CN109161583A - The method for the cycle index augmentation detection alkaline phosphatase that primer dephosphorylation causes - Google Patents
The method for the cycle index augmentation detection alkaline phosphatase that primer dephosphorylation causes Download PDFInfo
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Abstract
The invention discloses the methods for the cycle index augmentation detection alkaline phosphatase that primer dephosphorylation causes, it is made of using sensor DNA primer, hairpin probe 1 and hairpin probe 2, the end the 5' base of hairpin probe 1 and hairpin probe 2 modifies fluorophor, the base modification quenching group with the end the 5' base complementrity of hairpin probe 1；With the base modification quenching group of the end the 5' base complementrity in hairpin probe 2, the base sequence complementary of base sequence and DNA primer between quenching group in hairpin probe 1 and the end 3', base sequence complementary between 1 restriction endonuclease recognition sequence of the end 5' and hairpin probe of hairpin probe 1 between base sequence and the quenching group in hairpin probe 2 and the end 3', the base sequence complementary of base sequence and DNA primer between 2 restriction endonuclease recognition sequence of the end 5' and hairpin probe of hairpin probe 2.Method of the invention is capable of the activity of quick, overdelicate detection of alkaline phosphatase.
Description
Technical field
The invention belongs to bioassay techniques, are related to the cycle index augmentation detection alkaline phosphatase of primer dephosphorylation initiation
The method of enzyme.
Background technique
Alkaline phosphatase (ALP) is a kind of widely distributed hydrolase, is catalyzed various phosphorylated substrates (such as albumen
Matter, carbohydrate and nucleic acid) dephosphorylation, in cell cycle regulation, growth, apoptosis, signal transduction pathway etc. are multiple
It plays an important role in intracellular processes.The abnormal level of ALP and bone disease (such as osteitis deformans, malacosteon and at
Osteocytic osteocarcinoma), liver diseases (such as obstructive jaundice, hepatitis and liver cancer), diabetes, breast cancer and prostate cancer etc. it is each
Kind human diseases is closely related.Further, since its high catalytic activity, good stability and extensive substrate specificity, ALP are
Enzyme immunoassay (EIA), gene expression analysis, histochemical stain and biomolecule monitoring in most popular label tracer it
One.Therefore, develop a kind of effectively reliable ALP activity test method have for clinical diagnosis and medicament research and development it is highly important
Meaning.
So far, a variety of methods have been developed to measure activity/level of ALP, including based on radiolabeled
Electrophoresis, chromatography, Surface enhanced Raman scattering method, colorimetric method, chemoluminescence method and electrochemical determination.Although these methods
All there is respective characteristic, but they will receive that such as harmful radiation, poor sensitivity, complicated for operation, program is time-consuming, at sample
It manages complicated, expensive equipment and protein antibody is needed to be fixed on the limitation on solid support.Fluorescent method, which has, quickly to be divided
Analysis, highly sensitive, high-throughput, instrument is simple, the analysis excellent remarkable advantage of performance, so by fluorescent method and noble metal nano cluster
(such as copper nano-particle and ag nano-cluster), inorganic semiconductor (such as carbon quantum dot, copper sulfide indium quantum dot, carbon dots and quantity of graphite
Sub- point), fluorescent polymer (such as polyethyleneimine polymers, pet fiber and dicyandiamide compound) and
Fluorescer (such as Fmoc-K FITC FFYP) combines, and can be used for the active measurement of ALP.Such as utilize copper ion (Cu2+) and burnt phosphorus
Strong interaction between sour (PPi), it is the copper nano particles (CuNPs) of template that PPi, which can inhibit double-stranded DNA (dsDNA),
Fluorescence and realize the signal-off fluorescence detection to ALP.And utilize Cu2+The single stranded DNA (ssDNA) of mediation is the silver nanoparticle of template
Cluster is quenched, and can also realize the signal-off fluorescence detection to ALP.The PPi-Cu mediated based on PPi dephosphorylation2+- PPi is multiple
It closes object to split, realizes the signal-off fluorescence detection to ALP.PNP can be converted by nitro phenyl ester (PNPP) using PPi, from
And being quenched for carbon dots (CD) is induced, realize the signal-off fluorescence detection of ALP.And a phosphoric acid is contained by ALP induction
The increase of the fluorescence intensity ratio (F378/F488) of the pyrene derivatives (Py-P, aliphatic phosphate ester) of group, develops for ALP
The ratio fluorescent method of measurement.But fluorescent method described above all refers to the complicated synthesis and time-consuming expense of fluorescent nano material
The experimental implementation of power, and since signal-off (signal-off) measurement causes accuracy poor, cause due to lacking signal amplification
Poor sensitivity.Meanwhile in clinical diagnosis, since ALP is 40~190 every liter of units in adult body, content is lower, thus existing
Technology can not carry out high sensitive detection to the activity of low concentration ALP.
Summary of the invention
In order to solve the deficiencies in the prior art, an object of the present invention is to provide a kind of cycle index augmentation detection alkalinity
The sensor of phosphatase is capable of the activity of quick, overdelicate detection of alkaline phosphatase using the sensor.
To achieve the goals above, the technical solution of the present invention is as follows:
A kind of sensor of cycle index augmentation detection alkaline phosphatase, by DNA primer, hairpin probe 1 (HP1) and hair clip
Probe 2 (HP2) composition；
DNA primer is single-stranded DNA sequence, and the end 3' of DNA primer is phosphorylated ends, hairpin probe 1 and hairpin probe 2
All have the end 3' outstanding, the end the 5' base of hairpin probe 1 and hairpin probe 2 modifies fluorophor, the 5' with hairpin probe 1
Hold the base modification quenching group of base complementrity；
With the base modification quenching group of the end the 5' base complementrity in hairpin probe 2, quenching group in hairpin probe 1 and
The base sequence complementary of base sequence and DNA primer between the end 3', the end 5' of hairpin probe 1 and 1 digestion of hairpin probe identification
Base sequence complementary between sequence between base sequence and the quenching group in hairpin probe 2 and the end 3', the 5' of hairpin probe 2
The base sequence complementary of base sequence and DNA primer between end and 2 restriction endonuclease recognition sequence of hairpin probe.
Hairpin probe 1 and hairpin probe 2 of the present invention serve not only as the template of cycle index amplification, are also used as letter
Number output generator.In the presence of ALP, the 3'- phosphorylated ends of DNA primer are hydrolyzed into 3'- hydroxylating end.Dephosphorylation
DNA primer can hybridize with the 3' protruding terminus of HP1 to start first strand displacement amplification (SDA), generate first triggering
Device simultaneously generates fluorescence signal.First trigger of release is complementary with the 3' jag of HP2 to start second strand displacement amplification
(SDA), it generates second trigger and generates fluorescence signal simultaneously.Second trigger is complementary with the 3' jag of HP1 to open
Above-mentioned two continuous strand displacement amplification (SDA) reaction is moved, cycle index amplification is made low abundance ALP information can be converted to finger
The fluorescence signal of number amplification, to realize the activity of quick, overdelicate detection of alkaline phosphatase.
The second object of the present invention is to provide a kind of kit of cycle index augmentation detection alkaline phosphatase, including it is above-mentioned
Sensor.
The third object of the present invention is to provide a kind of the sensor or kit in cycle index augmentation detection alkalinity phosphorus
Application in phytase activity.
The application is for the purpose of the Clinics and Practices of non-disease.
The fourth object of the present invention is to provide a kind of cycle index augmentation detection alkaline phosphatase that primer dephosphorylation causes
The method of enzyme, using the sensor or kit, process are as follows:
(1) phosphate group at the end alkaline phosphatase catalytic dna primer 3' in prepare liquid is hydrolyzed to hydroxyl；
(2) DNA primer after hydrolyzing hybridizes with the 3' jag of hairpin probe 1 carries out first time strand displacement amplification (SDA)
Reaction obtains first trigger and generates fluorescence signal；
(3) first triggers carry out second of strand displacement amplification (SDA) with the hybridization of the 3' jag of hairpin probe 2 and react
It obtains second trigger and generates fluorescence signal；
(4) second triggers are hybridized with the 3' jag of hairpin probe 1, to be repeated in step (2)~(4)；
According to the activity for carrying out the alkaline phosphatase in the fluorescence signal acquisition prepare liquid that step (1)~(4) generate afterwards.
The invention has the benefit that
1. the active high sensitivity of detection of alkaline phosphatase of the present invention: compared with ALP measuring method before, the present invention
Than the sensitivity (8.4 × 10 of colorimetric method-7Every microlitre of unit) 4200 times high, the electrochemical process of the substrate than using Ferrocene-Derived
(4×10-7Every microlitre of unit) it is 2000 times high, than based on DNA bracket (scaffolded) silver nanoclusters fluorimetry (5 ×
10-6Every microlitre of unit) it is 25000 times high, than the fluorescence method (1.5 × 10 of polyethylene terephthalate (PET) optical fiber-6Unit
Every microlitre) it is 7500 times high, than Photo-induced electron transfer fluorescence analysis (9 × 10-7Every microlitre of unit) it is 4500 times high.Of the invention is super
High sensitivity is attributable to three factors: (1) high specific of the dephosphorylation reaction of ALP catalysis makes 3' terminal phosphate group
It is hydrolyzed into 3' terminal hydroxyl group, the high amplification efficiency for the circulation isothermal duplication that (2) difunctional hairpin probe mediates makes a small amount of
DNA primer can be converted into a large amount of trigger, and (3) shorter oligonucleotides (i.e. trigger 1 and trigger 2) induces hair clip to visit
Needle opens hairpin probe by polymerization extension.
2. detection method of the invention is easy to operate: the present invention carries out in the solution of homogeneous, be not related to it is any washing and
Separating step；Cycle index of the present invention amplification is to carry out under isothermal conditions, avoids complicated thermal cycle and accurate
Temperature control.
3. the present invention can effectively eliminate experiment false positive, the accuracy of detection is provided.
Detailed description of the invention
The accompanying drawings constituting a part of this application is used to provide further understanding of the present application, and the application's shows
Meaning property embodiment and its explanation are not constituted an undue limitation on the present application for explaining the application.
Fig. 1 is the active schematic diagram of detection of alkaline phosphatase of the present invention；
Fig. 2 is the phenogram for the cycle index amplified reaction that the difunctional hairpin probe that ALP dephosphorylation causes mediates；A
Figure is tested and analyzed for the native polyacrylamide gel electrophoresis of first step strand displacement amplification reaction product, swimming lane 1 is trigger
1, swimming lane 2 is hairpin probe HP1, and swimming lane 3 is amplified reaction product in the presence of ALP and HP1, and B is that cycle index amplification is anti-
The native polyacrylamide gel electrophoresis of product is answered to test and analyze, swimming lane 1 is trigger 1, and swimming lane 2 is primer strand, and swimming lane 3 is
Hairpin probe HP1；Swimming lane 4 is hairpin probe HP2；Swimming lane 5 is the amplified reaction product in the presence of ALP, HP1 and HP2.C is
Fluorogram under different time；
Fig. 3 is various concentration ALP Fluorescent Characterization figure, and A is different fluorescence intensity figures, and B is fluorescence intensity in various concentration ALP
Under its linear analysis figure；
Fig. 4 is cell experiment phenogram, and A is the fluorescence intensity of different cells, and B is that fluorescence intensity is thin in different HeLa
Situation of change and its linear analysis figure under born of the same parents' number.
Specific embodiment
It is noted that described further below be all exemplary, it is intended to provide further instruction to the application.Unless another
It indicates, all technical and scientific terms used herein has usual with the application 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 application.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.
Herein described hairpin probe refers to hair clip DNA probe.
Trigger described herein is to refer to the DNA sequence complementary with the 3' jag of the hairpin probe in next step
Column.
The purpose that the application modifies quenching group is to be able to the fluorescence for issuing the fluorophor at the end hairpin probe 5'
Extinguish.
As background technique is introduced, the prior art, which exists in the prior art, to carry out the activity of low concentration ALP
The deficiency of high sensitive detection, in order to solve technical problem as above, the circulation caused present applicant proposes primer dephosphorylation refers to
The method of number augmentation detection alkaline phosphatase.
A kind of exemplary embodiment of the application provides a kind of sensing of cycle index augmentation detection alkaline phosphatase
Device is made of DNA primer, hairpin probe 1 (HP1) and hairpin probe 2 (HP2)；
DNA primer is single-stranded DNA sequence, and the end 3' of DNA primer is phosphorylated ends, hairpin probe 1 and hairpin probe 2
All have the end 3' outstanding, the end the 5' base of hairpin probe 1 and hairpin probe 2 modifies fluorophor, the 5' with hairpin probe 1
Hold the base modification quenching group of base complementrity；
With the base modification quenching group of the end the 5' base complementrity in hairpin probe 2, quenching group in hairpin probe 1 and
The base sequence complementary of base sequence and DNA primer between the end 3', the end 5' of hairpin probe 1 and 1 digestion of hairpin probe identification
Base sequence complementary between sequence between base sequence and the quenching group in hairpin probe 2 and the end 3', the 5' of hairpin probe 2
The base sequence complementary of base sequence and DNA primer between end and 2 restriction endonuclease recognition sequence of hairpin probe.
Restriction endonuclease recognition sequence described herein is that the DNA sequence dna being complementary is capable of the DNA sequence dna of incision enzyme shearing.
The application hairpin probe 1 and hairpin probe 2 serve not only as the template of cycle index amplification, are also used as letter
Number output generator.In the presence of ALP, the 3'- phosphorylated ends of DNA primer are hydrolyzed into 3'- hydroxylating end.Dephosphorylation
DNA primer can hybridize with the 3' protruding terminus of HP1 to start first strand displacement amplification (SDA), generate first triggering
Device simultaneously generates fluorescence signal.First trigger of release is complementary with the 3' jag of HP2 to start the second strand displacement amplification
(SDA), it generates second trigger and generates fluorescence signal simultaneously.Second trigger is complementary with the 3' jag of HP1 to open
Above-mentioned two continuous SDA reaction is moved, makes cycle index amplification that low abundance ALP information can be converted to the fluorescence of exponential amplification
Signal, to realize the activity of quick, overdelicate detection of alkaline phosphatase.
In one or more embodiments of present embodiment, fluorophor FAM, quenching group BHQ1.
In order to increase the sensitivity of sensor, in one or more embodiments of present embodiment, DNA primer has 19~21
A base.
In one or more embodiments of present embodiment, sequence of the DNA primer from the end 5' to the end 3' is T CGA TTG
ATG AAC ACT GCG A。
In one or more embodiments of present embodiment, sequence of the hairpin probe 1 from the end 5' to the end 3' isACT ATA
CAA CCT ACT ACC TTT CAG ACT CAC GTA GTA GGT TGT ATA GTG TTT GTC ATC GCA GTG
TTC ATC A (wherein underscore base A and T modifies fluorescent molecule FAM and quencher molecule BHQ1 respectively).
In one or more embodiments of present embodiment, sequence of the hairpin probe 2 from the end 5' to the end 3' isTCG CAG
TGT TCA TCA ATC GAC TTT CAG ACT CAC TCG ATT GAT GAA CAC TGC GAA TCT GAG AAC
TAT ACA ACC TAC T (wherein two base T of underscore modify fluorescent molecule FAM and quencher molecule BHQ1 respectively).
In one or more embodiments of present embodiment, including archaeal dna polymerase and DNA nickase.
The another embodiment of the application provides a kind of kit of cycle index augmentation detection alkaline phosphatase,
Including the sensor.
In one or more embodiments of present embodiment, including 10 × ThermoPol buffer, 10 × NEBuffer
3.1 and dNTP solution mixture.
Embodiment there is provided a kind of the sensors or kit in cycle index augmentation detection for the third of the application
Application in alkaline phosphatase activities.
The application is for the purpose of the Clinics and Practices of non-disease.
Embodiment there is provided the cycle index augmentation detection alkali that a kind of primer dephosphorylation causes for the 4th kind of the application
The method of acid phosphatase, using the sensor or kit, process are as follows:
(1) phosphate group at the end alkaline phosphatase catalytic dna primer 3' in prepare liquid is hydrolyzed to hydroxyl；
(2) DNA primer after hydrolyzing hybridizes with the 3' jag of hairpin probe 1 carries out first time strand displacement amplification (SDA)
Reaction obtains first trigger and generates fluorescence signal；
(3) first triggers carry out second of strand displacement amplification (SDA) with the hybridization of the 3' jag of hairpin probe 2 and react
It obtains second trigger and generates fluorescence signal；
(4) second triggers are hybridized with the 3' jag of hairpin probe 1, to be repeated in step (2)~(4)；
According to the activity for carrying out the alkaline phosphatase in the fluorescence signal acquisition prepare liquid that step (1)~(4) generate afterwards.
Since hairpin probe is in the nature single stranded DNA, in order to make the single-stranded formation hairpin structure of DNA of hairpin probe, this implementation
In one or more embodiments of mode, with the mixed liquor of magnesium chloride and three (methylol) aminomethane-hydrochloric acid by hairpin probe 1
DNA is single-stranded or the single-stranded dilution dissolution of the DNA of hairpin probe 2, be then heated at high temperature room temperature and be incubated for.The condition of high-temperature heating
It is 95 ± 2 DEG C, 5~6min；Incubation at room temperature condition is 30min.The room temperature is 15~30 DEG C.Three (methylol) aminomethanes-
The pH of hydrochloric acid is 8.0.The molar ratio of magnesium chloride and three (methylol) aminomethane-hydrochloric acid is 1~2:10.It presss from both sides and visits after dilution dissolution
The concentration that the DNA of needle 1 is single-stranded or the DNA of hairpin probe 2 is single-stranded is 1 ± 0.01 μm of ol/L.
In one or more embodiments of present embodiment, step are as follows: 36 after mixing DNA primer and alkaline phosphatase
After carrying out incubation a period of time at~37.5 DEG C, heats to eliminate the activity of alkaline phosphatase, obtain part A；By hairpin probe
1, after hairpin probe 2, archaeal dna polymerase, DNA nickase and dNTP solution mixture are mixed, obtain part B, by part A and
After being incubated for a period of time at 55~60 DEG C after part B mixing, fluorescence intensity.
In one or more embodiments of present embodiment, archaeal dna polymerase is Vent archaeal dna polymerase.
In one or more embodiments of present embodiment, DNA nickase is Nt.BstNBI.
In one or more embodiments of present embodiment, part B there are also 10 × ThermoPol buffer and 10 ×
NEBuffer3.1。
In one or more embodiments of present embodiment, when detecting fluorescence, excitation wavelength 492nm.Spectrum occurs to exist
Within the scope of 500~600nm.When excitation and transmite slit width are all 5nm, maximum fluorescence emission is at 520nm.
The cycle index amplification caused with primer dephosphorylation includes two continuous reactions to detect the active principle of ALP
Step: (1) the 3'- phosphorylated ends dephosphorylation of ALP catalytic dna primer is 3'- hydroxylating end, and (2) difunctional hair clip is visited
The cycle index amplified reaction that needle mediates.Respectively in the terminal modified hydroxyl of the 5' of DNA primer and 3' (OH) group and phosphoryl (PO4)
Single-stranded (ss) DNA sequence dna of 20 bases of group, function function not only as the DNA primer of ALP, and as opening
Move the primer for the cycle index amplification that subsequent difunctional hairpin probe mediates.The stem of two hairpin probes of HP1 and HP2 contains
There is two complementary strands (sense strand and antisense strand).It is repaired respectively on the thymidine of the adenine sum of the end 5'- of HP1 and HP2
Decorations FAM, and the gland on the thymidine matched with the end the 5'- adenine of HP1, with thymidine pairing in the end 5'- in HP2
Purine modifies quencher BHQ1 on the thymidine of 2 bases respectively.In addition, the stem in HP1 and HP2 all designs protrusion
The end 3' DNA sequence dna (23 bases) so that its respectively with 1 partial hybridization of single-stranded primer and trigger.In the ring of HP1 and HP2
Middle design nickase (Nt.BstNBI) identifies sequence, so that generating nicking sites when forming double-stranded DNA (dsDNA).In ALP
In the presence of, the 3'-PO of primer4Group is hydrolyzed into 3'-OH group.Primer with 3'- hydroxylating end will have as one
The primer of effect and the end the protrusion 3' DNA sequence dna partial hybridization of HP1 are extended with causing polymerization in the presence of archaeal dna polymerase and dNTP
Reaction.The extension of primer opens the hairpin structure of HP1, results in the complete double-strand with Nt.BstNBI nicking sites
DNA, while by BHQ1 and FAM separation to restore FAM fluorescence.Then, Nt.BstNBI is cut at specific recognition nicking sites
DNA chain.At new replication site, first SDA reaction is started in the presence of archaeal dna polymerase and nickase.It is repeating
Extend, after cutting and release cycle, finally generates many short oligonucleotides (trigger 1).The trigger 1 of release can be made
For primer and with the 3' distal process of HP2 go out DNA sequence dna partial hybridization initiation polymerize extension, cause HP2 hairpin structure opening,
And the recovery of Nt.BstNBI nicking sites and FAM fluorescence is formed in HP2.HP2 notch position is then cut by Nt.BstNBI
Point generates the new replication site of archaeal dna polymerase, causes second of SDA reaction.In the case that archaeal dna polymerase and nickase there are,
Start another circulation for extending, cutting and discharging, to generate a large amount of triggers 2.It is worth noting that, trigger 2 and drawing
Object all has an identical sequence of 5'-T CGA TTG ATG AAC ACT GCG A-3', thus trigger 2 can be used as primer with
The 3' jag hybridization of free HP1, first SDA reaction of starting in the presence of archaeal dna polymerase and nickase.Therefore, it connects
Two SDA reaction constitute cyclic annular chain reaction, cause trigger 1 and trigger 2 largely to generate with exponential form, and finally lead
Cause the exponential amplification of fluorescence signal.On the contrary, in the case where ALP is not present, the 3'-PO of primer4Group is not hydrolyzed into 3'-OH
Group, therefore cycle index amplified reaction cannot be started and cannot detect fluorescence signal.It is anti-using ALP catalysis dephosphorylation
The high efficiency for the cycle index amplified reaction that the high specific answered and difunctional hairpin probe mediate, this method can be used for highly sensitive
Detect ALP activity.
In order to enable those skilled in the art can clearly understand the technical solution of the application, below with reference to tool
The technical solution of the application is described in detail in the embodiment of body.
The cycle index amplified reaction that difunctional hairpin probe mediates: first with 1 × tri- (methylol) aminomethane-second two
Amine tetraacethyl (Tris-EDTA) buffer dilutes all oligonucleotides to prepare stock solution.With containing 1.5 mMs every liter
Magnesium chloride (MgCl2) and 10 mMs every liter three (methylol) aminomethane-hydrochloric acid (Tris-HCl) (pH 8.0) buffers
Hairpin probe HP1 and HP2 are diluted to 1 every liter of micromole, and heated 5 minutes at 95 DEG C, is then slowly cooled to room temperature, is incubated for
30min, so that probe forms hairpin structure completely.The mixture of amplified reaction is divided into part A and part B to prepare.In part A
In, by 10 microlitres of reaction mixtures (DNA primer of 50 every liter of nanomoles, ALP and 1 microlitre of 10 × CutSmart of various amounts
Buffer 37 DEG C be incubated for 30 minutes, be then heated to 65 DEG C 5 minutes with inactivate ALP activity.It is anti-by 10 microlitres in the B of part
Answering mixture, (HP1 of 100 every liter of nanomoles, the HP2 of 300 every liter of nanomoles, 2 unit Vent (exo-) archaeal dna polymerases, 1 is micro-
10 × ThermoPol the buffer risen, the Nt.BstNBI of 8 units, 1 microlitre of 10 × NEBuffer3.1,500 micromoles are every
The dNTP solution mixture risen) it is added in the A of part, incubate 60 minutes at 58 DEG C then to carry out cycle index amplified reaction.
The measurement of fluorescence intensity: 20 microlitres of amplified productions are diluted to 60 microlitres of final volume with ultrapure water.Use Hitachi F-
Quartz cuvette on 7000 sepectrophotofluorometers (Tokyo, Japan) measures all fluorescence spectrums.Excitation wavelength is 492nm,
Emission spectrum is all 5 nanometers for excitation and transmite slit width in the wave-length coverage of 500~600nm.Maximum fluorescence emission
At 520nm.There will be no the fluorescence intensities of the reaction product obtained when ALP to be defined as background signal.
The preparation of cell culture and cell extract.Human cervical cancer cell lines (HeLa cell) and human colon cancer cell line
(HT-29 cell) culture medium is that the Dulbecco containing 10% fetal calf serum (FBS) and 1% Pen .- Strep improves her lattice
That culture medium (DMEM).Cell is containing 5% carbon dioxide (CO2), it cultivates in 37 DEG C of incubator.In exponential phase of growth, use
Trypsin digestion collects cell, and with glacial phosphoric acid salt buffer, (137 mMs of sodium chloride solutions, 2.7 mMs of potassium chloride are molten
Liquid, 10 mMs of phosphate buffers, pH 7.4) it washes twice, then it is centrifuged 4 minutes in 4 DEG C, 800 rpms.It will obtain
Cell be suspended in 100 microlitres of lysis buffers (10 mMs every liter three (methylol) aminomethane-hydrochloric acid (Tris-HCl)
(pH 8.0), 150 mMs every liter of sodium chloride, the Nonidet P40 (NP-40) of 1% (mass/volume), 0.25 milli
Mole every liter of NaTDC, the glycerol of 1% (mass/volume), 0.1 mM every liter 4- (2- aminoethyl) benzene sulfonyl fluorine
Hydrochloride) in, it is then incubated for 30 minutes on ice, while every 5 minutes are vortexed 30 seconds.After cracking, by cell fragment 4 DEG C with
12000 rpms are centrifuged 20 minutes, and gained supernatant is transferred in new pipe and is immediately available for ALP measurement.
Experimental principle (such as Fig. 1):
The cycle index amplification caused with primer dephosphorylation includes two reaction steps to detect the active principle of ALP:
(1) it is 3'- hydroxylating end that ALP, which is catalyzed the 3'- phosphorylated ends dephosphorylation of primer, what (2) difunctional hairpin probe mediated
Cycle index amplified reaction.Respectively in the terminal modified hydroxyl of 5' and 3' (OH) group and phosphoryl (PO4) group 20 bases
Single-stranded (ss) DNA primer (SEQ ID NO.1).Two hairpin probes of HP1 (SEQ ID NO.2) and HP2 (SEQ ID NO.3)
In, FAM is modified respectively on the thymidine of the adenine sum of the end 5'- of HP1 and HP2, and in the end the 5'- gland with HP1
Thymidine of the adenine matched on the thymidine that purine matches, with the end 5'- thymidine in HP2 at a distance of 2 bases
It is upper to modify quencher BHQ1 respectively.In addition, the stem in HP1 and HP2 all designs the end 3' outstanding DNA sequence dna (23 bases),
So that its respectively with 1 partial hybridization of primer and trigger.Sequence is identified containing nickase (Nt.BstNBI) in the ring of HP1 and HP2
Column, so that generating nicking sites when forming double-stranded DNA (dsDNA).In the presence of ALP, the 3'-PO of ssDNA primer4Group quilt
It is hydrolyzed into 3'-OH group.SsDNA primer with 3'- hydroxylating end is by the end the protrusion 3' DNA sequence dna as primer and HP1
Partial hybridization causes polymerization extension in the presence of archaeal dna polymerase and dNTP.The extension of primer opens the hair clip knot of HP1
Structure results in the complete double-stranded DNA with Nt.BstNBI nicking sites, while by BHQ1 and FAM separation to restore FAM
Fluorescence.Then, Nt.BstNBI cutting DNA chain at specific recognition nicking sites.At new replication site, start
One SDA reaction.It is repeating to extend, after cutting and release cycle, is finally generating many short oligonucleotides (trigger 1).
The trigger 1 of release, which can be used as primer and go out the initiation of DNA sequence dna partial hybridization with the 3' distal process of HP2, polymerize extension,
Cause HP2 hairpin structure to be opened, and forms the recovery of Nt.BstNBI nicking sites and FAM fluorescence in HP2.Then pass through
Nt.BstNBI cuts the new replication site that HP2 nicking sites generate archaeal dna polymerase, causes second of SDA reaction.It polymerize in DNA
In the case of enzyme and nickase exist, start another circulation for extending, cutting and discharging, to generate a large amount of triggers 2.And it touches
Hair device 2 all has the identical sequence in part with primer, therefore trigger 2 can be used as primer and hybridize with the 3' jag of free HP1,
First SDA reaction of starting in the presence of archaeal dna polymerase and nickase.Therefore, concatenated two SDA reaction constitutes closed chain
Formula reaction, causes trigger 1 and trigger 2 largely to generate with exponential form, and eventually lead to the exponential amplification of fluorescence signal.Phase
Instead, in the case where ALP is not present, the 3'-PO of primer4Group is not hydrolyzed into 3'-OH group, therefore cannot start circulation and refer to
It counts amplified reaction and cannot detect fluorescence signal.Utilize the high specific and difunctional hair of ALP catalysis dephosphorylation reaction
The high efficiency for the cycle index amplified reaction that probe mediates is pressed from both sides, this method can be used for highly sensitive detection ALP activity.
The experimental verification of principle
The present invention is followed dependent on what the 3'- phosphorylated ends dephosphorylation of primer and subsequent difunctional hairpin probe mediated
The starting of chain index amplified reaction.In order to study whether the dephosphorylation of ALP catalysis can trigger cycle index amplification, with non-change
Property first SDA of Polyacrylamide Gel Electrophoresis reaction product (Fig. 2A) and entire cycle index amplified reaction (Fig. 2 B)
Product.As shown in Figure 2 A, 19-nt band is observed there are ALP (Fig. 2A swimming lane 3), the trigger with synthesis
The band of 1 (19nt) is identical (Fig. 2A, swimming lane 1), shows that first SDA, which has occurred, to react and produce trigger 1.In addition,
A large amount of bands are observed in the presence of archaeal dna polymerase and dNTPs (Fig. 2A, swimming lane 4), this band is greater than 70nt (size of HP1)
(Fig. 2A, swimming lane 2) shows that, using dephosphorylized DNA primer as primer, extending expansion HP1 by polymerization can produce completely
Or part dsDNA double-strand (i.e. reaction intermediate).It is worth noting that, by the way that 5 microlitres of above-mentioned reaction products are added 15 microlitres
Comprising HP, archaeal dna polymerase, Nt.BstNBI and dNTP solution mixture reaction system in, it can be observed that have one because of ALP
In the presence of and generate 22nt band (Fig. 2 B, swimming lane 5), show HP2 mediate second of SDA reaction beginning and trigger 2
Generation.Furthermore, it was further observed that the band (Fig. 2 B, swimming lane 5) of > 76nt, this band be greater than HP1 (70nt) (Fig. 2 B, swimming lane 3) or
HP2 (76nt) (Fig. 2 B, swimming lane 4) shows that the cycle index amplified reaction that difunctional hairpin probe mediates occurs.In addition, carrying out
Fluorescence measurement uses the HP1 and HP2 of FAM modification as fluorescence indicator with the entire reaction process of real-time monitoring.Such as Fig. 2 C institute
Show, in the presence of ALP, fluorescence intensity is increased in a manner of S-shaped, shows the cycle index amplified reaction that difunctional hairpin probe mediates
Generation and HP1 and HP2 expansion.And in the control group of not ALP, it only detects low background fluorescence signal, shows do not have
The cycle index amplified reaction that difunctional hairpin probe mediates occurs.It can thus be clearly illustrated from Fig. 2, ALP can be effective
Ground is by the 3'-PO of DNA primer4Group is hydrolyzed into 3'-OH group, and the cycle index that the difunctional hairpin probe of subsequent start-up mediates expands
Increase.
Sensitivity experiment.
Under the experiment condition optimized, the variation of various concentration ALP fluorescence intensity is measured.As shown in Figure 3A, with
ALP concentration is from 1 × 10-9Increase to 5 × 10-3(concentration from the bottom to top is followed successively by control (concentration 0), 1 × 10 to U/ μ L-9U/μ
L、1×10-8U/μL、1×10-7U/μL、1×10-6U/μL、5×10-6U/μL、1×10-5U/μL、5×10-5U/μL、1×10- 4U/μL、5×10-4U/μL、1×10-3U/μL、2.5×10-3U/μL、5×10-3U/ μ L), fluorescence intensity is with enhancing.In order to comment
Estimate its quantitative analysis ability, logarithm is taken to the concentration of ALP, observes fluorescence intensity and its log concentration value in a certain concentration range
Good linear relationship is inside showed, and detects limit up to 2.0 × 10-10Every microlitre of unit (Fig. 3 B).Therefore the present invention has super
High detection sensitivity.
Cell sample experiment.
It can be used for the detection of clinical complex sample to verify the present invention, select two kinds of human cancer cells, human cervical carcinoma
The positive and negative sample of cell (HeLa) and human colon adenocarcinoma cell (HT-29) respectively as experiment.As shown in Figure 4 A, only
There is the fluorescence signal of enhancing in human cervical carcinoma cell (HeLa) sample.In order to assess its quantitative analysis ability, to HeLa cell
Number take logarithm, observe fluorescence intensity and its number logarithm show good linear pass within the scope of a certain concentration
System, and detect and limit up to 1 cell (Fig. 4 B).Therefore it present invention can be suitably applied to the detection of clinical complex sample.
The foregoing is merely preferred embodiment of the present application, are not intended to limit this application, for the skill of this field
For art personnel, various changes and changes are possible in this application.Within the spirit and principles of this application, made any to repair
Change, equivalent replacement, improvement etc., should be included within the scope of protection of this application.
SEQUENCE LISTING
<110>Shandong Normal University
<120>method for the cycle index augmentation detection alkaline phosphatase that primer dephosphorylation causes
<130> 2018
<160> 3
<170> PatentIn version 3.3
<210> 1
<211> 20
<212> DNA
<213>artificial sequence
<400> 1
tcgattgatg aacactgcga 20
<210> 2
<211> 70
<212> DNA
<213>artificial sequence
<400> 2
actatacaac ctactacctt tcagactcac gtagtaggtt gtatagtgtt tgtcatcgca 60
gtgttcatca 70
<210> 3
<211> 76
<212> DNA
<213>artificial sequence
<400> 3
tcgcagtgtt catcaatcga ctttcagact cactcgattg atgaacactg cgaatctgag 60
aactatacaa cctact 76
Claims (10)
1. a kind of sensor of cycle index augmentation detection alkaline phosphatase, characterized in that by DNA primer, hairpin probe 1 and hair
Probe 2 is pressed from both sides to form；
DNA primer is single-stranded DNA sequence, and the end 3' of DNA primer is phosphorylated ends, the tool of hairpin probe 1 and hairpin probe 2
There is an end 3' outstanding, the end the 5' base of hairpin probe 1 and hairpin probe 2 modifies fluorophor, the end the 5' alkali with hairpin probe 1
The base modification quenching group of base complementation；
With the base modification quenching group of the end the 5' base complementrity in hairpin probe 2, quenching group and the end 3' in hairpin probe 1
Between base sequence and DNA primer base sequence complementary, 1 restriction endonuclease recognition sequence of the end 5' and hairpin probe of hairpin probe 1
Between base sequence complementary between base sequence and the quenching group in hairpin probe 2 and the end 3', the end 5' of hairpin probe 2 and
The base sequence complementary of base sequence and DNA primer between 2 restriction endonuclease recognition sequence of hairpin probe.
2. sensor as described in claim 1, characterized in that fluorophor FAM, quenching group BHQ1.
3. sensor as described in claim 1, characterized in that DNA primer has 19~21 bases.
4. sensor as described in claim 1, characterized in that sequence of the DNA primer from the end 5' to the end 3' is T CGA TTG
ATG AAC ACT GCG A；
Or, sequence of the hairpin probe 1 from the end 5' to the end 3' isACT ATA CAA CCT ACT ACC TTT CAG ACT CAC
GTA GTA GGT TGT ATA GTG TTT GTC ATC GCA GTG TTC ATC A；
Or, sequence of the hairpin probe 2 from the end 5' to the end 3' isTCG CAG TGT TCA TCA ATC GAC TTT CAG ACT
CAC TCG ATT GAT GAA CAC TGC GAA TCT GAG AAC TAT ACA ACC TAC T。
5. sensor as described in claim 1, characterized in that including archaeal dna polymerase and DNA nickase.
6. a kind of kit of cycle index augmentation detection alkaline phosphatase, characterized in that including any institute of Claims 1 to 5
The sensor stated.
7. kit as claimed in claim 6, characterized in that including 10 × ThermoPol buffer, 10 × NEBuffer
3.1 and dNTP solution mixture.
8. a kind of Claims 1 to 5 any sensor or any kit of claim 6~7 are in cycle index
Application in augmentation detection alkaline phosphatase activities.
9. a kind of method for the cycle index augmentation detection alkaline phosphatase that primer dephosphorylation causes, characterized in that in use
State sensor or kit, process are as follows:
(1) phosphate group at the end alkaline phosphatase catalytic dna primer 3' in prepare liquid is hydrolyzed to hydroxyl；
(2) DNA primer after hydrolyzing hybridize progress first time strand displacement amplification reaction acquisition the with the 3' jag of hairpin probe 1
One trigger simultaneously generates fluorescence signal；
(3) first triggers hybridize second of strand displacement amplification reaction of progress with the 3' jag of hairpin probe 2 and obtain second
A trigger simultaneously generates fluorescence signal；
(4) second triggers are hybridized with the 3' jag of hairpin probe 1, to be repeated in step (2)~(4)；
According to the activity for carrying out the alkaline phosphatase in the fluorescence signal acquisition prepare liquid that step (1)~(4) generate afterwards.
10. method as claimed in claim 9, characterized in that use the mixing of magnesium chloride and three (methylol) aminomethane-hydrochloric acid
Then liquid is heated at high temperature the DNA of hairpin probe 1 is single-stranded or the single-stranded dilution of the DNA of hairpin probe 2 is dissolved room temperature and is incubated for；
Or, step are as follows: after carrying out incubation a period of time at 36~37.5 DEG C after mixing DNA primer and alkaline phosphatase, add
Heat obtains part A to eliminate the activity of alkaline phosphatase；By hairpin probe 1, hairpin probe 2, archaeal dna polymerase, DNA nickase
And after dNTP solution mixture is mixed, part B is obtained, is incubated for one section at 55~60 DEG C after part A and part B are mixed
After time, fluorescence intensity；
Preferably, archaeal dna polymerase is Vent archaeal dna polymerase；
Preferably, DNA nickase is Nt.BstNBI；
Preferably, there are also 10 × ThermoPol buffers and 10 × NEBuffer 3.1 for part B；
Preferably, when detecting fluorescence, excitation wavelength 492nm.
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Cited By (4)
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| CN109988822A (en) * | 2019-04-26 | 2019-07-09 | 山东师范大学 | Controllable self-catalysis cutting mediates fluorescence to restore to detect the sensor and method of hAAG |
| CN110408680A (en) * | 2019-06-28 | 2019-11-05 | 山东师范大学 | For detection of alkaline phosphatase, based on ligase amplified reaction catalysis assembling single quantum dot nano-sensor and application |
| CN111979295A (en) * | 2020-08-13 | 2020-11-24 | 山东师范大学 | Tyrosine phosphatase biosensor and detection method and application thereof |
| CN114517225A (en) * | 2021-12-08 | 2022-05-20 | 山东师范大学 | Single-molecule fluorescent biosensor for detecting alkaline phosphatase and method thereof |
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109988822A (en) * | 2019-04-26 | 2019-07-09 | 山东师范大学 | Controllable self-catalysis cutting mediates fluorescence to restore to detect the sensor and method of hAAG |
| CN109988822B (en) * | 2019-04-26 | 2023-06-02 | 山东师范大学 | Sensor and method for detecting hAAG through controllable autocatalysis cleavage mediated fluorescence recovery |
| CN110408680A (en) * | 2019-06-28 | 2019-11-05 | 山东师范大学 | For detection of alkaline phosphatase, based on ligase amplified reaction catalysis assembling single quantum dot nano-sensor and application |
| CN111979295A (en) * | 2020-08-13 | 2020-11-24 | 山东师范大学 | Tyrosine phosphatase biosensor and detection method and application thereof |
| CN111979295B (en) * | 2020-08-13 | 2023-05-09 | 山东师范大学 | Tyrosine phosphatase biosensor and detection method and application thereof |
| CN114517225A (en) * | 2021-12-08 | 2022-05-20 | 山东师范大学 | Single-molecule fluorescent biosensor for detecting alkaline phosphatase and method thereof |
| CN114517225B (en) * | 2021-12-08 | 2024-04-05 | 山东师范大学 | Single-molecule fluorescent biosensor for detecting alkaline phosphatase and method thereof |
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