and MECP mutated gene segment concentration, drawing curve.
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